Posts Tagged ‘Turmeric’

Disorders of central nervous system | Tags: Amaranth, Ashwagandha, Brahmi, Calamus, Ginseng, Gotu kola, Lebbeck, Neem, Sage, Staff tree, tulasi-cognition-memory-nootropic-human-trial-or-study-or-clinical-alloxan, Tulsi, Turmeric

Herbal medicinal products affecting memory and cognitive disorders

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October 11, 2010 at 7:45 am

Effect of herbal medicinal products on neurotransmission and enhancing cognition

Herbal medicinal products have played a pivotal role in development of CNS-active drugs that affect neurotransmissions in the brain. The interest of CNS-active herbal medicinal products originated from opioid alkaloids, e.g. morphine from Papaver somniferum and the tropane alkaloid cocaine from Erythroxylon coca. Anticholinesterase agents such as physostigmine from Physostigma venosum have shown significant activity on the CNS. This chapter deals with various medicinal plants, and compounds derived from them, which affect neurotransmission related to behaviour and memory dysfunction.

Acorus calamus L. (sweet flag, calamus)

Acorus calamus L. (Araceae) is a semi-aquatic, perennial, aromatic herb with creeping rhizomes. In Ayurveda, herbal medicines with rasayana effects (plants having adaptogen-like properties) are believed to be restorative, to attain longevity, intelligence and freedom from age-related disorders. Acorus calamus is regarded in Ayurvedic medicine as promoting rasayana effects and has been used to treat memory loss. Acorus calamus is used in Ayurvedic medicine on a regular basis for the treatment of loss of memory and other mental disorders. Acorus calamus extract has also been used as a traditional Chinese prescription and its beneficial effects on memory disorder, on learning performance, lipid peroxide content and anti-ageing effect in senescence have been reported. The in-vitro acetylcholinesterase inhibitory effect of hydroalcoholic extract and essential oil of Acorus calamus rhizomes has been reported, based on Ellman’s method. The essential oil showed stronger inhibition than the hydroalcoholic extract. Methanol extracts of Acorus calamus showed significant acetylcholinesterase enzyme inhibition at the concentration 200 µg/mL. Mukherjee et al. (2007) reported the in-vitro acetylcholinesterase inhibitory effect of β-asarone and α-asarone from the Acorus calamus. β-Asarone is at least an order of magnitude more active than its trans isomer α-asarone, so the acetylcholinesterase-inhibitory activity of the oil can be ascribed to β-asarone. Since cognitive performance and memory are related to acetylcholine levels, the acetylcholinesterase-inhibitory effect of the plant may account for its traditional use.

Albizia lebbeck Benth. (lebbeck)

Albizia lebbeck Benth. (Mimosaceae), is a well-known Indian medicinal plant and it has been reported to possess nootropic activity. The saponin-rich n-butanol fraction separated from leaves of Albizia lebbeck has been shown to affect the normal and impaired memory function in rats. Semi-purified saponins at doses of 10, 25 and 50 mg/kg, when administered orally, enhanced the learning and memory of normal and amnesic rats induced by scopolamine. Administration of 10 and 25 mg/kg of the saponin-containing butanol fraction increased the step-down latencies in acquisition and retention period as measured by inflexion ratio on the second and ninth day at levels comparable with the standard nootropic agent piracetum (100 mg/kg) tested in both the passive avoidance ‘step-through test’ using a passive avoidance chamber and the elevated plus maze test.

The nootropic effect of the natural saponins was correlated with concentrations of various neuro-chemicals of the rat brain, since dopamine and GABA levels decreased, and serotonin and noradren-aline increased in the Albizia lebbeck-tteated mice. The memory-enhancing property of the saponin fraction from Albizia lebbeck is considered to be due to inhibition of GABA and enhancement of noradrenaline in the brain. Three active albizia saponins A, B, and C were isolated and identified.

Amaranthus paniculatus L. (amaranth)

Amaranthus paniculatus L. (Amaranthaceae) is said to overcome the problems of psychological stress and affordability and its effects have been tested in stress-induced memory dysfunction. Stress was induced by gamma radiation in mice and methanolic extract of Amaranthus paniculatus at a dose of 600 mg/kg and 800 mg/kg was administered orally for 15 days. It was observed that mice supplemented with the extract, and trained in Hebb William’s maze model D, took less time to reach the goal than those without any treatment. The mice treated with Amaranthus paniculatus were further exposed to gamma radiation by ⁶⁰Co-beam therapy; the surviving mice took less time to reach their goals than those without plant extract. This finding is explained by presuming that mice supplemented with Amaranthus paniculatus have a lower concentration of free radicals formed by the radiation stress and so less damage occurs to the relevant parts of the brain, therefore leading to sustenance of the learning ability even after irradiation. The study has been correlated with the antioxidative property of nutrients and their effect on maintaining cholinergic neurone integrity, which is essential for maintaining the learning and memory process.

Azadirachta indica A. Juss. (neem)

Azadirachta indica (Meliaceae) is a well-known traditional herb in India and is reported to exert therapeutic effects relating to the CNS. Azardirachitin is a major constituents found in neem. The aqueous extract of leaves at the dose of 500 mg/kg for 7 days significantly improved the memory, which was impaired by cerebral hypoperfusion inducing ischaemic insult. The memory task of the ischaemic rats was tested after 2 weeks of hypoperfusion period in the Morris water maze. The aqueous extract from Azadirachta indica significantly prevented the delay in escape latencies and increased the acquisition memory of rats.

Bacopa monniera L. (brahmi)

Bacopa monnieri L. (Scrophulariaceae) has a long history of use in India as an anti-ageing and memory-enhancing ethnobotanical therapy. It has been mentioned in religious, social and medical treatises of India since the time of Atharvan Ved (800 BC); the first clear reference to its CNS effect is to be found in Charak Sambita, written in the first century AD. It is mentioned in the authentic Ayurvedic treatise, Susrutu Sambita, which describes brahmi as efficacious in the loss of intellect and memory.

The alcoholic extract of brahmi showed a beneficial effect on the acquisition, consolidation and retention of three newly acquired behavioural responses in albino rats. Alcoholic extract of brahmi (40 mg/kg) and its two important chemical constituents, bacosides A and B (10 mg/kg), were investigated for shock-motivated brightness discrimination reaction, active conditioned avoidance and conditioned taste aversion response. Preadministration for 3 days with bacosides A and B significantly improved the acquisition, consolidation and retention in all three behavioural paradigms. Beside this effect bacosides attenuated the retrograde amnesia produced by immobilisation-induced stress, and scopolamine.

In another experiment, standardised Bacopa monniera extract was concluded to be beneficial in animal models of Alzheimer’s disease and elevated levels of their central cholinergic markers such as choline acetyltransferase and acetylcholine. Chronic administration of Bacopa monniera given orally for 12 weeks improved the speed of early information processing, verbal learning rate and memory consolidation in humans. This finding supported previous preclinical animal studies and clinical studies in children and patients with anxiety neurosis. Bacopa monniera interferes with cholinergic transmission and also has some serotonergic modulation.

Celastrus paniculatus Willd. (staff tree)

Celastrus paniculatus (Celastraceae) seeds and seed oil have been used in Ayurvedic medicine for stimulating intellect and sharpening the memory. It has been reported to have beneficial effects in psychiatric patients. Administration of the seed oil, rich in sesquiterpenes, to rats also reversed a scopolamine-induced memory deficit assessed in navigational memory performance, but this effect was not associated with acetylcholinesterase activity. The seed oil (3 g/kg) significantly improved the retention ability of the drug-treated rat passive avoidance paradigm and decreased levels of noradrenaline, dopamine, serotonin and their metabolites. Beside this, the memory-enhancing effect of Celastrus paniculatus was correlated with the antioxidant-enhancing effect of the drug on brain tissue. These data indicate that Celastrus paniculatus oil causes an overall decrease in the turnover of the three central monoamines and implicates the involvement of these aminergic systems in the learning and memory process.

Centella asiatica L. (gotu kola)

Centella asiatica (Umbelliferae) is a reputed ancient Ayurvedic remedy to enhance memory and longevity. The pharmacological basis to explain the reputed anti-amnesic effects of Centella asiatica has been explored experimentally. Studies have shown that the alcoholic extract has a tranquillising effect in rats, which was attributed to α-triterpene and brahmoside. Centella asiatica ethanolic extract was also found to elicit a marked increase in neurite outgrowth in human SH-SY5Y cells in the presence of nerve growth factor. Asiatic acid in Centella ethanolic extract showed marked activity at 1 µg/mL. Neurite elongation by Asiatic acid was completely blocked by the extracellular-signal-regulated kinase (ERK) pathway inhibitor PD 098059 (10 urnol/L). Male Sprague-Dawley rats given Centella ethanolic extract in their drinking water (300-330 mg/kg daily) demonstrated more rapid functional recovery and increased axonal regeneration (larger-calibre axons and greater numbers of myelinated axons) compared with controls, indicating that the axons grew at a faster rate. Further studies showed that the extract of Centella asiatica leaf possessed cholinomimetic action in vivo and that it may also influence cholinergic activity, and thus cognitive function.

Cognitive-enhancing effects have been observed in rats following oral administration of an aqueous extract of Centella asiatica, this effect being associated with an antioxidant mechanism in the CNS. The essential oil from Centella asiatica leaf contains monoterpenes, e.g. α-pinene, β-pinene and γ-terpinene, which are reported to inhibit acetylcholinesterase. However, monoterpene acetylcholinesterase inhibitors are weak compared with the anticholinesterase alkaloid, physostigmine. In view of the relatively weak anticholinesterase activity of monoterpenes reported to date, it is unlikely that they would be therapeutically effective in cognitive disorders. Asiatic acid, a triterpene from Centella asiatica (L.) has been patented as a treatment for dementia and an enhancer of cognition by Hoechst (EP 0 383 171 A2).

Clitoria ternatea L.

The root of the Indian medicinal plant Clitoria ternatea (Fabaceae) has a reputation for promoting intellectual behaviour. Clitoria ternatea contains the triterpenes taraxerol and taraxerone as major phytoconstituents. Administration of Clitoria ternatea root extract to rats showed an increase in acetylcholine and choline acetyltransferase in rat brain and they were shown to increase the acetylcholinesterase activity in cortical regions. An aqueous extract of the root also increased acetylcholine levels in rat hippocampus, and it was hypothesised that this effect may be due to an increase in acetylcholine synthesis.

Coptis chinensis Franch.

Coptis chinensis (Ranunculaceae) has been used in traditional Chinese medicine for several conditions. Studies have shown that methanol extract fraction of Coptis chinensis improved scopolamine-induced learning and memory deficit in rats. The contained alkaloids berberine and palmatine have been shown to possess acetylcholinesterase inhibition in vitro.

Curcuma longa L. (turmeric)

Curcuma longa (Zingiberaceae) has also been used for culinary purposes. Turmeric has several components with immunomodulatory and antioxidant properties. Curcumin, an antioxidant present in turmeric, has been shown to protect the brain in vivo from ethanol-induced oxidative stress. It modulated glutathione-linked detoxification enzymes and reduced the lipid peroxidation in rat brain under oxidative stress. Some compounds from C. longa, including curcumin, demethoxycurcumin, bisdemethoxycurcumin and calebin-A (and some of its synthetic analogues), were shown to protect PCI 2 cells from β-amyloid insult in vitro, and this activity was suggested to be due to an antioxidant effect.

In another study using a rat intraventricular Aβinfusion model, curcumin at a dose of 25 mg/kg reduced the isoprostane index of oxidative damage, amyloid plaque burden and Aβ-induced spatial memory deficits in the Morris water maze in rats. Curcumin has been shown to lower the oxidised proteins and interleukin-1β in the transgenic mouse model of Alzheimer’s disease.

Ocimum sanctum L. (tulsi)

In Ayurveda, Ocimum sanctum (Lamiaceae) is described as rasayana. These Ayurvedic rasayanas have been reported in literature to improve physical and mental health, increase non-specific resistance of body, promote physiological functions and augment cognition. The aqueous extract of leaves of Ocimum sanctum at a dose of 500 mg/kg for 7 days significantly improved memory in rats, which was impaired by cerebral hypoperfusion-induced ischaemic insult. The memory task of the ischaemic rats was tested after 2 weeks of hypo-perfusion period in the Morris water maze and those treated with Ocimum sanctum extract had delayed escape latencies. This effect was correlated with their ability to reduce the lipid peroxidation, superoxide dismu-tase and increase in tissue sulphydryl groups and ascorbic acid contents of the hypoperfused brain tissue.

Panax ginseng C.A. Mey. (ginseng)

Interest in the use of Panax ginseng (Araliaceae) comes from its purported ‘adaptogen’ or ‘tonic’ activity, which is thought to increase the body’s capacity to tolerate external stresses, leading to increased physical or mental performance. Panax ginseng alone was tested in young (3 months) and old (26 months) rats, on a battery of negatively reinforced learning tests (two-way active avoidance; passive avoidance/step-down; passive avoidance/step-through), and on the Morris water maze. Ginseng (17, 50, 150 mg/kg), administered orally to young rats, increased the number of avoidance responses in the two-way passive avoidance test at all doses tested.

Although an extensive literature documenting adaptogenic effects in laboratory animal systems exists, results from human clinical studies are conflicting and variable. However, there is evidence that extracts of ginseng can have an immunostimulatory effect in humans, and this may contribute to the adaptogen or tonic effects of these plants. The major secondary products present in ginseng roots are an array of triterpene saponins, collectively called ginsenosides. The ginsenosides, of which there are at least 30, glycosylated derivatives of two major aglycones, panaxadiol and panaxatriol, are considered to be the most relevant for pharmacological activity. From laboratory studies, it has been suggested that the pharmacological target sites for these compounds involve the hypothalamus-pituitary-adrenal axis, owing to the observed effects on serum levels of adrenocorticotrophic hormone and corticosterone.

Salvia species (sage)

Several species of Salvia (Lamiaceae) have been reported to have potential activity in CNS.

Al-Yousuf et al. (2002) reported that Salvia aegyptiaca L. is used for treating various unrelated conditions that include nervous disorders, dizziness and trembling. This work examines some effects of the crude acetone and methanol extracts of the plant given at single oral doses of 0.25, 0.5, 1 or 2 g/kg, on the CNS in mice. It is concluded that the crude methanol and acetone extracts of Salvia aegyptiaca have CNS depressant properties, manifested as antinociception and sedation.

Perry et al. (2002) reported that Salvia lavandulaefolia Vahl. (Spanish sage) extracts and constituents have demonstrated anticholinesterase, antioxidant, anti-inflammatory, oestrogenic and CNS depressant (sedative) effects, all of which are currently relevant to the treatment of Alzheimer’s disease. The essential oil inhibits the enzyme acetylcholinesterase from human brain tissue and bovine erythrocyte and individual monoterpenoid constituents inhibit acetylcholinesterase with varying degrees of potency.

In a study in healthy volunteers, essential oil administration produced significant effects on cognition. In a pilot open-label study involving oral administration of the essential oil to patients with Alzheimer’s disease, a significant increase in diastolic and systolic blood pressure was observed in two patients; however, this may have been due primarily to pre-existing hypertension and there were no abnormalities in other vital signs or blood samples during the trial period.

Salvia elegans Vahl, popularly known as mirto, is a shrub that has been widely used in Mexican traditional medicine for the treatment of different CNS diseases, principally anxiety.

The antidepressant and anxiolytic-like effects of hydroalcoholic (60%) extract of Salvia elegans (leaves and flowers) have been reported in mice. The extract, administered orally, was able to increase the percentage of time spent and the percentage of arm entries in the open arms of the elevated plus maze, as well as to increase the time spent by mice in the illuminated side of the light-dark test, and to decrease the immobility time of mice subjected to the forced swimming test. The same extract was not able to modify the spontaneous locomotor activity measured in the open-field test. These results provide support for the potential antidepressant and anxiolytic activity of Salvia elegans. Wake et al. (2000) also reported that S. elegans displayed differential displacement at nicotinic and muscarinic acetylcholine receptors, with the highest [3H](N)-scopolamine displacement.

In a double-blind, placebo-controlled, crossover study, 30 healthy participants received a different treatment in counterbalanced order on each occasion (placebo, 300, 600 mg dried sage leaf). On each day mood was assessed before the dose and at 1 h and 4 h afterwards. Both doses of sage led to improved ratings of mood in the absence of the stressor (that is, in pre-DISS mood scores) post-dose, with the lower dose reducing anxiety and the higher dose increasing ‘alertness’, ‘calmness’ and ‘contentedness’ on the Bond-Lader mood scales. Task performance was improved for the higher dose at both post-dose sessions, but reduced for the lower dose at the later testing session.

Withania somnifera L. (ashwagandha)

Withania somnifera (Solanaceae) root is one of the most highly regarded herbs in Ayurvedic medicine. Withania somnifera, an Ayurvedic rasayana (memory-facilitating drug), was shown to attenuate amnesic effects in animal models of Alzheimer’s disease by reversal of cholinergic dysfunction induced by ibotenic acid. Ayurvedic formulations based on Withania somnifera induced a similar amnesia-reversal effect in rats. The steroidal derivatives sitoindosides IX and from Withania somnifera, augmented learning acquisition and memory in both young and old rats. The root extract of Withania somnifera reversed scopolamine-induced disruption of acquisition and attention and attenuated amnesia following electroconvulsive shock in mice. These effects are attributed to nootropic activity.

The mechanism of this memory-enhancing effect is attributed to enhanced acetylcholinesterase activity and reversed the ibotenic acid altered cholinergic marker such as acetylcholine and choline acetyl transferase. Therefore preferential action is on cholinergic neurotransmission in the cortical and basal forebrain areas involved in cognitive function. In another experiment Withania somnifera (50 mg/kg) which contains sitoindosides VII-IX and withaferin A as the major bioactive entities, the relative abundance of these compounds in the extract being responsible for 28-30% significant enhancement of leaning as tested in passive avoidance test in chronically stressed rats.

Disorders of central nervous system | Tags: Amaranth, Ashwagandha, Brahmi, Calamus, cognition-herbal-treatment, Ginseng, Gotu kola, Lebbeck, Neem, Sage, Staff tree, tulasi-cognition-memory-nootropic-human-trial-or-study-or-clinical-alloxan, Tulsi, Turmeric

Neurochemistry of cognition and cognitive dysfunction

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October 8, 2010 at 7:39 am

The human brain is very complex and is based on specialised cells designed to transmit information, called neurones. Neurones are an integral part and basic functional unit of the brain, which contains almost one billion of these cells. The neurones consists of a cell body containing a nucleus and an electricity-conducting fibre called an axon, which also gives rise to many branches before ending at nerve terminals. Neurones send signals by transmitting electrical impulses along their axons. When the signals reach the end of the axons, they trigger the release neurotransmitters, which then bind to receptors in adjacent neurones. This point of vital contact is called the synapse. The synaptic response involves the closing and opening of ion channels, which pass through the cell membranes and enable the ions to flow through them. This phenomenon creates an electrical current that provides tiny voltage changes across the membrane which leads to altered synaptic connectivity.

This network is capable of controlling a vast array of activities, including heart rate, body movement, perception, sexual function, emotions, learning and memory. The organisation and neurotransmitter content of intrinsic cerebral cortical and hippocampal neurones, and those of extrinsic inputs to these regions, are described below with respect to neuronal systems known to be affected in Alzheimer-type dementia. For the intrinsic neurones, particular attention is focused on the neuropeptides such as cholecystokinin, vasoactive intestinal polypeptide, somatostatin and neuropeptide Y, which are apparently stable post mortem and provide biochemical markers that can be used to judge the integrity of neuropeptide-containing cells in dementia.

Neurochemistry of cognition and cognitive dysfunction: cholinergic hypothesis

The first neurochemical was identified 70 years ago as acetylcholine. The neurones that release acetyl-choline are called cholinergic neurones and control the heartbeat and voluntary muscles, causing them to contract. Acetylcholine also serves as a neurotrans-mitter in many regions of the brain and plays an important role in learning and memory function. Mammalian brain contains several groups of cholinergic projection neurones located within the basal forebrain and brainstem. Cholinergic axons exert their neurotransmitter effect through the mediation of nicotinic and muscarinic receptors. Cholinergic neurones and cholinergic neurotransmitter pathways are highly implicated in cognition and cognitive dysfunction. The cholinergic neurones are centred on the medial septum, around the vertical limb of the diagonal band of Broca, around the horizontal limb of the diagonal band of Broca, and are also found around the nucleus basalis of Meynert.

All cholinergic neurones of the human basal fore-brain and brain stem contain the cholinergic enzymes choline acetyltransferase and acetylcholinesterase. Much of the research on the participation of neurotransmitter systems in cognitive decline associated with ageing and Alzheimer’s disease has concentrated on the role of acetylcholine, because of its correlation with the degree of cognitive dysfunction, and learning and memory deficits produced in humans, even though the role of interaction between acetylcholine and other neurotransmitters such as noradrenaline, dopamine, serotonin, γ-aminobutyric acid (GABA) and several neuropeptides affecting cognition are also important. To improve cholinergic transmission, different strategies have been suggested including increased acetylcholine synthesis, the augmentation of pre-synaptic acetylcholine release, and stimulation of postsynaptic acetylcholine muscarinic and nicotinic receptors and reduction of acetylcholine synaptic degradation with cholinesterase inhibitors. Several aspects of the functional features of the cholinergic system are shown in Figure: Functional features of the cholinergic system.

Figure: Functional features of the cholinergic system

Assessment of neurodegeneration

Animal models used for the assessment

Drugs effective in neurodegenerative disease should have several aims: to improve the cognitive impairment, control the behavioural and neurological symptoms, delay the progression of the disease and to prevent the onset. To attain these targets, cell and animal models are needed in which pathogenetic hypothesis and potential effectiveness of new drugs are to be tested, exploiting links between the molecular and biochemical studies on the disease and the reality of human pathology. Animal models of Alzheimer’s disease can provide insight into the neurological and pathological mechanisms of cognitive and behavioural changes in patients. Monitoring of behavioural changes in animal models could both provide insight into the neurobiology of these behavioural changes and help validate the felicity of the model to Alzheimer’s disease. Animal models will play a critical role in further defining the events and processes underlying the final phenotypic expression.

Aged animals models

Aged animals (rats, mice and monkeys) have been investigated on a variety of learning and memory tasks. Aged rodents have been shown to have memory impairment on tasks such as the Morris water maze and passive avoidance tests. This behavioural impairment has provided a model that resembles the neuropsychiatric symptoms commonly observed in Alzheimer’s disease. The senescence-accelerated mouse (SAM) exhibits age-related deficits in learning and memory in the Morris water maze and radial arm maze and decreased acetylcholine synthesis in hippocampus pyramidal neurones. The effect of nerve growth factor (NGF) on cognition of aged rats has been assessed by intraven-tricular infusion of NGF, delayed alternation, Morris water maze, and sensory motor tasks. NGF has been most thoroughly assessed in this setting and has shown effects in rats, which have provided a model for the assessment of cholin-ergic neurones and the cholinoprotective effects of compounds potentially useful in the treatment of Alzheimer’s disease.

Brain lesion models

In concert with the recognition of the importance of the cholinergic deficit in Alzheimer’s disease, early models of the disease concentrated on surgical or chemical lesions of the basal forebrain. Transection of the fornix results in degeneration of cholinergic cells in the basal forebrain. These experiments involved primarily rats, monkeys and baboons, which demonstrated deficits in attention and memory, tested in various maze paradigms such as passive active avoidance, Morris water maze and the eightarm radial arm maze. Lesion studies have focused primarily on the behavioural changes of the animals, with only limited attention to the pathological to the neuropsychiatric symptoms commonly observed in Alzheimer’s disease. Ageing studies are all likely to contribute information important to our understanding of the disease, and none are likely to represent a completely isomorphic model that is fully predictive of the pathogenesis, course, and treatment of human Alzheimer’s disease.

Amyloid beta protein infusion induced model of Alzheimer’s disease

Artificially created amyloid (AS) deposits in normal rats, and transgenic mice overexpressing amyloid precursor protein (APP) are the models in which possible treatments are tested. They are aimed at preventing formation of AS deposits or its transformation in neuritic plaques. Synthetic amyloid beta protein (Aβ1-42) application in vitro, using neuroglial and astrocytes, has also been used to screen various neuroprotective drugs. The injection of synthetic Aβ peptides β12-28, β25-35, and β1-40 into the septum of adult rats induced a marked decrease in basal and potassium-evoked acetylcholine release in the hippocampus. These findings confirmed in vivo the neurotoxic effects of Aβ observed in primary neuronal cell cultures exposed to Aβ peptides.

The multiple mechanisms through which Ap peptides, involving oxidative stress, loss of cellular calcium homeostasis and mitochondrial dysfunction have been reviewed and, in both in-vitro and in-vivo experiments, it has been shown that Aβ neurotoxicity depends on its fibrillary aggregation forming a sheet. These preclinical experiments give support to the hypothesis of the pivotal pathogenetic role of AS deposit in Alzheimer’s disease, throw some light on the molecular mechanisms of AS toxicity, and offer an experimental model for testing potentially useful drugs. Undoubtedly, there are limitations in the validity of intracerebral Ap injections as a model of Alzheimer’s disease.

Amyloid precursor protein transgenic mouse model

The development of transgenic mice, mimicking the genetic mutations occurring in familial Alzheimer’s disease and showing some of the neurochemical and morphological alterations of the disease, is another example of the interactions between clinical and preclinical investigations into this disease. The clinical and genetic investigations have identified the early-onset, familial forms of the disease, and the genes in which autosomal dominant mutations take place. There is a clear recognition of autosomal dominant cases induced by mutations of APP (chromosome 21), presenilin 1 (chromosome 14), or presenilin 2 (chromosome 1), which allowed development of transgenic mouse models of Alzheimer’s disease. These transgenic animals exhibit some of the pathological hallmarks of the disease, including neuritic plaques, although they have not evidenced neurofibrillary tangles and have limited cell death. These models facilitate investigation of the relationship of amyloid deposition to other aspects of the pathology of Alzheimer’s disease, including inflammation, hormonal levels, trophic factor influences, calcium metabolism, amino acid toxicity and apoptosis. However, there has been limited behavioural testing of transgenic mice, but impairments of memory have been reported on the Morris water maze, spatial reference memory and Y-maze alternation tasks.

Apolipoprotein knockout models

Cognitive tasks analogous to the deficits observed in human Alzheimer’s disease need to be developed for application to transgenic, knockout and other models currently used to investigate pathogenesis. Tests of language are obviously not applicable but assessment of attention, memory, spatial orientation and executive function are feasible. A variety of transgenic and knockout apolipoprotein models are available to screen novel molecules for the treatment of the disease. The E-4 allele of apolipoprotein (ApoE-4) confers an increased risk for Alzheimer’s disease and a decreased age of onset. Review of the cognitive testing and behavioural measures of the various available animal models reveals the impoverished state of these assessments and the need to develop new evaluation technologies.

Brain inflammation models

Epidemiological and clinical studies, reporting the efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) in reducing the incidence and progression of Alzheimer’s disease, provided strong support for the critical involvement of inflammatory processes in the pathogenesis of the disease. Brain inflammation is considered to be a pathogenetic link in many neurodegenerative diseases, including Alzheimer’s disease. The long-term lipopolysaccharide (LPS) infusion into the fourth ventricle was followed by astrocyte activation, an increase in microglia cells, an increase in the levels of interleukin (IL)-1β, tumour necrosis factor (TNF)-α, APP mRNA and the degeneration of hippocampal neurones.

In-vitro screening methods for acetylcholinesterase inhibition

Several methods have been reported for the screening of acetylcholinesterase inhibitory activity from herbal medicinal products (HMPs). Acetylcholinesterase inhibition was initially detected by the use of gut-bath pharmacological methods with isolated tissue preparations such as guinea pig ileum. These methods are costly in several respects, including time, animal tissue and amounts of compound needed, so they have been replaced by more sensitive chemical methods. Consideration of the relative merits of various methods that might be useful in studying the time course of acetylcholinesterase activity in very small tissue samples use a combined method reported by Koelle (1951) with a sulphydryl reagent studied by Ellman (1959). This Ellman method is extremely sensitive and is applicable to either small amounts of tissue or to low concentrations of enzyme.

Figure: The detection of acetylcholinesterase activity by Ellman’s method.

The principle of this colorimetric method is the measurement of the rate of production of thio-choline, as acetylthiocholine is hydrolysed by the acetylcholinesterase enzyme. Thiocholine reacts with Ellman reagent [5,5'-dithiobis-(2-nitro-benzoic acid) (DTNB)] to produce 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate (Figure: The detection of acetylcholinesterase activity by Ellman’s method). This product has a yellow chromophore that can be detected at 405 nm. The reaction with the thiol has been shown to be sufficiently rapid so as not to be rate limiting in the measurement of the enzyme, and in the concentrations used does not inhibit the enzymatic hydrolysis. The absorbance obtained using a standard volume of a known concentration of the substrate with a fixed dose of acetylcholinesterase is compared with that in the presence of an added compound or extract, a significant reduction indicating an inhibitory role for the substance added.

This visible spectroscopy procedure requires several millilitres of reaction mixture and it was sometimes difficult to obtain enough material to show an effect. The development of the method for use on a smaller scale, using microtitre well plates and a microplate reader, has been introduced and has enabled determinations to be performed with a much higher throughput. Microtitre plate assay method requires smaller amounts of reagents and test substances.

The Ellman reaction has also been adapted for thin-layer chromatography (TLC) bioautography assay for acetylcholinesterase inhibitory activity. The TLC plate is developed in the usual way. After development, enzyme-inhibitory activities of the developed spots were detected by spraying the substrate, dye and enzyme based on Ellman’s method. After incubating the plates, white spots on a yellow background showed inhibition of acetylcholinesterase. A false-positive test is carried out, to confirm any acetylcholinesterase-inhibiting activity arising from inhibition of thiocholine hydrolysis caused by the enzyme. To detect false-positive reactions, the plate is sprayed with substrate, dye and enzyme without test compounds. After a few minutes incubation, a yellow background appeared, the occurrence of white spots indicating false-positive reactions.

A similar method for TLC detection has been introduced by Marston et al. (2002), which uses acetylnaphthol as the substrate and measures the amount of naphthol, the reaction product formed, by its chromogenic reaction with Fast Blue B salt.

High-performance liquid chromatography (HPLC) with online coupled ultraviolet, mass spectrometric and biochemical detection methods for the identification of acetylcholinesterase inhibitors has also been developed. This uses a reverse-phase column with the column eluate being split into two streams, one with an ultraviolet detector and the other connected to a biochemical detection system. This latter system consisted of the eluant being mixed with acetylcholinesterase and DTNB before the online introduction of acetylthiocholine. The intensity of the reaction product was measured at 405 nm by means of a spectrophotometer as an indication of the amount of thiocholine-DTNB product formed. A HPLC method for the detection of acetylcholinesterase inhibition on immobilised acetylcholinesterase column and high-performance liquid chromatography with online coupled ultraviolet, mass spectrometric and biochemical detection for acetylcholinesterase inhibitory activity has also been reported.

Various diseases | Tags: Ashwagandha, Basil, Brahmi, Garcinia, Garlic, Ginkgo, Mango, pal-sawmetto-and-pregnancy, Pomegranate, Sage, Turmeric

Herbal antioxidant products

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October 5, 2010 at 12:56 pm

The ethnomedical literature contains a large number of plants that can be used against diseases, in which ROS are thought to play a major role. A large number of plants and phytoconstituents possess antioxidant properties (Table: Potent antioxidant plants and their phytoconstituents) and many of them are now articles of commerce, claiming to prevent or reduce diseases associated with high levels of ROS. Antioxidants can also be used in the preservation of food products. An important source of antioxidants is the diet, which contains numerous plants with antioxidant activity, including the spices and condiments. The traditional medical literature describes the potential role of spices as a source of many vitamins and as domestic remedies for many human diseases and the consumption of fruits and vegetables, olive oil, red wine and tea is inversely correlated with rates of incidence of many diseases.

Table: Potent antioxidant plants and their phytoconstituents

Plants	Family	Part used	Phytoconstituents
Artemisia monata	Asteraceae	Aerial	Luteolin-7-O-rutinoside and Esculctin
Andrographis paniculata	Acanthaceae	Arial parts	Andrographolide 14-deoxy-11-oxo-andrographolide, neo-andrographolide
Allium sativum	Liliaceace	Bulb	Garlicin, allicin, S-allylcysteine, S-allylmercaptocysteine, allin, allixin, N-acetyl-S-allylcysteine
Anoectochilus formosanus	Orchidaceae	Whole plant	Kinsenone (diarylpentanoid) andflavonoid glycosides
Asparagus racemosus	Liliaceae	Rhizomes	Shatavarin, coniferin and undecanyl cetanoate
Broussonetia papyrifera	Moraceae	Leaf	Broussoflavonols
Bacopa monniera	Scrophulariaceae	Whole plant	BacosideA₃, bacosaponin C
Bulbine capitata	Asphodelaceae	Root	Isofuranonaphthoquinones
Burkea africana	Leguminosae	Bark	Proanthocyanidins
Cedrus decodara	Pinaceae	Heartwood	Matairesinol
Crocus sativus	Iridaceae	Stigmas	Crocin
Curcuma longa	Zingiberaceae	Rhizhomes	Curcumin, turmeric antioxidant protein
Chrysophyllum cainito	Sapotaceae	Seeds, fruits	Quercetin and other polyphenols
Corylus colurna	Betulaceae	Leaves	Flavonoids
Crataegus monogyna	Rosaceae	Leaves, flowers and fruits	Flavonoids, proanthocyanidins, catechins
Cyanchum wilfordii	Asclepiadaceae	Roots	Cynandione A and a biacetophenone
Dalbergia odorifera	Fabaceae	Root	Benzophenone derivative and flavonoids
Dirca palustris	Thymelaeaceae	Twigs	Five novel phenolic glycosides
Daphniphyllum calycinum	Daphniphyllaceae	Leaf	Flavonoid glycoside
Dracaena cinnabari	Ruscaceae (Dracaenaceae)	Whole plant	Homoisoflavonoids
Ephemerantha lonchophylla	Orchidaceae	Stem	Dihydrostilbene, phenantherene
Eucalyptus globulus	Myrtaceae	Leaves	Ellagic acid
Eriobotrya japonica	Rosaceae	Leaves	Flavonoids; chlorogenic acid, quercetin-3-sambubioside, methyl chlorogenate kaempferol, quercetin-3-rhamnoside
Emblica officinalis	Euphorbiaceae	Fruits	Emblicanin A and B, gallic acid, punigluconin and pedunculagin
Ficus bengalensis	Moraceae	Bark	Rhamnoside and cellobioside
Garcinia subelliptica	Clusiaceae	Wood	Three prenylated xanthones
Garcinia kola	Clusiaceae	Seeds	Kolaviron (biflavones)
Camellia sinensis	Theaceae	Leaf	Epigallocatechin, gallocatechin and epigallocatechin gallate
Hordeum vulgare	Poaceae	Leaves	Isovitexin derivatives
Glycyrrhiza glabra	Leguminosae	Roots	Glabridin
Ginkgo biloba	Ginkgoaceae	Leaf	Ginkgolides, bilobalide, sciadopitysin, ginkgetin, bilobetin
Helenium aromaticum	Compositae	Whole plant	Sesquiterpene lactones; helenalin, mexicanin-l, linifolin A, geigerinin
Hedyotis diffusa	Rubiaceae	Fresh aerial	New acylflavonol diglycoside; kaempferol and quercetin derivatives, flavonol and iridoid glycosides
Hierochloe odorata	Poaceae	Aerial	Benzophenanthrone derivatives
Hibiscus syriacus	Malvaceae	Root bark	Lignans: hibiscuside, syringaresinol, E&Z feruloyltyraminesand isoflavonoids
Hypericum erectum	Hypericaceae	Aerial	Flavonoids-quercetrin, hyperoside, isoquercetrin, orientin
Helichrysum picardii	Asteraceae	Aerial	Gnaphalin
Iryanthera lancifolia	Poaceae	Pericarps	Two dihydrochalcones and two flavonolignans
	Amaranthaceae	Aerial	Waxes, β-sitosterol, cam, pestrol and methoxy flavone
Iberis amara	Brassicaceae	Seeds	6-O-sinapoyl sucrose
Lavandula angustifolia	Lamiaceae	Aerial	Phenolics-romarinic acid, caffeic acid, luteolin and methyl carnosoate
Larix gmelini	Pinaceae	Wood	Dihydroquercetin
Mahonia aquifolium	Berberidaceae	Root and leaf	Alkaloids
Muscari racemosum	Liliaceae	Flower	homoisoflavonoids
Magnolia coco	Magnoliaceae	Stem	Lignans-sesamin, fargesin, syringaresinol
Myrica gale	Myricaceae	Fruit	Flavonoids-C-methylated dihydrochalcones, myrigalone A & B
Mangifera indica	Anacardiaceae	Leaves, bark, fruits	Mangiferin, myricetin, protocatechuic acid, quercetin, friedelin, gallic acid, homomangiferin, kaempferol and lupeol
Nelumbo nucifera	Nymphaeaceae	Rhizomes, seed, leaves, flower	Gallic acid
Panax pseudoginseng	Araliaceae	Roots	Trilinolein
Phyllostachys edulis	Poaceae	Leaves	Chlorogenic acid derivatives
Punica granatum	Punicaceae	Fruits	Flavonoids
Prunus cerasus	Rosaceae	Fruits	Cholorgenic acid methylester derivatives
Prunus amygdalus	Rosaceae	Fruit skin	Catechin, protocatechinic acid and flavonoids
Phaseolus aureus	Fabaceae	Seeds	Flavonoids
Pteleopsis hylodendron	Combretaceae	Stem bark	Ellagic acid derivatives
Psoralea corylifolia	Leguminosae	Seeds	Monoterpene phenol-bakuchiol
Pistacia weinmannifolia	Anacardiaceae	Leaves	Pistafolia A
Picrorhiza kurroa	Scrophulariaceae	Roots and rhizomes	Picrovil
Panax ginseng	Araliaceae	Roots	Ginsenosides
Palm spp.	Arecaceae	Oil	Tocotrienols
Podocarpus nagi	Podocarpaceae	Root bark	Totarane diterpenoids
Rosmarinus officinalis	Lamiaceae	Leaves	Diterpenoids
Salvia officinalis	Lamiaceae	Leaves	Phenolics-abietane diterpenes, caffeoyl glycosides, rosmarinic acid
Saururus chinensis	Sauruaceae parts	Underground	Machilin-D
Saururus cernuus	Saururaceae	Whole plant	Feruloylgeraniol derivative
Silybum marianum	Asteraceae	Seeds	Silybin, silymarin
Spinacia oleracea	Amaranthaceae	Leaves	p-Coumaric acid derivative and flavonoids
Terminalta catappa	Euphorbiaceae	Leaves	Punicalagin and punicalin
Terminalia bellerica	Com beta ceae	Fruits	Gallic acid, ellagic acid, ethyl gallate, galloyl glucose, chebulagic acid, bellericanin
Telekia speciosa	Asteraceae	Leaves and Roots	Sesquiterpene lactones
Vaccinium myrtillus	Ericaceae	Leaves	Anthocyanosides
Vitex rotundifolia	Verbenaceae	Fruits	Labdane and abietane-type diterpenoids
Withania somnifera	Solanaceae	Root	Glycowithanolides
Triticum aestivum	Poaceae	Leaves	Ferulic acid dehydrodimers
Vitis vinifera	Vitaceae	Fruits and seeds	Proanthocyanidinsand resveratrol
Zingiber cassumunar	Zingiberaceae	Rhizome	Cassumunin A & B (cucurminoids)
Zingiber officinalis	Zingiberaceae	Rhizome	6-gingerol

Spices and herbs, particularly from the Lamiaceae family, demonstrate strong antioxidant properties and a shortage of antioxidants in the diet might enable diseases caused by reactive oxygen species to arise. Many vegetables and fruits used as foods are particularly rich in natural antioxidant nutrients, e.g. including vitamin C, the tocopherols and carotenoids. Phenolic antioxidants such as flavonoids, tannins, coumarins, xanthenes and more recently, procyanidins, have been shown to scavenge radicals in a dose-dependent manner and therefore are viewed as promising therapeutic potential for free-radical pathologies. A sufficient supply of antioxidants from the diet might help to prevent or delay the occurrence of pathological changes associated with oxidative stress. When diet fails to meet the antioxidant requirements, dietary supplement might be used to enhance health but more needs to be done to test this hypothesis by good clinical studies. Consumer demand for healthy food products provides an opportunity to develop food rich in antioxidants as new functional foods or nutraceuticals.

Gingko biloba (ginkgo)

Gingko biloba extract is widely used in traditional medicine for a great number of therapeutical properties. A large number of studies confirm the antioxidant nature of the extract and its phytoconstituents.

A study by Butnaru et al. (1997) in rats showed that the treatment with this extract before stress inhibited the post-stress growth MDA concentration and the process of stress ulcer formation. Gingko protects against cardiac ischaemia and reperfusion injury and these effects are shown to be dependent on its antioxidant properties. Its in-vivo free-radical scavenging action and proof of its haematological properties in rats was confirmed. Gingko biloba extract and kaem-ferol isolated from it were demonstrated to be antioxidant in a lipid peroxidation assay. Gingko biloba extracts have properties indicative of potential neuroprotective ability. From the n-butanol extract of Gingko biloba leaves, flavonoids were isolated, which showed strong antioxidant activities in DPPH and cytochrome-c reduction assays using the HL-60 cell culture system.

Bacopa monniera (brahmi)

Bacopa monniera is a component of several popular drugs of the Ayurvedic system of medicine. Its ethanol extract showed strong protection against lipid peroxidation induced by ferrous sulphate and cumene hydroperoxide. Bacopa monniera alcohol extract exerted a hepatoprotective effect against morphine-induced liver toxicity, which was found to be related to its antioxidant nature. The effect of a standardised extract of Bacopa monniera was assessed on rat brain frontal cortical, striatial and hippocampal reactive oxygen species, catalase and glutathione peroxi-dase activities and the results indicated a significant antioxidant effect.

Mangifera indica (mango)

The standardised aqueous extract of crude mango stem bark showed a powerful scavenger activity of hydroxyl radicals and hypochlorous acid and acted as an iron chelator. The extract also showed a significant inhibitory effect on the peroxidation of rat brain phospholipid and inhibited DNA damage by bleomycin or copper phenanthrolin system. Oral administration of an M. indica extract (QF 808) was found to reduce ischaemia-induced neuronal loss and oxidative damage in gerbil brain. QF 808 has the ability to scavenge free radicals involved in microsome peroxidation. An aqueous decoction of mango bark has been developed in Cuba on an industrial scale to be used as a nutritional supplement, cosmetic and phytomedicine. It is useful in preventing the production of reactive oxygen species and oxidative tissue damage in vivo. Polyphenols including mangiferin were found to be the major constituents. Mangiferin has shown to be able to maintain the cellular oxidant/antioxidant balance.

Curcuma longa (turmeric and curcumin)

Curcuma longa and a large number of its constituents exhibit potent antioxidant properties in several models. Curcumin and its sodium salt have been shown to have a strong antioxidant activity. Curcumin exhibited a significant time- and concentration-dependent effect on lipid peroxidation induced by radiation and other curcumin analogues exhibited an antioxidant activity stronger than α-tocopherol. Turmeric antioxidant proteins isolated from the aqueous extract of turmeric were found to prevent Ca²⁺-stimulated ATPase from inactivation in the presence of promoters of lipid peroxidation, as well as the depletion of the thiol content during peroxidation.

Withania somnifera (ashwagandha)

Witbania somnifera is used as an antistress adaptogen. Its glycowithanolides showed antioxidant effects in chronic footshock stress-induced perturbations of oxidative free-radical scavenging enzyme and lipid peroxidation in rats. Administration of plant extract, along with equivalent doses of lead acetate for 20 days, significantly decreased lipid peroxidation and increased reactive oxygen species and catalase, thus retaining normal peroxida-tive status of the tissues. The antioxidant effects depend on the presence of steroidal lactones, the withanolides.

Allium sativum (garlic)

The inhibitory property of garlic on reactive oxygen species generation and lipid peroxidation has been reported in a number of in-vitro studies. Banerjee et al. (2002) showed that chronic garlic intake dependently augmented endogenous antioxidants, which might have important direct cytoprotective effects on the heart, especially in the event of oxidative stress-induced injury. Diallyl sulphide is a flavour component from garlic and is found to attenuate lipid peroxidation in mice infected with Tricbinella spiralis. Antioxidant activity of the diallyl sulphide garlicin was due to its ability to scavenge peroxyl or alkoxyl radical intermediates of lipid peroxidation. Allicin, another component, may have multiple mechanisms of action, acting both as a stronger chain-breaking antioxidant and as an inhibitor of first chain reaction by scavenging an initiating radical species.

Punica granatum (pomegranate)

Punica granatum fermented juice and seed oil flavonoids exhibited antioxidant activities and the methanol extract of pomegranate demonstrated potent antioxidant activity using various in-vitro models. Three major anthocyanidins isolated from Punica granatum fruits showed free-radical scavenging activity and inhibitory effects on lipid peroxidation in rat brain homogenates.

Ocimum sanctum (tulsi, holy basil)

An aqueous extract of the leaves of Ocimum sanctum has been found to protect mice against radiation lethality and bone marrow damage and had strong radical scavenging activity in vitro. The extract also protected against radiation-induced lipid peroxidation, where GSH and antioxidant enzymes play an important role in protection. The hydroalcoholic extract, investigated against isoproterenol-induced myocardial infraction in rats, caused a significant reduction in GSH, SOD, LDH and TBARS levels, thus demonstrating antioxidant and cardioprotective effects.

Garcinia

Garcinia kola, Garcinia indica, Garcinia subelliptica, Garcinia atroviridis and several other Garcinia species were found to possess strong antioxidant activity and a large number of active constituents isolated from Garcinia kola fruits possess inhibitory activity against lipid peroxidation. Kolaviron, a mixture of Garcinia biflavonoids 1 and 2 and kolaflavonone isolated from Garcinia kola seed extract, acts as an in-vivo natural antioxidant and effective hepatoprotective and is as effective as BHA in rats. Garcinol, a polyisoprenylated benzophenone derivative isolated from Garcinia indica fruit rind, has shown potent free-radical scavenging activity and was able to scavenge both hydrophilic and hydrophobic reactive oxygen species, the activity being stronger than that of DL-α-tocopherol. Oral administration prevented acute ulceration in rats induced by indometacin and water-immersion stress caused by radical formation.

Emblica officinalis

The fruits of Emblica officinalis contain polyphenolic compounds such as emblicanin A and B which have been reported to exhibit antioxidant activity in vitro and in vivo. A study showed that emblicanin A and B preserve erthrocytes against oxidative stress induced by asbestos, a generator of superoxide radical. The active tannoids administered intra-peritoneally for 7 days showed augmentation of brain SOD, catalase and reduction in lipid peroxidation. The results indicate that the antioxidant activity may reside in these tannoids rather than vitamin C itself. Pretreatment with the butanol extract of the water fraction of Emblica officinalis fruits, orally administrated to rats for 10 consecutive days, was found to prevent indometacin-induced gastric ulcer; this activity was attributed to its antioxidant property.

Salvia officinalis (sage)

Salvia is an important genus, widely cultivated and used in flavouring and folk medicine. It is a rich source of polyphenols, and a large number of these are apparently constructed from the caffeic acid building block. Salvia officinalis 50% methanol extract demonstrated considerable inhibition of lipid peroxidation in both enzyme-dependent and enzyme-independent systems and supercritical fluid extracts of Salvia officinalis also showed antioxidant activity. Salvia officinalis leaves and terpenoids and flavonoids showed strong antioxidant properties using DPPH and by the oil stability index method. Various constituents such as rosmarinic acid, abietene diterpenes and caffeoyl-glycosides have been identified as antioxidant principles. The extracts of other Salvia species also displayed considerable concentration-dependent antioxidative effects that were comparable with those of Salvia officinalis.

Vitis vinifera (grapevine)

Resveratrol is a polyphenolic stilbene occurring in grapes and various other medicinal plants and has been the subject of a considerable amount of recent research. It has been identified as a potential cancer chemopreventive agent and its presence in red wine has been suggested to be linked to the low incidence of heart diseases in France. It acts as a powerful antioxidant, both by classic hydroxyl radical scavenging and also via a novel glutathione-sparing mechanism. Various studies have demonstrated the effects of resveratrol on biological mechanisms involved in cardioprotec-tion. These include modulation of lipid turnover, inhibition of eicosanoid production, prevention of low-density lipoprotein oxidation and inhibition of platelet aggregation.

Based on the quantity and diversity data available on the biological activity of resveratrol, it has to be considered to be a very promising chemoprotector and chemotherapeutic. Urgent investigation on its bioavailability and effects on in-vivo systems, especially in humans, are necessary. Martinez and Moreno (2000) showed that resveratrol treatment caused a significant impairment of COX-2 induction, stimulated by lipopolysaccharides and phorbol esters or by O₂^- or H₂O₂ exposure. It also significantly decreased [³H]arachidonic acid release induced by these agents. These results support the anti-inflammatory action of resveratrol.

Silybum marianum (milk thistle)

Silybum marianum fruits are reported to exert antioxidant and free-radical scavenging action. Silymarin and silybin, the flavonolignans present, were found to be the active constituents. Silymarin prevents doxorubicin-mediated damage to rat heart membrane primarily through free radical scavenging. Oral administration of silybin protected against iron-induced hepatotoxicity in vivo and can be used in chelation therapy of chronic iron overload.

Conclusions

It is obvious that a large number of plants possess strong antioxidant potential and these include commonly used fruits, vegetables and spices. Concentration of total phenols in the plant show close correlation with the antioxidant activity, so it is useful to determine the total phenol content of the plants before antioxidant screening. A large number of plants have been tested, based on their uses in folklore and have been found to be active, and still there is scope for antioxidant screening of a large number of plants belonging to families rich in antioxidants. Fermented extracts have been found to be more potent and the production of low-molecular-weight compounds during fermentation is responsible for the action. There is scope for evaluation of antioxidant properties of fermented extracts of plants known to possess strong antioxidant properties.

Various diseases | Tags: Ashwagandha, Basil, Brahmi, Garcinia, Garlic, Ginkgo, Mango, Pomegranate, Sage, Turmeric

Antioxidants

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October 2, 2010 at 12:49 pm

Free radicals are chemical species possessing one or more unpaired electrons and can be considered as a fragment of molecules that are extremely reactive and short lived. They are produced continuously in cells, either as accidental byproducts of metabolism or deliberately (for example, during phagocytosis). Unpaired electrons usually make a molecule more reactive than the corresponding non-radical. The molecule acts as an electron acceptor and essentially ‘steals’ electrons from other molecules. Free radicals are referred to as oxidising agents since they cause other molecules to donate their electrons. Free radicals can be formed by the homolytic cleavage of a covalent bond of a normal molecule, with each fragment retaining one unpaired electron; by the loss of a single electron from a normal molecule; or by the addition of a single electron to a normal molecule.

The most common cellular oxygen free radicals are superoxide radical (0₂^-), hydroxyl radical (OH^·) and nitric oxide (NO). Other molecules, such as hydrogen peroxide (H₂0₂) and peroxynitrate (ONOO^·) are not free radicals themselves but can lead to their generation through various chemical reactions.

Oxygen free radicals and related molecules are often classified together as reactive oxygen species (ROS), to signify their ability to promote oxidative changes within the cell.

All aerobic organisms produce free radicals, predominantly superoxide, formed as a side product during the reduction of molecular oxygen by mitochondria. An average cell utilises 10¹³ molecules of 0₂ per day. It is estimated that 1 % of respired molecular oxygen will form ROS, thus approximately 10¹¹ ROS are produced by each cell in a day. Cells normally employ a number of defence mechanisms against damage induced by free radicals. Oxidative stress is the term referring to the imbalance between generation of reactive oxygen species and the activity of the antioxidant defences.

There is increasing evidence to support the involvement of free-radical reactions in several human diseases since reactive oxygen species play a role in a variety of normal regulatory systems, the de-regulation of which may play an important role in inflammation. ROS and other free radicals have long been known to be mutagenic and have more recently emerged as mediators of other phenotypic and genotypic changes causing mutations and neoplasia.

In the last decade, evidence has accumulated that the free-radical process known as lipid peroxidation plays a crucial and causative role in the pathogenesis of atherosclerosis, cancer, myocardial infarction and also in ageing. Participation of free-radical oxidative interactions in promoting tissue injury in conditions such as brain trauma, ischaemia, toxicity and also in neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s dementia, multiple sclerosis and lipofuscinosis are now well documented. The involvement of ROS in the pathogenesis of several lung diseases has also been suggested while the pioneering studies on the role of reactive oxygen species reactions in the genesis and the expression of cellular and tissue damage has been carried out mainly in the liver, using acute rat poisoning with carbon tetrachloride.

Studies in experimental models have incriminated ROS as primary mediators in the pathogenesis of renal injury. Diabetes mellitus is also associated with oxidative reactions, particularly those that are catalysed by decompartmentalised transition metals, but their causative significance in diabetic tissue damage remains to be established.

In 1956, Harman proposed the free-radical theory of ageing, the assumption that ageing results from random deleterious effects of tissue brought about by ROS and it is very likely that reactive oxygen species contribute considerably to the development of stochastic disorders observed during the progress of ageing.

In recent years, increasing experimental and clinical data have provided compelling evidence for the involvement of oxygen free radicals in the three main disorders of prematurity — chronic lung disease, retinopathy of prematurity and intraventicular haemorrhage, the hypothesis being that oxygen-centred radical and related reactive oxygen metabolites are formed too rapidly to be detoxified by antioxidant defence mechanisms.

Defence against free radicals: antioxidants

Antioxidant defences fall in to two main categories, those whose role is to prevent the generation of free radicals and those that intercept any radicals that are generated. They exist in both the aqueous and membrane compartments of cells and can be enzymes or non-enzymes. Various animal studies have shown that antioxidants delay or protect against the oxidative damage produced by the free-radical reaction and a protective role against ailments mediated by free radicals is now well established.

Antioxidants are exogenous (natural or synthetic) or endogenous compounds acting in several ways, including removal of O₂, scavenging reactive oxygen/ nitrogen species or their precursors, inhibition of reactive oxygen species formation and binding metal ions needed for catalysis of ROS generation and up-regulation of endogenous antioxidant defences. The protective efficacy of antioxidants depends on the type of reactive oxygen species that is generated, the place of generation and the severity of the damage. The natural antioxidant system can be classified into two major groups: endogenous enzymes and low-molecular-weight antioxidants.

Endogenous enzymes include extensively studied enzymes such as superoxide dismutase (SOD), cata-lase, glutathione peroxidases, DT diaphorase, and glutathione-regenerating enzyme systems. Some enzymatic systems such as SOD and catalase act specifically against reactive oxygen species, while certain other enzyme systems reduce thiols. The low-molecular-weight antioxidants can be further classified into directly acting antioxidants (e.g. scavengers and chain-breaking antioxidants) and indirectly acting antioxidants (e.g. chelating agents). The directly acting antioxidants are extremely important for defence against oxidative stress. Direct scavenging of ROS is one of the many antioxidant actions required to restore oxidative equilibrium once it is lost in different pathologies. This subgroup of antioxidants currently contains several hundred compounds including ascorbic acid (vitamin C), retinoic acid (vitamin A), melatonin, lipoic acids, polyphenols, and carotenoids, being derived from dietary and herbal sources. The hypothesis that restoring redox equilibrium through activation of intracellular signals is also an important step of the antioxidation process is gaining increasing support. It is likely that the trapping of excess free radicals could restore redox equilibrium in the initial states of cellular oxidative stress.

Free radicals in various diseases

According to Halliwell and Gutteridge (1999), oxidative stress occurs in most human diseases, although this is not the same as saying that it is the cause of most diseases. The increase in free radicals may be secondary to the disease process. Free radicals are very short lived and difficult to study in vivo. Direct detection of free radicals is possible with electron spin resonance, but it is very expensive and complex, so a variety of surrogate markers to ascertain free-radical activity must be used. Developing accurate methods to measure biomarkers for DNA damage and lipid peroxidation is challenging and methods in the current literature include urine levels of F2-isoprostanes as a biomarker for lipid peroxidation, measurement of oxidised low-density lipoprotein (LDL), use of a chemical mutagenic product of fat oxidation, and 8-oxo-deoxyguanosine, associated with a decline in mitochondrial function. There have also been efforts to detect changes in the levels of antioxidants such as SOD, glutathione or vitamin E in the body in response to oxidative stress, to identify many conditions associated with free-radical formation, but results have not been consistent. The implications of the presence of ROS in cardiovascular, pulmonary, carcinogenesis, diabetes and neurological diseases as well as inflammation are currently under intense investigation.

It is easy to appreciate that the lungs are vulnerable to inhaled agents, e.g. ozone, nitrogen dioxide, sulphur dioxide and other toxins, that stimulate reactive oxygen species production. ROS can stimulate lipid peroxidation and oxidation of DNA bases in the lungs. The irritant effect of smoke also activates lung macrophages and neutrophils with resultant production of additional ROS. Chronic lung inflammation such as asbestosis, asthma and cystic fibrosis is also associated with elevated markers of oxidative stress so reactive oxygen species may contribute to the ongoing pathology.

The brain may be especially sensitive to oxidative damage. Oxidative stress can damage neurones and glial cells in a manner similar to other issues: via products of lipid peroxidation that are neurotoxic, DNA damage, etc. Reper-fusion injury also occurs in the brain after a stroke and superoxide produced during reperfusion results in abnormalities of cerebral vascular responses and blood-brain barrier permeability. Extracellular gluta-mate levels in the brain increase rapidly during ischaemia, leading to increased production of OH radicals, calcium ion imbalance and increased neurotoxicity. If bleeding occurs with the stroke, normally sequestered iron molecules are released and may initiate harmful free-radical chain reactions. Neurodegenerative diseases associated with ROS include Parkinson’s, Alzheimer’s, and many others. It is possible that although the initiators of the disease state vary, free radicals are involved in a common pathway that leads to neural cell death.

The acute inflammatory response is typically beneficial to the organism, being a major defence against microorganisms and normally self-limited. However, the superoxide-producing neutrophil itself is destroyed in the process and healthy surrounding cells may also be damaged. With chronic inflammation, such as in rheumatoid arthritis, the overall impact of the continued generation of free radicals is deleterious. Degradation of hyaluronic acid (synovial fluid) is driven by the presence of OH. These radicals may be produced by phagocytic cells in the joint, by changes in tissue oxygenation caused by swelling, followed by reperfusion, or by some of the drugs used to treat RA. A role for reactive oxygen species in the endothelial dysfunction associated with diabetes was proposed and levels of manganese superoxide dismutase have been reported to be decreased in streptozotocin-induced diabetes in rats. Normalising mitochondrial O₂ has been shown to block pathways involved in hyperglycaemic damage. Consistent with these observations, SOD pretreatment improved vasodilation in isolated aortic rings from streptozo-tocin diabetic rats. Levels of O₂ are also increased in hyperinsulinaemic rats, which is believed to be related to activation of NAD(P)H oxidase.

The progression of heart failure is associated with programmed cell death or apoptosis, which studies suggest occurs in response to ischaemia, reperfusion, pressure overload and in dilated cardiomyopathies. Oxidative stress may also be critical for the activation of apoptosis in dilated cardiomyopathies.

Free radicals in cardiovascular diseases

Cardiovascular disease is a heterogeneous group of disorders that affects the heart and blood vessels. The diseases are characterised by angina pectoris, hypertension, congestive heart failure, acute myocardial infarction (heart attacks), stroke and arrhythmia. There is now considerable biochemical, physiological and pharmacological data to support a connection between free-radical reactions and cardiovascular tissue injury. Evidence shows that these disease conditions are directly or indirectly related to oxidative damage and share common mechanisms of molecular and cellular damage. As these mechanisms are elucidated, it may be possible to improve the techniques for clinical and pharmacological intervention.

Ischaemia-reperfusion myocardial injury

Exposure of myocardial tissue to a brief, transient ischaemia, followed by reperfusion, has attracted much attention in recent years as an explanation for some cardiac diseases. Myocardial ischaemia occurs when myocardial oxygen demand exceeds oxygen supply. Unless reversed, this situation results in cell injury and, clinically, myocardial infarction. Logically, reperfusion of ischaemic myocardium is recognised as potentially beneficial, because mortality is directly proportional to infarct size, and this latter to the severity and duration of ischaemia. Reperfusion of the ischaemic myocardium can restore oxygen and substrates to the ischaemic myocardial cells, but this process may create another form of myocardial damage termed ‘reperfusion injury’. Thus, restoration of a normal blood flow in the heart by methods such as angioplasty, thrombolytic agents or cardiopulmonary bypass can lead to specific lesions (arrhythmias, deficit in contractility, necrosis), the importance of which also depends on the duration of ischaemia.

Evidence suggests that this may be due, in part, to the generation of toxic reactive oxygen species. The active involvement of ROS in the ischaemia-reperfusion damage is demonstrated by direct and indirect experimental evidences. Direct evidence arises from the possibility of measuring radicals in myocardial tissue by electron spin resonance (ESR) and spin trapping methodology; indirect evidence by the measurement of the products of free-radical attack on biological substrates (e.g. malondialdehyde as a measure of lipid peroxidation extent), and intracellular and extracellular antioxidant capacity. Experimental findings suggest that in ischaemic tissue there is an impairment of antioxidant mechanisms. Evidence to support this statement comes also from the cardioprotective effects of agents capable of inducing antioxidant enzymes in the heart and from the beneficial effects of several enzymatic free-radical scavengers, anti-oxidants and iron chelators in reperfused myocardium.

Free-radical hypothesis of atherosclerosis

Considerable in-vivo evidence, animal and human, supports the important role of reactive oxygen species in atherosclerotic coronary heart disease. While the exact mechanisms for atherogenesis are not completely understood, recent studies suggest that oxidative modification of low-density lipoproteins (LDL) is a critical factor. LDL may be oxidatively modified by all major cell types of the arterial wall via their extracellular release of reactive oxygen species (ROS). Hydroxyl radicals (thus formed) may initiate the peroxidation of long-chain polyunsaturated fatty acids within LDL, giving rise to conjugated dienes and lipid hydroperoxy radicals (LOO^·). This process is self propagating, since LOO^· can attack adjacent fatty acids until complete fatty acid chain fragmentation occurs. A number of highly reactive products then accumulate in the LDL particle, including malondialdehyde and lysophos-phatides, which interact with the amino side chain of the apoprotein B 100 and modify it to form new epitopes that are not recognised by the LDL receptor.

Hypertension

Essential hypertension (EH) appears associated with increased superoxide anion and hydrogen peroxide production, as well as decreased antioxidant capacity. The involvement of reactive oxygen species in EH is also suggested by the observation of increased level of lipid peroxides and decreased concentrations of antioxidant vitamin E in plasma of EH patients. Recently, Simi et al. (1998) have shown that patients with EH have plasma concentrations of free-radical scavengers lower than healthy normotensive subjects. The elevated consumption of plasma antioxidants was accompanied by increased activity of extracellular antioxidant enzymes (glutathione peroxidase and SOD), suggesting that ROS production in EH overwhelms antioxidant defence capacity. Oxidative stress in patients with EH is accompanied with the decreased red blood cell counts and decreased SOD and glutathione peroxidase activity in neutrophils.

Chronic heart failure

Chronic heart failure is a state characterised by a number of processes that may promote reactive oxygen species generation in vivo, including cytokine activation, recurrent hypoxia-reperfusion, possibly genetic susceptibilities and activation of the renin-angiotensin system. There are a number of potential cellular sources implicated in enhanced ROS generation in chronic heart failure. It has recently been demonstrated that patients with chronic heart failure may have increased leucocyte 0₂^- production, which is, in turn, related to severity of disease. Other sources of enhanced ROS generation in human chronic heart failure are both the myocardium and peripheral blood vessels. Increased activity of myocardial NADPH oxidase has been reported in heart failure.

Myocardial damage

Reactive oxygen species (ROS) have direct effects on cellular structure and function and may be integral signalling molecules in myocardial remodelling and failure. ROS result in a phenotype characterised by hypertrophy and apoptosis in isolated cardiac myocytes. ROS have also been shown to activate matrix metalloproteinase (MMP) in cardiac fibroblasts. Myocardial MMP activity is increased in the failing heart and an MMP inhibitor has been shown to limit early left ventricular dilatation in a murine model of myocardial infarction (MI). Hayashidani et al. (2003) showed significant improvement in the survival after MI in MMP-2 knockout mice, which was mainly attributable to the inhibition of early cardiac rupture and the development of subsequent LV dysfunction. Because MMP can be activated by reactive oxygen species, one proposed mechanism of ventricular remodelling is the activation of MMPs secondary to increased ROS production. Sustained MMP activation might influence the structural properties of the myocardium by providing an abnormal extracellular environment with which the myocytes interact. Kinugawa et al. (2000) demonstrated that the OH scavenger, dimethylthiourea, inhibits the activation of MMP-2 in association with the development of ventricular remodelling and failure. These data raise the interesting possibility that increased ROS after MI can be a stimulus for myocardial MMP activation, which might play an important role in the development of HF.

Left ventricular hypertrophy

In animal models of heart failure, levels of ROS are elevated and cardiac protection is observed with antioxidant treatment. The increase in ROS associated with left ventricular hypertrophy appears to be NAD(P)H oxidase-dependent. Myocardial NAD(P)H oxidase activity is elevated and expression of p22phox, gp91phox, p67phox and p47phox is increased in left ventricular tissue from guinea pigs after aortic banding. The gp91phox containing NAD(P)H oxidase has been shown to play an important role in the cardiac hypertrophic response to Ang II in mice. It has been suggested that the increase in ROS is responsible for impaired endothelial regulation of left ventricular relaxation observed in moderate pressure overload left ventricular hypertrophy.

Cardiac hypertrophy occurs in response to a sustained increase in cardiac work. The mechanisms underlying this progression from compensated hypertrophy to decompensated heart failure remain poorly understood and incompletely explored. There are data supporting at least a contributory role for alterations in ROS production in the pathophysiology of cardiac hypertrophy. There is substantial evidence from animal studies indicating that reactive oxygen species, and particularly O₂, production is increased in cardiac hypertrophy. Recently, Date et al. demonstrated attenuated cardiac hypertrophy in mice subjected to pressure overload following treatment with the free-radical scavenger, N-2-mercaptopropionyl glycine. This is the first evidence in an experimental model suggesting a causal role for ROS in the development of pressure overload hypertrophy. The precise source of reactive oxygen species in this study was not apparent. In a similar study using a guinea pig model of pressure overload, an attenuation of LV hypertrophy was observed in animals treated with vitamin E. Taken together, these data support an important functional role for reactive oxygen species, in particular NADPH oxidase derived ROS, in the development of pressure-overload hypertrophy.

Free radicals in hypercholesterolaemia

Increased levels of O₂ generation and attenuated NO mediated responses have been demonstrated in aortic rings from cholesterolfed rabbits. Treatment of the animals with polyethylene glycolated SODs improved endothelium-dependent vasodilation. Supplementation with L-arginine has also been shown to reduce O₂ levels and restore NO-mediated responses in cholesterol-fed animals (Boger et al., 1995). O₂ levels are also raised in WHHL (Watanabe heritable hyperlipidaemic) rabbits. Multiple mechanisms appear to be involved in O₂^-production in association with hypercholesterolaemia. Stepp and colleagues provided evidence that in canine carotid arteries eNOS, mechanisms dependent on xanthine oxidase and possibly NAD(P)H-oxidase were involved. Further evidence for the involvement of NAD(P)H oxidase was obtained in WHHL rabbits. In monkeys with atherosclerosis, disease severity is related to O₂levels, and regression of atherosclerosis is associated with decreases in O₂ levels and NAD(P)H oxidase activity.

Free radicals in skeletal muscle dysfunction

Oxidative stress could be the mechanistic basis also for muscle fatigue and reduced exercise tolerance in patients with heart failure. This notion is supported by a positive correlation between ROS and exercise intolerance in these patients. Further, Tsutsui et al. (2001) demonstrated that the production of reactive oxygen species was increased in the skeletal muscle homogenates obtained from a murine model of HF and increased ROS were identified as OH originating from O₂, which was associated with a concomitant increase in the oxidation of lipids. These results are consistent with the previous studies that the oxidative capacity is reduced and O₂utilisation is inadequate in skeletal muscle mitochondria from patients with heart failure. Skeletal muscle mitochondria from heart failure are associated with a decrease in the activities of complex I and complex III. As has been shown in the failing hearts, the defects in electron transfer function may lead to reactive oxygen species production. ROS may play an important role in the muscle atrophy commonly seen in patients with heart failure through the induction of apoptosis. In addition, ROS impair myoplasmic Ca²⁺ homeostasis and inhibit the oxidative energy production in the mitochondria, both of which may contribute to the muscle contractile dysfunction. An attempt to attenuate oxidative stress would improve, to some extent, the exercise capacity of patients with heart failure.

Tests for antioxidant activity

Antioxidant activity can be evaluated both in vitro and in vivo. There are potential models for evaluation of the antioxidant activity. Animals such as mice, rats, guinea pigs and rabbits can be used for in-vivo evaluation with the oxidative stress induced by some external chemical agent (e.g. carbon tetrachloride), physical, emotional, mental or environmental stress (e.g. torturing the animals, depriving animals from food, water and sexual activity, increasing noise or temperature of the animal housing). Even surgery can be performed for inducing oxidative stress in rats, e.g. cerebral ischaemia/reperfusion induced oxidative stress in which the induction of ischaemia in rats was performed by occluding bilateral common carotid arteries with clamps for 30 min followed by 24 h reperfusion. Following any of the methods whereby the oxidative stress can be induced in the animals, they should be grouped as treated (at least two or more doses), control and normal animals. In the end of study the animals can be sacrificed to isolate the vital organs. Enzymes such as SOD, catalase and glutathione can be measured in these tissues, together with the extent of lipid peroxidation caused by the oxidative stress, using assays such as barbituric acid reactive substances (TBARS).

In-vitro methods consist of chemical methods in which free radicals can be generated using chemical reactions, e.g. nitric oxide method or chemicals which themselves act as the source of free radicals such as DPPH (2,2-diphenyl-1-picrylhydrazyl). In-vitro methods are also available in which generated free radicals can attack tissues isolated from the animal body leading to the oxidation of lipids present in the tissues, e.g. thiobarbituric acid-reactive substances (TBARS) assay. Details on some of the in-vitro methods used for the evaluation of antioxidant activity are given below.

DPPH radical scavenging assay

The antioxidant activity of the plant extract and pure compounds was assessed on the basis of radical scavenging effect of the stable DPPH free radical, which is purple. Antioxidants react with DPPH, and convert it to 1,1-diphenyl-2-(2,4,6-trinitrophenyl) hydrazine, which is colourless. Reaction mixtures containing test samples (dissolved in DMSO) and 300 µmol/L DPPH ethanolic solutions in 96-well microtitre plates are incubated at 37°C for 30 min, and absorbances measured at 515 nm. The degree of discolouration indicates the scavenging potentials of the antioxidant compounds and IC50 values can be calculated, i.e. the concentration of sample required to scavenge 50% DPPH free radicals. DPPH reagent (0.5% in methanol) can be sprayed on to preparative TLC plates to identify active antioxidant compounds in plant extracts. Active radical scavengers give yellow colour zones against a purple background.

Nitric oxide radical scavenging assay

Nitric oxide (NO^·) is a free radical and scavengers of nitric oxide compete with oxygen, leading to reduced production of nitric oxide. NO is generated from sodium nitroprusside and measured by the Griess Illosvoy reagent (Garratt, 1964), which can be modified by using naphthylethylenediamine dihydro-chloride (0.1% w/v) instead of 1-naphthylamine (5%). The extent of NO radical scavenging can be assessed by colorimetry whereby reaction mixtures containing 10 mmol/L sodium nitroprusside, phosphate buffer saline and extracts or standard solution are incubated at 25°C for 150 min. After incubation, 0.5 mL of the reaction mixture is mixed with 1 mL of sulphanilic acid reagent (0.33% in 20% glacial acetic acid) and allowed to stand for 5 min to complete diazotisation. Naphthyl ethylenediamine dihydrochloride is then added, mixed and allowed to stand for 30 min at 25°C and a pink coloured chro-mophore is formed in diffused light whose intensity is measured at 540 nm.

Scavenging of superoxideanion radicals assay

Various cellular enzymes can catalyse chemical reactions involving molecular oxygen, including admission formation of superoxide radicals, which can inactivate vital cell components. Superoxide can be generated by enzymatic oxidation of hypoxanthine with xanthine oxidase and can be detected colori-metrically by nitroblue tetrazolium (NBT) reduction. The reaction is started by adding 100 µL of phenazine methosulphate (PMS) solution (60 µmol/L PMS in 100 mmol/L phosphate buffer, pH 7.4) to the mixture, incubating at 25°C for 5 min, and measuring the absorbance at 560 nm. Decreased absorbance of the reaction mixture indicates increased superoxide anion scavenging activity.

Deoxyribose degradation assay

In this method hydroxyl radicals are generated by incubating a mixture containing KH₂PO₄-KOH, H₂O₂, FeCl₂-EDTA and deoxyribose. The extent of deoxyribose degradation by the formed hydroxyl radical can be assessed by the thiobarbituric acid method. The typical reaction is started by adding Fe(II) at a final concentration of 6 µmol/L to a 0.5 mL final volume of 20 mmol/L phosphate buffer, 5 mmol/L of 2-deoxyribose, Cu(II) (5 µmol/L) (pH 7.2) and 100 µmol/L H₂O₂ with and without 10 µmol/L of ascorbate as an iron chelator. Reactions were carried out for 10 min at 25 °C ± 1°C and were stopped by adding of 0.5 mL of 50 mmol/L NaOH containing 4% (w/v) phosphoric acid. After boiling for 15 min, the absorbance of the solution containing the oxidation products is measured at 532 nm.

Thiobarbituric-acid-reactive substances assay

In this method the lipid peroxidation is measured in terms of malondialdehyde (MDA) content following the thiobarbituric acid method of Ohkawa et al. (1979). MDA is formed in vivo and in vitro through oxidation of unsaturated lipids by ROS, and other oxidative agents. Thiobarbituric acid reacts with MDA to form a pink chromogen, which can be detected spectrophotometrically at 532 nm.

β-Carotene-linoleic acid (linoleate) assay

The antioxidant activity is measured by the ability of a compound to minimise the coupled oxidation of linoleic acid and β-carotene in an emulsified aqueous system. β-carotene loses its orange colour when reacting with reactive oxygen species, so colorimetery can be used to investigate the decline in colour caused by oxidative stress. In this method a stock solution of β-carotene and linoleic acid is prepared by dissolving 0.5 mg of β-carotene in 1 mL of chloroform and adding 25 µL of linoleic acid together with 200 mg of Tween 40, evaporating the chloroform and adding 100 mL of aerated water to the residue. To 2.5 mL of this mixture, 300 µL of extract is added and the mixture incubated in boiling water for 2 h together with two blanks, one containing the antioxidant BHT and the other without antioxidant, before measuring the absorbance at 470 nm.

DNA nicking assay

The ability of a test drug to prevent the DNA damage caused by agents such as 2,2′-azobis (2-methylpropionamide) dihydrochloride (APPH) is measured in this method. The test substance is mixed with DNA and APPH, dissolved in phosphate-buffered saline, is added to start the reaction. The resultant mix is developed on agarose gel, elec-trophoresis carried out and then staining with ethidium bromide. DNA bands are visualised under illuminated ultraviolet light and examined for DNA breakage.

Diseases of circulatory system | Tags: Ashwagandha, Ginseng, Green tea, Red grapes, St. John’s Wort, Turmeric

Herbal products with angiogenesis-inhibitory activity

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September 16, 2010 at 8:11 am

Angiogenesis modulators are present in a wide range of plant products, some of which are also consumed on a daily basis through diets in certain ethnic populations. In addition, herbal products derived from specific medicinal plants known for their curative properties on chronic angiogenesis-dependent conditions are also gaining recognition for their principal active agents.

Curcuma longa (turmeric)

The staple in India’s armoury of wound-healing plants is the common spice plant Curcuma longa (turmeric), used for injuries, burns, and as an all-purpose, topical anti-inflammatory. The principal active substance is curcumin. The use of curcumin as an inhibitor of angiogenesis has only recently been appreciated, despite great interest in this natural product for cancer chemoprevention. We showed that local delivery of curcuminoid pellets (2 mg), implanted in the cornea of rabbits, blocked angiogenesis induced by fibro-blast growth factor 2, and even oral delivery of curcuminoids to mice blocked angiogenesis induced by the same growth factor in the mouse corneal model of neovascularisation.

The anti-angiogenic activity of this class of inhibitor was demonstrated as acting through the targeting of gene expression of MMP-9, a critical proteolytic enzyme that cleaves gelatinous substrates of the vascular basement membrane. This gene expression blockade of MMP-9 was found to occur through the inhibition of AP-1 and NF-kB transcription factors, two critically important activators of proliferative and inflammatory cytokine genes. The use of turmeric in promoting growth of blood vessels to heal wounds has also been remarkable. Contrary to the anti-angiogenic activity of curcumin, its wound-healing properties are mediated through promotion of angiogenesis.

The mechanism of this natural product is believed to be dependent on disease contexts. For instance, it was shown that one of curcumin’s targets is the kinase that is responsible for activating the multipurpose signalling complex, the COP9 signalosome. This complex lies at the interface of a number of divergent stress signalling cascades, acting as a central modulator of stress response. The COP9 signalosome activates the expression of vascular endothelial growth factor (VEGF) in tumour cells providing the cells with survival advantage by stimulating blood vessels. In this manner, curcumin’s anti-angiogenic activity causes the inhibition of VEGF expression. On the other hand, cyclooxygenase (COX)-2 is also shown to associate with COP9 signalosome, where this enzyme is targeted for proteosomal degradation.

Yet another interesting finding is that curcumin regulates the expression of the Id proteins through their association with the COP9 signalosome. Thus, complex, broad and effective activities of curcumin fall into a category of compounds that would best be described as ‘homeostatins’, which would be agents that act on stressors of dishomeostasis but do not perturb cellular balances under homeostasis. The non-pungent flavour of turmeric has also made this spice broadly appealing for oral ingestion, albeit the compound is not readily bioavailable to target organs at doses that would be necessary for severe conditions. A Phase 1 study of oral daily dose of 8 g curcumin consumed for 4 months showed no toxic effects, other than nausea and diarrhea, but higher doses were not acceptable to patients because of the bulk substance. Since a daily oral dose of 3.6 g of curcumin in the clinical setting is found to be detectable in colorectal tissues, the proposed protective effect of curcumin is largely limited to organ tissues which are exposed to the drug. Thus, the rather poor pharmacokinetic and dynamic characteristics resulting possibly from sulphation and glucuronidation of curcumin has precluded this otherwise highly effective agent to be developed for other cancers. However, novel advances in nanoparticle formulation have succeeded in making this natural product more bioavailable. It remains to be seen whether the clinical benefits of such formulations of curcumin will advance to angiogenic-dependent disease which could benefit from the therapeutic action of this homeostatin.

Panax ginseng (ginseng)

The roots of Panax ginseng are highly revered in the Far East for their medical properties. The main active principles that target blood vessels are the ginsenosides. Unlike turmeric, whose dual actions of angiomodulatory activity can be shown to result from a single compound (curcumin), the activity of ginseng is attributed to different subclasses of ginsenosides such as Rb1 and Rg1. At doses of 1 nmol/L to 1 µmol/1, 20(R)-Rg₃ showed dose-dependent inhibition of endothelial cell proliferation and inhibition of VEGF-induced chemo-invasion and tube formation. Additionally, in the Matrigel plug assay in mice, 600 nmol/L of Rg₃reduced blood vessel growth by fivefold compared with controls. Rg₃ also reduces the expression of MMP-2 and MMP-9, metalloproteinases that are involved in tube formation and invasion. Like Rg₃, the ginsenoside Rb1 also demonstrates anti-angiogenic activity.

Notwithstanding the important anti-angiogenic activities of ginseng, it is shown that when Rb1 is combined with Rg1 in differing amounts these mixed ginsenosides can either induce or restrict blood vessel growth based on their compositional ratios. This is because the panaxatriols represented by Rg1 and Rb1 have proangiogenic activity. The proangiogenic mechanism of Rg₃ which induces endothelial cell proliferation, is related to stimulation genes involved in cytoskeletal dynamics, cell-cell adhesion and migration. It would appear that the cognitive supportive activity of ginseng derives from promotion of angiogenesis, or at least the stabilisation of blood vessels that are diseased in ageing brains of humans, while that of its use in the treatment of cancer would result from the anti-angiogenic activity of Rb1 or Rg3. Panax ginseng, which is rich in Rb1, is reported to exert preventative activity in diverse cancer models, whereas Sanqi ginseng, which is rich in Rg1 ginsenoside, has been employed in treatment of trauma injuries that require the promotion of capillary growth. Given these very interesting findings on the mechanism of ginseng varieties, it is imperative that the individual bioactive agents and their abundance be characterised in formulation of ginseng extracts.

Withania somniferia (ashwagandha)

This herb plant has invigorating and tonic uses in Ayurvedic medicine. Some of the popular uses of the roots of this plant are for the treatment of arthritic conditions and for bleeding disorders that result from menstrual dysfunction. Hypothesising that an underlying angiogenic mechanism is targeted by the extracts of Withania somniferia (ashwagandha), we investigated the extracts of this plant for the presence of angiogenesis inhibitors by exploiting the 3D-ECSA. The combination of bioactivity testing in the 3D-ECSA along with assessment in the Matrigel model of angiogenesis revealed that the angiogenic inhibitory activity present in the methanolic extracts was enriched about fivefold upon further fractionation into chloroform-soluble substances. In assessing the molecular mechanism targeted by the chloroform-enriched fraction, it was found that the DNA binding activity of transcription factor NF-kB was specifically and potently inhibited by the chloroform extract (IC₅₀ 10 µg/mL). Further fractionation of the chloroform extract using HPLC afforded isolation of discrete peaks, which were individually tested for inhibitory activity in the 3D-ECSA. We characterised two of these compounds as withaferin A and withanolide D. The anti-angiogenic activity of withaferin A and withanolide D result from potent targeting of NF-kB activity (IC₅₀ = 0.5 µM) via a mechanism linked to upstream interference with the critical protein quality control complex, the ubiquitin proteasome pathway (UPP), a therapeutic target for a range of angio-inflammatory diseases.

The UPP is a cytoplasmic proteolytic complex that regulates protein expression during signal transduction by causing the destruction of critical factors, which are involved in the cell cycle, apoptosis, differentiation and inflammatory response. Withaferin A exerts its cytostatic effect on endothelial cells at substantially lower doses, causing blockade of the cell cycle (IC₅₀ 12 nmol/L) via UPP-dependent down-regulation of the critical cell cycle regulator, cyclin D1. Based on these findings, it could be further demonstrated that the in-vivo inhibition of angio-genesis by withaferin A was also significantly lower in the basic-fibroblast growth factor stimulated Matrigel plug model, being highly effective between 7 and 200 µg/kg/day. On the other hand, assessment of withaferin A in the corneal inflammatory model of neovascularisation revealed that doses between 500 µg/kg/day and 2 mg/kg/day reduced corneal angiogenesis by 50 and 80%, respectively (Mohan, unpublished data). In testing other genetic backgrounds of mice (129 SVEV) compared with previously used C57BL6 lines in the corneal inflammatory model of neovascularisation, we found that withaferin A at 2 mg/kg/day was highly effective, resulting in inhibition of 73%. Taken together, our strategy for isolation and investigation of anti-angiogenic natural products from medicinal plants has proven to be successful with discovery of withaferin A’s angiogenesis inhibitory activity.

Perturbation of the UPP is responsible for various diseases states. For example, tumour cells possess a highly active proteasome which results in over stimulation of cell proliferation. In addition, proteasome inhibition also results in blockade of angiogenesis by causing apoptosis of vascular endothelial cells and inhibition of vascular endothelial growth factor expression. Intriguingly, unlike proteasome inhibitors which directly target the enzymatic site of the 20S proteosome, withaferin A interferes with the UPP by an indirect mechanism. This UPP-targeting mechanism was recently shown to be due to binding by withaferin A to the type III intermediate filament protein vimentin. The antiangiogenic response to 2 mg/kg/day withaferin A treatment in the corneal inflammatory model of neovascularisation is found to be 3-fold-lower in vimentin-deficient mice than corresponding wild-type mice.

The multiple dose-related activities of withaferin A, and structurally related withanolides that possess anti-angiogenic activity, can be distinguished. At low nanomolar concentrations withaferin As anti-angiogenic activity is related to cytostatic blockade of the cell cycle in G1 phase, whereas at sub-to-low micromolar concentrations, withaferin A targets cell differentiation associated with tubule formation and inflammatory activation of NF-kB. At doses higher than 2 micromolar, withaferin A induces apoptosis via a mechanism linked to cleavage of vimentin and F-actin aggregation. Given such differences in the mechanisms of withaferin A with respect to its dose, one has to be careful in how extracts from this plant are prepared and of the exact amounts and proportions of the bioactive withanolides present. Studies have shown that extracts obtained from different cultivars of Withania somniferia (ashwagandha), or from different geographical locations have a wide range in amounts of withanolides. Thus it is imperative not only that there be standardising criteria to provide exact concentrations of the major chemical substances present in withania extracts but that these extracts also be biologically tested for efficacy for their intended use. Due to the heavy demand for Withania somniferia (ashwagandha), scientific attempts to produce these desirable compounds under defined laboratory conditions are being attempted. It may soon be possible to then use such techniques to produce metabolites under highly controlled environments. In addition, the application of genetic engineering approaches to modify bio-synthetic pathways in plants and plant cells so that desired metabolites are preferentially generated is another modern technology now being used to solve some of the issues of seasonal influences on natural product biosynthesis.

Hypericum perforatum (St John’s wort)

The widely used herb for depression, Hypericum perforatum (St John’s wort), is also the source of anti-angiogenic agents, hypericin and hyperforin. Attention to the angiogenesis-inhibitory activity of hyperforin has attracted attention not only to the broader uses of this plant in human diseases, but also to the potential side-effects, especially so in patients who may have other vascular complications where an anti-angiogenic agent would have contradiction. The mode of action of hyperforin is due to inhibition of MMP-9 expression, an enzyme that is responsible for basement membrane degradation during blood vessel growth. In addition, hyperforin inhibits microtubules which prevent endothelial cells from forming capillary tubes. Also, in other models hyperforin was shown to target component(s) within G-protein signalling cascades that regulate Ca²⁺ homeostasis, and inhibit neutrophil invasion and block inflammatory activation, suggesting that the target of this natural product is present on both vascular and inflammatory components that act in synergy during many angiogenic diseases. Interestingly, a dose of Hypericum extract 900 mg/day used as an antidepressant (which supplies 0.4 µmol/L of hyperforin) was shown to down-regulate production of the angiogenic cytokine interferon-gamma in activated T-cells with concomitant inhibition of MMP-9 expression. On the other hand, hypericin is also a potent angiogenesis inhibitor that targets activity of a related proteinase, MT1-MMP and is also responsible for inhibiting signalling events that trigger MAP kinase. Hypericin administered at 2 mg/kg intraperiteoneally, blocks activating phosphorylation of ERK1/2, which is required for the transactivation of hypoxia-inducible factor 1 alpha (HIF-1a) and in VEGF-induced blood vessel growth in models employing photodynamic therapy. Additionally, hypericin 3-50 µmol/L inhibits the activity of the proteasome complex in a dose-dependent manner. This upstream activity is shown to block activation of transcription factor NF-kB at doses of between 6 and 50 µmol/L. It is noteworthy to point out some of the adverse effects of this plant, which include sensitivity to sunlight and drug interactions with selective serotonin reuptake and protease inhibitors, as well as intermenstrual bleeding or altered menstrual bleeding in users of oral contraceptives, which may result from inherent proteasome inhibitory activity of hypericin-containing extracts of St John’s wort.

Camellia sinensis (green tea)

Epidemiological evidence has raised the interest in green tea consumption for prevention of cancers and cardiovascular diseases, and invigorated scientific research to identify the biologically active substances of tea extracts. One of the major ingredients of green tea, (—)epigallocatechin gallate (EGCG), a flavonoid, was shown to inhibit angiogenesis and have chemopreventive activity. Using data derived from rodent studies, a Phase 1 study of green tea extract was performed. Cohorts of adults with cancer were administered oral GTE with water with doses provided one or three times daily for 4 weeks. The maximum-tolerated dose was 4.2 g/m² once daily or 1.0 g/m² three times daily. Thus, a dose for anti-angiogenic activity in humans was calculated to be 1 g/m² three times per day (equivalent to 120 mL or 7-8 Japanese cups) for human consumption. As much as 200-500 mg of green tea consisting of 50% (—)EGCG is believed to be the pharmacological dose for angiogenesis prevention. Dose-limiting adverse effects of (—)EGCG are gastrointestinal and neurological, for which the coadministered presence of caffeine in green tea extracts is thought to be responsible for these side-effects. (—)EGCG has also been shown to inhibit COX-2 activity, an enzyme that is well known to be a target for anti-angiogenesis. The angiopreventive activity of (—)EGCG is also believed to result from inhibition of MMP-2 and MMP-9 activities. Furthermore, unlike other bioactive flavonoids that show inhibition of NF-kB activation, (—)EGCG is found to inhibit the DNA binding activity of inflammatory cytokine interleukin-1α-induced NF-kB, whereas flavonoids such as genistein do not produce this effect. It is likely that angio-inflammatory pathways that up-regulate IL-1β may be targets of this class of natural product, differentiating EGCG products from other flavonoids. Thus, this class of flavonoid may be more suitable for use in inflammatory angiogenic diseases.

Vitis vinifera (red grapes)

Red wine consumption is believed to be protective of the cardiovascular system, as evidenced in the prevention of the progression of atherosclerosis even in people who consume high amounts of red meat and cholesterol-containing foods. This was thought to be due to the major cardioprotective polyphenolic compounds found in skins and seeds of red grapes. One of these red wine polyphenolic compounds (RWPC), is the natural product resveratrol. The antiangiogenic mechanisms of resveratrol are known to be complex; since it inhibits proliferation of endothelial cells at 25 µmol/L, with inhibitory effects on cell migration and vessel tube formation occurring at 25 to 50 µmol/L.

Interestingly, the inhibitory activity of resveratrol on metalloproteinases MMP-9 was observed at 6.25 µmol/L, whereas on MMP-2 activity was at 25 µmol/L. Resveratrol inhibits VEGF-induced angiogenesis by interfering with reactive oxygen species-dependent Src kinase activation, and down-regulates the expression of angiogenic cytokines, including interleukin-8 and VEGF. It is interesting that RWPCs also show dose-dependent opposite effects on angiogenesis. In rats, 0.2 mg/kg/day of red wine polyphenolic compounds caused a pro-angiogenic effect while higher daily doses of 2 mg/kg of RWPC (equivalents found in seven glasses of red wine) showed anti-angiogenic activity in the post-ischaemic model of hind limb neovascularisation. It was found that the low-dose (1/10 glass) angiogenic effect occurs through overexpression of PI3 kinase-AKT-NOS pathway leading to increased VEGF production without affecting MMP production. Intriguingly, in the non-ischaemic leg, neither the low nor high dose of RWPC affected angiogenesis or blood flow. Thus, it appears that a prior disease condition needs to manifest, to observe these pharmacological effects of red wine polyphenolic compounds. Since normal tissues did not appear to be responsive to either high or low dose effects of red wine polyphenolic compounds, it cannot be inferred that these extracts are safe. For instance, others have shown that RWPCs at high doses can induce hypotension, decreased cardiac reactivity in rats. Interest in pharmacological activity of RWPCs has led to isolation of other principal active agents. Delphinidin, an abundant anthocyanin from RWPCs at high dose has been shown to inhibit vascularisation and blood flow at 0.6 m/kg per day, suggesting that the anti-angiogenic activity of red wine polyphenolic compounds is derived, in part, from delphinidin.

Conclusions

The field of antiangiogenesis has greatly benefitted from discoveries of targets for therapeutic development from which angiogenesis-inhibitory drugs such as Avastin have emerged for treatment of colon cancer. However, the literature is also beginning to see the emergence of undesirable side-effects of angiogenesis inhibitors. While it was once believed that adult tissues do not remodel their vasculature, it is now known that the microvasculature of the trachea and digestive system is not in a state of quiescence. Indeed, in mice, Avastin has been observed to cause normal mucosal capillaries in the trachea to regress. However, this drug-induced side-effect is ameliorated by cessation of Avastin treatment, indicative of the plasticity of the microvasculature to drug effects, and that developing safer treatments should involve careful examination of these preclinical and clinical results. As witnessed with natural product drugs emerging from traditional medicines, the guide to finding and developing highly effective and safe treatments for angiogenic diseases will need to integrate traditional knowledge with modern analytical methods of assessment and molecular pathobiology.

As the population ages, we are beginning to see many more diseases that result from vessel diseases which could benefit from angiomodulation. In these cases, one has to also remember that the physical constitution of older patients to drug activity is poorer because of weaker metabolism, reduced blood flow and general cellular ageing processes. More and more, the older patient groups will move towards more palatable medicines as older people are increasingly becoming dependent on multiple medications to support their different chronic conditions. In this context, it is critical to know contradictions to antiangiogenesis drugs. Other clinical adverse effects of anti-angiogenic drugs include gastrointestinal perforations of the bowels, arterial blood clots, and hypertension. The clinical manifestation of drug resistance to anti-angiogenic agents draws attention to yet another facet of cumulative toxic effects. That is, while the endothelial cell which is genetically stable does not become resistant to drug action, the genetic alterations that decrease the vascular dependence of tumour cells can influence the therapeutic response of tumours to angiogenesis inhibitors.

Herbal products that strive to restore the angiogenic balance must demonstrate standardisation in material quality, biological/pharmacological efficacy, and safety principles because many of the active principles have opposite effects on blood vessel growth when their concentrations or compositions are altered.

Diseases of circulatory system | Tags: Ashwagandha, Ginseng, Green tea, Red grapes, St. John's Wort, Turmeric

Herbal medicinal products as antiangiogenic agents

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September 14, 2010 at 8:03 am

Angiogenesis is the morphogenic process by which new blood vessels develop from a pre-existing vasculature. This process occurs during embryonic development and feeds the oxygen and nutrient requirements of growing tissues and organs. Blood vessels are lined internally by vascular endothelial cells, of mesenchymal origin. These endothelial cells are tightly compacted as a monolayer and display a cobblestone-like appearance. The endothelial cells also fulfil a role in maintenance of blood-brain and blood-retinal barrier and restrict the passage of blood components to inner tissues. However, when the vasculature is inflamed or injured such as in an oedema, substances from the blood can leak out; and the ability of the endothelial cell to re-populate the damaged tissue and re-establish cell-cell contact is critical for vascular homeostasis. In adults, the vasculature is usually dormant, except in certain instances, such as, during ovulation and menstruation in the female, and upon injury when tissue repair is required.

The vascular endothelium is thus an important beacon of humoral homeostasis and continues to guide the clinical diagnosis of underlying stress and disease. In fact, the silent and debilitating disease diabetes is at times diagnosed during a visit to the ophthalmologist for poor vision, which results from diabetic retinal capillaries leaking and causing vision impairment.

Pathological angiogenesis is an underlying disease process in tumour growth where it supports the expansion of the tumour mass in size and also acts as a means to aid tumour cells to metastasise to distal organs. In fact, it is known that tumour masses can only grow to sizes where oxygen can diffuse through tissue (approximately 200 um), hence, without establishment of new blood vessels, tumour cells are limited in their microscopic volume. The revolutionary paradigm that blocking new blood vessel growth (antiangiogenesis) could be a therapeutic means to ‘starve’ a tumour and thus arrest tumour development was at first strongly opposed by the oncology research community, who believed that directly killing the tumour cells with potent cytotoxins was the most effective means to cure cancers.

However, targeted strategies to cause interference with blood vessel formation have begun to open up antiangiogenesis as a new modality in therapeutic discovery. Today this idea has changed the treatment of cancers, with the clinical success of the neutralising antibody drug Avastin (bevacizumab), an antiangiogenic agent which binds to vascular endothelial growth factor (VEGF), a critical survival and growth-stimulatory protein for new blood vessel growth. Because it is believed that existing blood vessels are not affected by angio-genesis inhibitors, the quest to find new treatment options that do not possess the undesirable side-effects of currently used anticancer cytotoxic drugs has turned patient awareness to this new class of anticancer drugs. In this respect, researchers and clinicians have also begun looking seriously to the wealth of herbal medicines, many of which tout efficacy without the side-effects of radiation and chemotherapy.

Although much of our knowledge about the basic mechanisms of angiogenesis and clinical practice in the field of antiangiogenesis has come about from work done in the oncology field, it should be pointed out that there is a wide range of non-oncological diseases of no less importance as burdens to society that are angiogenic-dependent. Furthermore, the list of angiogenic diseases awakens us to how critical blood vessel homeostasis is to organ functions, and it is also important to note that the promotion of angiogenesis is necessary in other clinical conditions that result from a lack of adequate vascularisation, such as ischaemic heart disease and diabetic ulcers.

From ground-breaking work done by Folkman, a whole range of diseases can now be classified as having excessive angiogenesis or insufficient vascularisation (Table: Angiogenesis in human diseases). He raised the hypothesis that tumour angiogenesis results when the net levels of stimulators of new blood vessel growth exceed that of inhibitors and devoted his laboratory in Boston, USA, to identifying the body’s endogenous activators (and inhibitors) of tumour angiogenesis. Thirty-five years later, there are over 20 such protein effectors, which have been identified and shown to possess angiomodulatory activity. By demonstrating validation in a wide range of cell culture, organ and animal models of angiogenesis, it is possible to provide therapeutic doses of angio-inhibitory molecules to block angiogenesis, and contrarily, pro-angiogenic molecules to stimulate new blood vessel growth. The ability to discover new angiomodulatory agents and assess their effectiveness in controlling angiogenesis was enabled by the development of a number of in-vitro and in-vivo angiogenesis assays (Table: In vitro and in vivo angiogenesis assays).

Table: Angiogenesis in human diseases

Organ	Disease manifestation	Comments
Blood vessels	Vascular malformations caused by abnormal remodelling, haemangioma and atherosclerosis from increased vascularisation	Localised lesions due to vascular malformation; congestive heart failure resulting from atherosclerosis
Eye	Diabetic retinopathy and wet age-related macular degeneration from increased vascularisation	Blindness due to leaky vessels in diabetic retina; blindness from proliferating choroidal blood vessels
Skin	Psoriasis from increased vascularisation that becomes tortuous and enlarged; decubitus (stasis) ulcers from insufficient vascularisation; Kaposi’s sarcoma, allergic oedema, and neoplasms from increased vascularisation	Psoriasis appears as scaly, raised red lesions as a common form of this disease; stasis ulcers are open surface wounds that fail to heal
Bone and joints	Increased vascularisation of synovial joints in arthritis and of bone tissue in cancers	Inflammation of synovium in rheumatoid arthritis leads to joint destruction; destruction of cartilage in osteoarthritis causes pain and impaired mobility
Heart, skeletal muscle	Increased vascularisation of heart due to work overload; ischaemic heart and limb disease from insufficient vascularisation	Contractile dysfunction of heart tissue leads to heart failure; coronary heart disease manifests as a result of occlusion of blood vessels and poor oxygen supply
Adipose tissue	Increased vascularisation of fat tissue	Fat cells accumulate around new blood vessels causing obesity
Uterus, ovary	Increased vascularisation of uterine tissue, endometrium, ovary	Uterine tissue becomes dysfunctional from excessive bleeding; endometriosis can cause ectopic pregnancy, miscarriage and also infertility
Brain	Increased vascularisation in brain tumours; insufficient vascularisation of brain can lead to strokes	Gliomas and glioblastomas are incurable diseases of brain; stroke can incapacitate the cognitive and functional aspects of the brain

Table: In vitro and in vivo angiogenesis assays

Assay	Measurement	Comments
Cell proliferation	Inhibition of cell doubling opposing stimulatory effect of a defined angiogenic factor	Cytostatic activity blocks cell proliferation without causing cell death
Cell migration	Inhibition of cell migration opposing stimulatory effect of a defined angiogenic factor such as VEGF or bFGF	The extension of endothelial cell processes allows cells to migrate over a substratum
Invasion	Inhibition of cell invasion opposing stimulatory effect of a defined angiogenic factor	The growth of endothelial cells through a porous membrane or matrix in response to a chemotactic factor
Sprouting	Inhibition of migration, invasion and tube formation in a 3D matrix of collagen 1 or fibrin opposing stimulatory effect of a defined angiogenic factor	An integrated assay which couples vascular invasion, tube formation and maturation in 3D matrix
Matrigel cord assay	Inhibition of cord assembly by endothelial cells on complex matrix derived from tumour stroma opposing stimulatory effect of a defined angiogenic factor	Endothelial cells assemble into cords over the matrix
CAM in vivo	Inhibition of blood vessel growth in the CAM of a fertilised developing chicken egg	The developing vasculature of the CAM is highly sensitive inhibitors of angiogenesis
Corneal angiogenesis in vivo	Inhibition of de novo capillary growth in cornea opposing stimulatory effect of a defined angiogenic factor	Blood vessels from surrounding scleral vessel supply invade the avascular cornea in response to slow-release growth factor implanted in cornea
Matrigel in vivo	Inhibition of blood vessel growth into a Matrigel plug implanted in abdominal region of mouse	Blood vessels invade the Matrigel plug in response to stimulus from growth factor impregnated plug

Tests to examine effects of plant extracts on angiogenesis

Growth of blood vessels within a matrix

In-vitro vessel formation is assessed by measuring the total length of new blood vessels formed in a matrix over a period of time. Typically, endothelial cells are cultured within a matrix of fibroblasts in the absence or presence of the test compound. The matrix is supplied with fresh medium every third day and, after 11 days, the cells are washed and fixed. A staining reagent is applied to show the blood vessels formed from the endothelial cells and then quantified using a scanner attached to a computer which is able to process the images captured.

Use of genetically modified zebra fish

The embryos of the zebra fish (Danio rerio) have been used extensively in recent studies for several types of biological activity. Transgenic lines are used which express the green fluorescent protein GCFP to visualise vasculogenesis in the tail region. The extent of vascularisation can be quantified by computer-aided image analysis and is taken as a model of angiogenesis.

Three-dimensional endothelial cell sprouting assay

Sprouting of endothelial cells is an early event in angiogenesis, which follows vasodilation and degradation of matrix and it represents a valuable target for therapies because it takes place so early in the angiogenic process. The degradation of matrix is accomplished by the family of matrix metalloproteinases (MMPs). The mechanisms by which sprouts progress to form a lumen and ultimately become competent to support blood flow are largely unknown. Therefore, the study of the early steps of vessel sprouting can point to new therapeutic directions once key targets in these pathways have been identified.

The most promising in-vitro assays for elucidating relevant molecules and pathways necessary for endothelial cell morphogenesis are those using three-dimensional extracellular matrices, because endothelial cells experience a richer, more complex physical environment than cells cultured on twodimensional surfaces. The collagen I and fibrin matrices represent the major matrix environments where angiogenic events take place. For example, during endothelial sprouting there is the induced expression of endogenous growth factors, transcription factors and signalling molecules, endothelial cell differentiation markers and adhesion molecules and a marked down-regulation of positive regulators of the cell cycle and ubiquitin-proteasome genes. In stark comparison, the angiogenesis-screening assay using the basement-membrane matrix Matrigel, which measures the ability of endothelial cells to form a meshwork of cords on a tumour cell-derived matrix is markedly independent of transcriptional events, and protein synthesis. These and other drawbacks with the Matrigel gel assay limit its scope for screening purposes. In the endothelial cell sprouting assay (3D-ECSA), endothelial cells are induced over a period of 24 h to form spheroids by aggregating. The spheroids are next seeded in suspension in a collagen I matrix by gelling at 37°C. Exogenous growth factors, such as vascular endothelial growth factor, when added to the three-dimenional culture, stimulate the growth of vessel-like structures that grow out from the spheroid. Extracts and drugs being tested for angiogenesis inhibition are added along with VEGF. The sprouting extent and its inhibition are observed after a period of 18-24 h, which allows one to readily identify agents that block vessel development. The assay has been used by our laboratory to identify several classes of angiogenesis inhibitors, one of which is withaferin A from the medicinal plant Witbania somnifera.

The angiogenic balance: a paradigm for herbal medicine

Several traditional medicine systems, such as those used in China and India (Ayurveda, Siddha and Unani) explain disease as the imbalances in the body’s humoral and local effectors of normal physiology. In contrast, Western medicine identifies targets and designs therapeutic agents to affect particular diseased proteins/factors. Traditional or ethnomedicines lay emphasis on multiple modalities, focusing, on the one hand, on reducing the disease burden with the use of complex mixtures of principal active agents, while, on the other hand, also laying equal emphasis on reducing the undesired effects of these principal active agents with secondary substances to alleviate drug-induced toxicities. Considering this complex paradigm, two important aspects regarding herbal products need to be considered. One is focused on understanding the molecular factors that contribute to the pathobiology of disease and the other to the toxicology of drug activity. Using modern tools of analytical chemistry, biochemistry and molecular biology, molecular descriptors (genomics, proteomics, metabolomics), the activity of plant extracts and their principal active components on cellular and animal models can be understood mechanistically and are providing a wealth of information that serve hypotheses on which targets are tractable and how best to affect their functions to reverse disease.

Neoplasms | Tags: Garlic, Ginseng, Turmeric

Therapy and prevention of hepatocellular carcinoma using herbal drugs

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August 31, 2010 at 7:56 am

Cancer is a condition characterised by the uncontrolled growth and spread of abnormal cells, causing their massive aggregation producing either tumours or dispersal in the vascular system such as blood and lymph. Owing to a deviation from normal genetic makeup, cancer cells acquire immortality and a capability to evade apoptosis, non-responsiveness to anti-growth signals, self-sufficiency in growth factors, the ability to metastasise and to form new blood vessels that can supply nutrition and oxygen to the growing tissues. The transformation of normal to cancer cell occurs through accumulation of a series of genetic alterations or mutations, especially of oncogenes.

Carcinogenesis

There are many aetiological factors leading to cancer through a multistep process called carcinogenesis, which involves initiation, promotion, progression and malignant conversion. Mutation in a single cell initiates clonal expansion to form a premalignant lesion. These initiated cells will have resistance to cytotoxicity, defects in maturation, escape from senescence and have altered dependence on growth factors and hormones. Tumour promotion involves activation of cell surface receptors, activation/inhibition of cytosolic enzymes and nuclear transcription factors, stimulation of proliferation and inhibition of apoptotic cell death. Progression is accelerated by additional exposure to genotoxic agents and it is due to genetic instability and nonrandom sequential chromosomal aberrations. Malignant conversion involves multifocal change in premalignant lesions. There will be up-regulation of transcriptional activity and expression of modified cell surface molecules, gene amplification, alterations in cell-cycle regulatory genes, secreted proteases and methylation of DNA. All these changes facilitate migration and invasion.

Inhibition of carcinogenesis

As the progression of carcinogenesis is through a multistep pathway, there are many possible intervention sites inhibiting this progression. The procarcinogen can be detoxified and eliminated from the system. The conversion of procarcinogen to ultimate carcinogen is through multiple mechanisms including metabolic activation by enzymes. These mechanisms can be inhibited by blocking those enzymes involved in the activation step and several natural compounds of plant origin are reported as blocking agents in the chemoprevention of cancer, including flavonoids, ellagic acid and sulforaphane. These either block the conversion of carcinogen to ultimate carcinogen, or prevent the action of active metabolites on the normal cell. They may also alter carcinogen metabolism, enhance carcinogen detoxification, scavenge electrophiles and reactive oxygen species or enhance DNA repair.

The conversion of normal cells from preneoplastic cells to neoplastic cells takes several years, either by a second exposure to the carcinogen or promoting agent and accumulation of genetic variations. These steps can be inhibited by compounds such as curcumin, resveratrol, carotenoids, retinoids and genistein, which inhibit the malignant transformation of initiated cells by scavenging reactive oxygen species, altering gene expression, decreasing inflammation, suppressing proliferation, inducing differentiation, encouraging apoptosis, enhancing immunity or inhibiting angiogenesis and metastasis. Several chemopreventive phytochemicals have been shown to interfere with the cell-cycle regulatory pathways, qualifying them as potential therapeutic agents. Some are powerful inhibitors of growth factor receptors, including epidermal growth factor receptor (EGFR), and a variety of flavonoids are inhibitory, e.g. apigenin, luteolin, quercetin, catechin, epigallocatechin gallate, hesperitin, anthocyanins, genistein, with potential use in preventive anticancer treatment. Some phytochemicals undergoing clinical trials in the inhibition of carcinogenesis are given in Table: Selected ongoing Phase 1 and II cancer prevention trials sponsored by the US National Cancer Institute.

Table: Selected ongoing Phase 1 and II cancer prevention trials sponsored by the US National Cancer Institute
Target organ	Agent
Phase 1 trials
Breast	Soy isoflavones
Colon	Curcumin
Prostate	Lycopene (3 trials); Soy isoflavones
Skin	Epigallocatechin gallate
Phase II trials
Anogenital warts, human papillomavirus, HIV	Indole-3-carbinol
Cervix	9-cis-Retinoic acid
Prostate	Soy (dietary); soy isoflavones

Hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common primary malignant tumour of the liver and is the fifth most common cancer in the world, ranking fourth in annual mortality rates. An estimated 564 000 new cases of HCC are diagnosed each year, with the highest incidence in eastern and southeastern Asia, some of the western Pacific islands and sub-Saharan Africa. Men are affected four to eight times more often than women and the incidence generally increases with increasing age, although there is a definite shift towards a younger age distribution in black African and ethnic Chinese populations.

Aetiological factors

There are some well-documented aetiological associations of HCC. The aetiological association between hepatitis-B virus (HBV) and HCC is well established. Chronic HBV infection is the leading risk factor and it has been estimated that 53% of cases worldwide are related to HBV. Malignant transformation occurs after a long period of chronic liver disease, frequently associated with cirrhosis. Chronic inflammation of the liver, continuous cell death and consequent cell proliferation might increase the occurrence of genetic alterations and risk of cancer. The long-term expression of regulator gene product of the X-gene and large envelope proteins (LHBs) are thought to play a major role in tumorigenesis. This viral oncoprotein behaves as a transcriptional transactivator, which activates oncogenes, cytokines and growth factors. A direct role of the virus through integration of viral DNA directly to host genome has also been hypothesised that may enhance chromosomal instability, large inverted duplications, deletions, amplifications or chromosomal translocations which lead to the activation of oncogenic pathways.

Chronic HCV (hepatitis C virus) infection is also associated with HCC. The HCC incidence rate in patients with HCV-related cirrhosis is about 3.7%.

Chemical carcinogens which are linked to HCC include nitrites, hydrocarbons, solvents, organochlorine pesticides, primary metals and polychlorinated biphenyls. Of all the chemicals linked to HCC, ethanol is the most important one that leads to HCC. Overconsumption of alcohol is one of the leading causes of liver cirrhosis which makes the patient more susceptible to HBV and HCV infection.

Aflatoxins produced by the fungi, Aspergillus flavus and A. parasiticus have also been linked to HCC. These fungal species grow on grains, peanuts and other food products and are the most common cause of food spoilage. These fungi also produce aflatoxins, aflatoxin Bl being the most hepatotoxic and chronic exposure to these mycotoxins will lead to HCC.

Some congenital conditions also lead to development of HCC. Genetic diseases such as haemochromatosis, Wilson’s disease, hereditary tyrosinaemia, type I glycogen storage disease and porphyria, have all been linked to a high incidence of HCC.

Symptoms and markers

The symptoms related to the early stages of HCC are poor. When HCC presents with clinical symptoms, the tumour is usually advanced and there are few therapeutic options. The current effective treatments available are only applicable in a relatively small proportion of early stage cases.

Serum a-fetoprotein is a useful tumour marker for the detection and monitoring of HCC development, but gives false-negatives in about 40% of patients. Serum Ύ-glutamyl transpeptidase (GGT) is frequently overexpressed in cancer cells. GGT activity is a sensitive marker of hepatobiliary disorders, exhibiting tissue-specific expressions under various physiological and pathological conditions. Other enzymes that are increased in the blood during HCC include alkaline phosphatase, alanine trans-aminase and aspartate transaminase, but they are non-specific.

The overexpression of transforming growth factor (TGF)-β1 and TGF-β1 messenger RNA is seen in most patients with HCC. The level of insulinlike growth factor (IGF)-II and IGF-II mRNA is also overexpressed in HCC. The analysis of telomerase activity in combination with α-fetoprotein increases the accuracy of HCC diagnosis to about 93%.

Even though tumours present limitations for cytogenetic analysis, there are some reports of cytogenetic analysis of HCC. They include chromosome 1p abnormalities and 8q amplification. Molecular studies have demonstrated frequent loss of heterozygosity on 1p, 4q, 8p, 11p, 13q, 16q and 17p and amplification of 8q areas in HCC.

Models

Rodents are usually studied as models of hepatic carcinogenesis. Many chemicals induce liver cancer in rodents since their livers are very sensitive to chemical carcinogens. Thus, a single experimental protocol can be used to understand the mechanisms of a number of carcinogens. The low cost of rodents and their potential for genetic studies and manipulation are also attributes. Apart from this, a fairly extensive understanding of liver biology has made rodent HCC a popular model. Other models used for HCC study include hamsters and other non-primates.

The chemicals used to study the initiation of HCC include nitrosamines, aromatic amines, vinyl chloride, polycyclic aromatic hydrocarbons, hetero-cyclic amines, aflatoxin and tamoxifen. The promoters which are used after initiation include phenobarbital, dioxin and polychlorinated biphenyl. The mechanism of action of these chemical carcinogens is combination with DNA to form adducts, either by direct binding to DNA, or after enzymatic activation in the liver to produce the carcinogen. Some agents that produce hepatic carcinogenesis are discussed below.

Polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons require metabolic activation to elicit their detrimental effect, e.g. benzo(a)pyrene is enzymatically activated to the 7,8-dihydrodiol, which induces both somatic mutations in crucial genes through DNA binding and subsequent outgrowth of irreversibly transformed cells.

Aryl amines/amides

In rodents these compounds induce tumours in the liver, e.g. acetamido-fluorine undergoes N-hydroxylation in liver cells. Additional tumour-promoting activities of acetamido-fluorine include the triggering of adaptive responses in mitochondria permeability transition pores and Bcl-2 production levels that increase resistance to apoptosis.

Alcohol

The mechanism of ethanol-induced cancer is closely related to its metabolism. Acetaldehyde, the end-product of ethanol metabolism, is the causative agent of cancer in chronic conditions. Oxidative stress and cirrhosis are important factors in ethanol-induced HCC.

Nitrosamines

These are the most widely used chemical carcinogen for animal experiments. N‘-nitrosodiethylamine (NDEA) is metabolised in the liver and its ethyl radical product is responsible for induction of HCC. This radical attacks the DNA and produces genetic changes which result in carcinogenesis. It also produces the conversion of certain proto-oncogenes to oncogenes.

Azo dyes

Para-dimethylaminoazobenzene (p-DAB) is metabolised to monoaminoazobenzene by N-demethylation and subsequently to aminoazobenzene or to N-hydroxy-N-methyl-4-aminoazobenzene. Covalent bindings of these metabolites with DNA are major carcinogenic factors.

Aflatoxins

Aflatoxins are highly mutagenic and are metabolised by cytochrome p450 to their epoxides, which results in formation of DNA adducts with the guanine N7, thus causing carcinogenicity.

Treatment

If detected early, suitable curative treatments include surgical resection, liver transplantation and percutaneous ablation. In patients with advanced stage of HCC, transarterial chemoembolisation has been proved to improve survival in selected candidates. Other therapeutic modalities such as intra-arterial chemotherapy and internal radiation offer promising results but have not been shown to improve survival. As HCC is usually chemoresistant, cytotoxic drugs are poorly tolerated in cirrhotic patients so many of the known anticancer agents, e.g. tamoxifen, octreotide, interferon, fail to produce any benefit in HCC patients. Promising results have been obtained with agents targeting receptor tyrosine kinase pathways. Since HCC is dependent on angiogenesis, molecules targeting the angiogenesis are currently under investigation. BAY 43-9006, which inhibits multiple pathways, mainly Raf kinase and VEGF, has been undergoing Phase II and III trials and has shown a partial response in a patient with advanced HCC.

The presence of unlimited chemical molecules, with diverse mechanisms, present in herbal drugs makes them interesting starting points in the search for newer drugs for cancer treatment.

Natural templates for treatment

The plant kingdom produces many potent pharmacologically active components, several of which have provided promising results to combat various diseases. Possible uses of herbal drugs in cancer are illustrated in Figure: Potential uses of herbal drugs in cancer treatment.

Figure: Potential uses of herbal drugs in cancer treatment.

The chemical basis of some anticancer plants has been elucidated and some are now used clinically. Plant extracts and their constituents which show significant activity against hepatic cancer are described in more detail below.

Curcuma longa (turmeric)

The rhizome of C. longa (Zingiberaceae) is described as an anti-inflammatory agent in Ayurveda and is widely used in foods and as a medicine thoughout India and other Asian countries as a treatment for liver disorders, including cancer. The most-studied ingredient in the rhizome is curcumin but several related compounds are present such as demethoxy-curcumin and bidemethoxycurcumin.

Curcumin treatment has been reported to reduce tumour incidence and inhibit the liver inflammation and hyperplasia in N-nitrosodiethylamine-induced liver-cancer-bearing animals. The chemopreventive effect of turmeric and curcumin against diethylnitrosamine-induced and phenobarbital-promoted hepatocarcinogenesis has been reported. Curcumin also suppressed diethylnitrosamine-induced development of altered hepatic foci in rat liver. The number of Ύ-GT positive foci induced by aflatoxin Bl was found to be reduced by curcumin treatment. Curcumin significantly protected the liver from oxidative stress-induced damage during chemically induced hepatocarcinogenesis in rats.

In a patient study, Curcuma aromatic oil showed a positive effect in treating primary liver cancer with longer survival time and myelosuppression. Curcumin treatment reduced the tumour incidence by inhibiting angiogenesis through down-regulating cyclo-oxygenase 2 and vascular endothelial growth factor in HepG2 cells. It was reported that curcumin induces mitochondrial and nuclear DNA damage, thereby inducing apoptosis through caspase 3 and 9 activation. Curcumin also suppressed intrahepatic metastasis mediated by the inhibition of MMP-9 and through alteration of cytoskeletal organisation. In-vitro studies showed that the production of p21(ras) was inhibited by curcumin. It also inhibited transcription factor NF-kB and IAP gene expression. External curcumin application has been tried as a palliative therapy for cancerous skin lesions. Clinical trials (Phase I and II) of curcumin are being carried out in several hospitals to find out its therapeutic role against colon cancer, pancreatic cancer, hepatocellular cancinoma and multiple myeloma. Curcumin was found to be non-toxic at doses up to 12 g/day in patients.

Silybum marianum (milk thistle)

The active ingredients present in Silybum marianum (Compositae) are the flavonolignans silymarin and silybinin. Silymarin has been proposed as a promising chemotherapeutic adjuvant for the treatment of liver cancer. N‘-nitrosodiethylamine-induced hepatocellular carcinoma was found to be inhibited by silymarin by modulating antioxidant defence status in rats. Silybin was found to inhibit the growth of Hep3B and HepG2 cells by G1 arrest. The apoptosis-inducing property of silybin has been shown to be through decreasing cyclin D1, cyclin D3, cyclin E and cyclin dependent kinases 2 and 4. Silymarin can suppress the proliferation of a variety of tumour cells through cell cycle arrest at the G1/S-phase, induction of cyclin-dependent kinase inhibitors (such as p15, p21 and p27), down-regulation of anti-apoptotic gene products (e.g. Bcl-2 and Bcl-xL), inhibition of cell-survival kinases (AKT, PKC and MAPK) and inhibition of inflammatory transcription factors (e.g. NF-kβ). Silymarin can also down-regulate gene products involved in the proliferation of tumour cells (cyclin D1, EGFR, COX-2, TGF-beta, IGF-IR), invasion (MMP-9), angiogenesis (VEGF) and metastasis (adhesion molecules). The anti-inflammatory effects of silymarin are mediated through suppression of NF- kβ -regulated gene products, including COX-2, LOX, inducible iNOS, TNF and IL-1. Treatment of patients with hepatitis B or C infection with silymarin seemed to be effective, although no effect in decreasing viral load was found.

Camellia sinensis (tea)

Constituents of Camellia sinensis (Theaceae) include flavonols, e.g. myricetin, kaempferol and quercetin; as well as caffeine and proanthocyanidins such as epigallocatechin gallate. Green tea was observed to have protective effect on liver cancer in population-based studies.

In multidose NDEA-induced HCC followed by carbon tetrachloride injection and partial heptoectomy studies, it was found that treatment with tea polyphenols and pigment showed significant reduction in number and area of GST-positive foci, which is a proliferative indicator of precancerous liver lesions by immunohistochemistry.

Green tea was reported to exert antiproliferative activity towards hepatoma cells. Green tea was also reported to possess chemopreventive activity against nitrosamine-initiated hepatocellular carcinoma. It was found that the production of p21(WAF1/CIP1) was significantly induced and that of cyclin Dl and cyclin-dependent kinase 4 were inhibited in tea-treated animals. Tea catechins, black tea extract and oolong tea extract are also reported to inhibit hepatocellular carcinoma.

Allium sativum (garlic)

The bulbs of Allium sativum (Alliaceae) have been described as useful against inflammation and tumours in Ayurveda. The anticarcinogenic activity of whole garlic, as well as its isolated ingredients, against NDEA-induced hepatocellular carcinoma in animals is well documented. Aged garlic extract inhibited the development of putative lesions in rat hepatocarcino-genesis involving a slowing in the proliferation rate of liver cells after partial hepatoectomy. Garlic powder inhibited the formation of preneoplastic foci during hepatocarcinogenesis initiated by diethylnitrosamine through suppression of CYP2E1. The organosulphur compounds isolated from garlic are highly active against liver cancer. There was a decrease in the number of preneoplastic, GST-positive foci of the liver and also a down-regulation of IGF-I and iNOS mRNA expression in the liver of organosulphur-treated animals which were induced with 2-amino-3,8-dimethylimidazo [4-5-f] quinoxaline. The protective effect of diallylsulphide isolated from garlic against HCC was reported by Singh et al. (2004). S-allylcysteine, an organosulphur compound, showed inhibition of tumour incidence and lipid peroxidation in NDEA-induced hepatic cancer animals with simultaneous elevation in antioxidants.

Benzo(a)pyrene-induced cancer was found to be inhibited by garlic constituents such as diallylsulphide (DAS), diallyldisulphide (DADS) and diallyltrisulphide (DATS). Diallylsulphide prevented DNA adducts induced by the carcinogen, thereby preventing the initiation of oestrogen-induced cancer. Allylthiopyridazine derivatives induced apoptosis in Sk-Hep-1 cells through a caspase-3-dependent mechanism and this also contributes to their chemopreventive function. The antiproliferative property of aqueous garlic extract was studied in HepG2 cells and it was found that these compounds induced a p53/p21-dependent cell cycle arrest in G2/M phase and apoptosis through activation of c-Jun-NH (2) terminal kinase (JNK)/c-Jun phosphorylative cascade. DAS, DADS and DATS also caused G2/M phase arrest in human liver tumour cells.

Emblica officinalis Gaertn. (emblica)

The extract of fruits of Emblica officinalis (Euphorbiaceae) was reported to give chemoprotection against chemically induced carcinogenesis. The fruits are rich in polyphenolic compounds such as gallic acid, tannic acid, emblicanin A and B and ellagitannins. Polyphenolic compounds, such as epigallo-catechin gallate, found in tea are also present in Emblica officinalis. Because of the presence of these compounds, emblica extract has been shown to possess significant antioxidant activity and is also antimutagenic, inhibiting DNA adducts produced by benzo(a)pyrene. The polyphenolic fraction of Emblica officinalis was found to modulate NDEA-induced hepatic cancer in rats. In-vitro experiments showed that it inhibited DNA topoisomerase I in Saccharomyces cerevisiae mutant cell culture and also inhibited the activity of cdc25 tyrosine phosphatase.

Phyllanthus amarus

The fresh root of Phyllanthus amarus (Euphorbiaceae) is said to be an excellent remedy for jaundice. The components present in Phyllanthus amarus are lignans, e.g. phyllanthin; tannins, e.g. phyllanthusiin D3, amariin and amarulone; alkaloids, e.g. entnorsecurinine, diarylbutanes; and neolignans, e.g. phyllnirurin. A variety of hydrolysable tannins purified from Phyllanthus amarus were reported to be potent inhibitors of rat liver cyclic AMP-dependent protein kinase catalytic subunit. Phyllanthus amarus extract was reported to significantly inhibit NDEA-induced hepatocar-cinogenesis in rats in a dose-dependent manner. In another study the lifespan of rats bearing NDEA-induced hepatocellular carcinoma was found to be significantly increased by the treatment with Phyllanthus amarus, from 33 weeks to 52 weeks. Phyllanthus amarus extract has been shown to have antiviral activity against hepatitis B virus. In a patient study, it was found that approximately 60% of the carriers of HBV lost the virus within 1 month of Phyllanthus amarus treatment.

Picrorhiza kurroa (kutki)

The root/rhizomes of Picrorhiza kurroa (Scrophulariaceae) are used in Ayurveda against jaundice. The components present in the root include the glycosides picrorhizin and kutkin, and sterols. Picroliv, an iridoid glycoside mixture prepared from this plant, contains equal concentrations of picroside and kutkoside as well as vanillic acid and sterols. Amelioration of NDEA-induced hepatocellular carcinoma was seen in animals treated with Picrorhiza kurroa extract, and there was a significant reduction in the levels of drug-metabolising enzymes such as glutathione-S-transferase (GST) and aniline hydroxylase (AH). Liver morphology and histopathology also revealed the protective effect of the extract against chemical carcinogenesis. Picroliv was found to inhibit HCC and was also reported to possess protective effect against 1,2-dimethylhydrazine-induced HCC in animals. Picroliv significantly down-regulated transcription factor API and thereby decreased the level of c-fos mRNA as well as c-jun and c-fos proteins in liver tissue. This would indicate a base for their potential anti-HCC activity.

Semecarpus anacardium (marking nut)

The rind of the fruit of Semecarpus anacardium (Anacardiaceae) is used in Ayurvedic medicine against inflammatory diseases. The active ingredient is usually reckoned as anacardic acid. Semecarpus anacardium nut extract affords anti-cancer activity by enhancing both phase I and phase II enzymes and it has been proposed that the anticancer activity may be mediated through the induction of hepatic biotransformation enzymes. It was found to modulate the carcinogenic effect of aflatoxin by enhancing anti-oxidant capacity in the system.

Andrographis paniculata (creat)

The root of Andrographis paniculata (Acanthaceae) is used in both Ayurvedic and Chinese medicine. The component present is a diterpene lactone andrographolide. The roots also contain a variety of compounds including the sesquiterpene andrographolide. The effectiveness of Andrographis paniculata was found to be through modulating hepatic and extra-hepatic carcinogen-metabolising enzymes and antioxidant status. Andrographis paniculata extract and andrographolide stimulated CTL production through enhanced secretion of IL-2 and IFN-Ύ by T cells and thereby inhibited the tumour growth. The species has been reported to modulate the immune response through enhancing natural killer (NK) cell activity and antibody-dependent cell-mediated cytotoxicity and antibody-dependent complement mediated cytotoxicity in tumour-bearing animals.

Glycine max (soybean)

Glycine max (Papilionaceae) contains isoflavone glycosides genistein and diadzin as active principles but certain saponin constituents were also found to be biologically active. The administration of 30% soybean to the rat diet was found to have protective effect against hepatocarcinogenesis induced by DL-ethionone. There was a 92.7% increase in lifespan in rats with primary liver cancer when treated with doxorubicin encapsulated with soybean-derived sterylglycoside mixture when compared with free doxorubicin. Genistein was found to inhibit diethyl-nitrosamine-induced and phenobarbital-promoted HCC. Genistein has been reported to inhibit lung metastasis in animals. Genistein was found to inhibit cell proliferation and induced apoptosis through caspase-3 induction and caspase-2 activation. In another study apoptosis was induced by genistein in Hep 3B cells through initiating endoplasmic reticular stress relevant regulators including m-calpain, GADD 153, GRP 78 and caspase-12.

Panax ginseng (ginseng)

Panax ginseng (Araliaceae) root (white and red) is extensively used in traditional Chinese medicine against various diseases. Ginseng contains polysaccharides and saponins, those known as ginsenosides are commonly considered to be the active constituents. The incidence of liver tumour development was lower in animals treated with red ginseng extract, and the average number of tumours per mouse was significantly reduced in the treated group. White ginseng was also found to possess anticarcinogenic properties both in vitro and in vivo. In another study, red ginseng was found to possess both preventive as well as curative properties against diethylamine-induced hepatic cancers in rats.

Terminalia arjuna (arjuna bark)

Terminalia arjuna (Combretaceae) bark is extensively used against tumours in Ayurvedic medicine. Terminalia arjuna was reported to possess chemopreventive activity in NDEA-induced HCC-bearing animals. In another study, diethylnitrosamine-induced HCC was inhibited by Terminalia arjuna bark extract through modulating the antioxidant status in tumour-bearing animals. The ingredients of Terminalia arjuna bark include flavonoids, e.g. arjunone, arjunolone and luteolin; phenols, e.g. gallic acid and ellagic acid; and terpenoids, e.g. oleanolic acid. The antitumour potential of luteolin and triterpenoids has been reported, while phenolic ingredients have significant chemopreventive activity.

Other plants

Baubinia variegata and Baubinia racemosa were reported to ameliorate NDEA-induced HCC in rats through modulation of antioxidant enzymes. Chemopreventive potential of extracts from Tamarix gallica, Paullina cupana, Butea monosperma, Lygodium flexuosum, Indigofera aspalatboides, Apium graveolens, Solanum trilobatum, Ardisia compressa, Calotropis procera, Amaranthus gageticus, Astragalus membranaceus, Beta vulgaris, Cymbopogon citrates, Asteracantha longifolia, Triantbema portulacastrum etc. has been reported in hepatic cancer models in animals.

Conclusions

There is no really effective treatment for hepatocellular carcinoma and so it stands high in global cause of mortality. Chronic hepatitis and lifestyle-induced oxidative stress are the major factors associated with hepatic cancer. It is detected in the later stages in many patients, and the current treatment modalities fail to keep the disease under control. Plants and plant-derived compounds have been found to be effective against hepatic cancer in animal models and through a few clinical studies. The antiviral and free-radical scavenging activities of the plant-derived constituents in many cases have proven to be beneficial. Many of the compounds, e.g. curcumin, are in the process of being testing in clinical trials and are giving promising results, while explorations for newer compounds are still progressing.

Disorders of digestive system | Tags: Anise, Peppermint, Turmeric

Herbal medicines for functional gastrointestinal disorders

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August 28, 2010 at 7:38 am

Gastrointestinal complaints rank among the most frequent reasons why people seek medical advice. The most common functional gastrointestinal disorders (FGIDs) seen in clinical practice are functional dyspepsia (FD) and irritable bowel syndrome (IBS), characterised by recurrent episodes of gastrointestinal symptoms, in the absence of structural lesions that explain symptoms. While symptoms of epigastric pain, bloating, nausea, belching, early satiety and heartburn are predominant in functional dyspepsia, abdominal discomfort, bloating and disturbed patterns of defecation (constipation, diarrhea or constipation/diarrhea) are common in patients with IBS. FGIDs reduce the health-related quality of life and account for an increased healthcare burden. The pathogenesis of functional gastrointestinal disorders is probably multi-factorial, genetic and environmental factors are thought to contribute to alterations in visceral sensory function, motility, bacterial overgrowth and central nervous system processing. Inflammation is considered a risk factor for the development of both functional dyspepsia and irritable bowel syndrome. Currently, the existing therapies to treat multiple symptoms of FGIDs are suboptimal, associated with restricted therapeutic potential and new agents are awaited that would improve global IBS symptoms. A wide variety of treatments have been used to manage functional gastrointestinal disorders and include Helicobacter pylori eradication, antacids, mucosal protectants, antisecretory agents, prokinetics, antidepressants and visceral analgesics. The fact that no single available therapy consistently provides relief to the majority of the patients validates the heterogeneity of these disorders. The emerging therapies are largely aimed to normalise pain perception and gastrointestinal motor and reflux function.

The therapy of functional gastrointestinal disorders is one of the domains of phytotherapeutic treatments. Traditionally, plants with a high tannin content, showing astringent properties, were particularly valued to treat diarrhea and dysentery whereas bitter, aromatic and bitter-aromatic plants were especially employed to treat gastrointestinal cramps and pain. Investigations on traditionally used plants have resulted in the isolation, and chemical and pharmacological characterisation of many different types of compounds. Most of these agents are helpful to prevent or arrest the progression, rather than to treat disease. Further, they exhibit pleotropic actions and therefore may serve as important leads for developing novel therapies for the treatment of functional gastrointestinal disorders.

So far, relatively few herbal medicines have been evaluated scientifically to prove their safety, potential benefits and effectiveness in gastrointestinal disorders (Table: Experimental studies on traditional herbs used in gastrointestinal disorders). This chapter specifically deals with the evaluation methods for the development of quality herbal medicines intended to be useful in FGIDs.

Functional dyspepsia

Functional dyspepsia is a clinical syndrome, whose origin is unknown, defined by chronic or recurrent pain or discomfort in the upper abdomen. On the basis of the Rome III diagnostic criteria for functional gastrointestinal disorders, patients who suffer from functional dyspepsia in the absence of any organic disease are categorised as having postprandial distress syndrome or epigastric pain syndrome for at least 3 months. About 15-30% of adult patients suffer from various different functional dyspeptic conditions. Several pathophysiological mechanisms are involved in functional dyspepsia, including visceral hypersensitivity, both in the stomach and the duodenum, impaired gastric accommodation, antral overdistention, delayed gastric emptying and abnormal duodenojejunal motility. Induction of gastric hypersensitivity by acid in the stomach seems to be important in a subset of patients. Many drugs can induce dyspepsia as a gastrointestinal side-effect, the major cause being the use of nonsteroidal anti-inflammatory drugs (NSAIDs) for arthritis and chemotherapeutic agents in cancer. Studies also suggest a possible link with G-protein polymorphisms in dyspepsia.

Currently, functional dyspepsia is classified into ulcer-like dyspepsia, dysmotility-like dyspepsia and non-specific dyspepsia, in which symptoms do not clearly fit into any of the above categories. While antacids, H₂-receptor antagonists and proton-pump inhibitors are useful in ulcer-like dyspepsia, pro-kinetic agents are more effective in dysmotility-like dyspepsia. Most therapies for patients with functional dyspepsia are intended to normalise pain perception and gastrointestinal motor and reflex function. Serotonin is the key mediator of gut function in relation to gastrointestinal motility, secretion and sensation of pain. There is limited evidence that the 5-hydroxytryptamine 5-HT₃ antagonist alosetron has potential efficacy in functional dyspepsia, perhaps via visceral analgesic effects. Animal studies also suggest that acid can induce serotonin release from enterochromaffin cells in the duodenum, which may in turn activate 5-HT₃ receptors and alter visceral sensation. Acid in the duodenum can also induce fundic relaxation. Thus, the interaction between acid and 5-HT₃ receptor antagonism is of interest in functional dyspepsia. A number of compounds may have visceral analgesic effects in the upper gastrointestinal tract, including 5-HT₃ antagonists and 5-HT₄ agonists. Approximately 25% of patients with functional dyspepsia have slow gastric emptying, and perhaps 10% have accelerated gastric emptying. It is, therefore, important to avoid the use of prokinetic therapy in patients who have accelerated gastric emptying, as presumably this would worsen symptoms.

No standard therapy is currently available for functional dyspepsia. From ancient times, bitter herbal drugs have played a role in the therapy of patients with dyspeptic symptoms. Studies point out that different plant-derived extracts and their constituents can give gastro/cytoprotection through several mechanisms (Table: Experimental studies on traditional herbs used in gastrointestinal disorders). Flavonoids are highly gastroprotective probably due to enhancement of the release of nitric oxide and neuropeptides, such as calcitonin gene-related peptide, released from sensory afferent nerves, which increase gastric microcirculation. These appear to stimulate, at even very small concentrations, the secretion of the stomach as well as the digestive glands and strengthen the smooth musculature of the digestive tract. Bitter substances are often combined with essential oils, which act primarily as spasmolytics and analgesics and possess anti-Helicobacter pylori effects. They exert anti-inflammatory action by:

Table: Experimental studies on traditional herbs used in gastrointestinal disorders

Name	Part used	Use	Constituents	Study type	Results
STW5 Iberogast® (herbal formula)	Ethanolic extracts of 9 plants	Functional dyspepsia (FD) and irritable bowel syndrome (IBS)	Methionine-like sulphur-compounds	Animal studies	Diminished binding affinity of 5-HT(4), muscarinic M(3), and opioid receptors in vitro
Artichoke (Cynara scolymus L.)	Leaf extract	Irritable bowel syndrome (IBS)	Caffeoylquinic acids and flavonoids	Animal studies	Protected animals from gastric ulceration
N-095 (crude drug containing red ginseng, polygala root, saffron, antelope horn and aloe wood)	Dried powder	Combat stress	Multi components	Experimental studies in rats	Prevented gastric ulceration — induced by restraint and water-immersion stress
Baishouwu (Chinese herbal drug)	Dried root tubers of 3 plants	Gastric diseases	Multicomponents	Experimental studies in rats	Offered gastroprotection against ethanol and indometacin-induced gastric lesions
Turmeric (Curcuma longa L.)	Rhizome extract	Ulcer and non-ulcer dyspepsia	Curcumin (diferuloyl methane)	Animal studies in vivo and anti-H. pylori in vitro	Demonstrated antiulcer, anti-inflammatory, anticancer and analgesic effects
Peppermint (Mentha piperita L.)	Leaf essential oil	Gastroprotective use	Menthol and menthonin	Animal studies	Antimicrobial, antispasmodic, antioxidant and analgesic effects
Anise (Pimpinella anisum L.)	Seed aqueous suspension	Gastroprotective use in Arab medicine	Volatiles	In vivo experimental ulceration in rats and anti-H.pylori in vitro	Gastro protection against necrotising agents and indometacin
Cardamom (Elettaria cardamomum Maton.)	Crude methanolic extract and fractions	Gastro protective use in Unani medicine	Terpenes and phenolic compounds	Experimental ulceration in rats	Gastro protection against ethanol and aspirin-induced injury
Fenugreek (Trigonella foenum graecum)	Aqueous extract of seed and gel fraction	Gastric complaints	Steroidal saponins and alkaloids	Rat study	Prevents ethanol-induced gastric lesions
Sangre de grado (Croton urucurana var. genuinus and related species)	Red sap from trunk wood of C urucurana	Gastric ulcer and diarrhoea	Proantocyanidins, taspine	Experimental study in rats and guinea-pigs	Antiulcer, antidiarrhoeal and visceral antinociceptive effects
Copaiba oil (Copaifera langs dorffii Desf.) and related species)	Oleo-resin from trunk wood	Gastrointestinal disorders	Volatiles and diterpenes that include kaurenoic acid	In vivo experimental studies with rats	Antiulcer, wound healing and anti-inflammatory effects
Aroeira (Myracrodruon urundeuva Engl.)	Aqueous extract from stem bark	Traditional use in gastric dyspepsia, anc diarrhoea	Tannins and Chalcones	Experimental studies with rats and guinea-pigs	Gastroprotection against ethanol, stress and histamine-induced lesions; acetic acid-induced colitis
Monkey puzzle (Araucaria araucana Mol.)	Resin	Mapuche Amerindian use to treat ulcers	Labdane diterpenes and lignans	Experimental studies with mice	Gastroprotection against ethanol-HCl-induced lesions
Macela (Egletes viscosa L.)	Ethanolic extract of flower buds and flavonoid, ternatin	Gastric dyspepsia, diarrhoea and constipation	Volatiles, flavonoid (ternatin) and diterpenes (centipedic acid and 12-acetoxy hawtriwaic acid)	Animal studies	Antiulcer, antidiarrhoeal and anti-inflammatory effects
Tsubaki (Camellia japonica L.)	Methanolic extract of flower buds	Blood vomiting and stomach ache	Camelliosides	Experimental studies in rats	Decreased the ethanol and indometacin-evoked gastric ulceration
Mango (Mangifera indica L.)	Aqueous decoction of mango flowers	Gastrointestinal disorders and arthralgias	Xanthone (mangiferin), flavonoids and triterpenes	Acute and subacute models of gastric ulceration in rodents	Decreased the acetic acid, ethanol and stress-induced gastric ulceration
Grape-seed (Vitis vinifera L.)	Seed extract	Gastro protection	Proanthocyanidins	Animal models of gastric ulceration	Decreased the ethanol, stress, and indometacin-induced gastric ulceration
Karela (Momordia charantiaL.)	Dried powdered fruits	Traditionally used in diabetes and for healing of peptic ulcer	Triterpenes and glycosides, karavilagenins and karavilosides	Animal models of gastric ulceration	Reduced ulceration
Almecega (Protium heptaphyllum March.)	Resin from the trunk wood	Traditionally used remedy in gastrointestinal disorders	Volatiles and triterpenes (alpha-and beta-amyrin)	Animal studies	Antiulcer, visceral antinociceptive and anti-inflammatory effects of α- and β-amyrin
Coptis (Coptis Chinensis Franch.)	Root extract	Traditionally used remedy in gastrointestinal disorders	Alkaloids (10% berberine)	Animal studies	Inhibits ulcer formation and acid secretion
Bone setter (Cissus quadrangularis L.)	Methanolic extract	Traditionally used for fracture healing	Vitamin C and β- carotene	Rat study	Prevents indometacin-induced ulceration
Red sanders (Pterocarpus santalinus L.)	Ethanolic extract	Traditional herbal drug for wound healing	3-keto-oleanane	Rat gastric ulceration model in vivo and anti-H. pylori in vitro	Cytoprotection antioxidant, anti-H. pylori and antiulcer effects

STW 5 is composed of ethanolic extracts from nine plants (Ibera Amara Totalis, Angelicae Radix, Cardui Mariae Fructus, Carvi Fructus, Chelidonii Herba, Liquiritae Radix, Marticariae Flos, Melissae Folium, Menthae Piperitae Folium). N-095 is a mixture of red ginseng, polygala root, saffron, antelope horn and aloe wood.

Sangre de grado is derived from several Croton species (Croton dracanoides, Croton palanostigma, Croton lecheleri, Croton urucurana).

Baishouwu is an appellative name of dried root tubers from three Asdepiadaceae plants: Cynanchum auriculatum Royle ex Wight, Cynanchum bungei Decne and Cynoctonum wilfordii Maxim.

• suppressing the neutrophil/cytokine cascade in the gastrointestinal tract

• promoting tissue repair through expression of various growth factors

• exhibiting antioxidant activity, scavenging reactive oxygen species

• inhibiting cytochome P450 2F1 activity, producing antinecrotic and anticarcinogenic activities.

However, experimental studies have aimed mostly to validate the traditional use of plants in gastrointestinal disease, but no attempt has been made to verify their potential in functional dyspepsia.

Irritable bowel syndrome

IBS is a potentially debilitating condition characterised by abdominal discomfort, bloating, and disturbed patterns of defecation with a lower health-related quality of life. According to Rome III criteria, irritable bowel syndrome is defined as recurrent abdominal pain or discomfort for at least 3 days per month. It affects approximately 15-30% of the general population. The prevalence is equally divided among three subtypes: irritable bowel syndrome with constipation, IBS with diarrhea, and irritable bowel syndrome with alternating constipation/diarrhea.

Conventional therapy includes the use of bulk laxatives and stool softeners for constipation, anti-motility drugs for diarrhea, and antispasmodics, antimuscarinics and antidepressants for pain and spasm. Chronic constipation is a very common disorder, and the goals in treating such patients are to improve the patient’s symptoms and to restore normal bowel function, aiming to achieve at least three bowel movements per week. Serotonin plays a prominent role in chronic constipation, since it affects the intestinal motility, fluid secretion and sensation through activation of receptors present in enterochromaffin cells.

The only FDA-approved agents for chronic idio-pathic constipation are tegaserod (5-HT₃ agonist) and lubiprostone, a type 2 chloride-channel activator, which both represent real therapeutic advances in the management of these patients. Lubiprostone draws chloride, sodium, and water into the lumen of the gut enhancing fluid secretion and facilitating increased motility and colonic transit. Lubiprostone, although significantly better than placebo in improving symptoms of constipation severity, stool consistency, straining, and abdominal discomfort, has been shown to be associated with adverse events such as nausea (30.2%), diarrhea (19.2%) and distention (9.3%). However, patients are refractory to these agents in the presence of pelvic floor dyssynergia, leaving the option for therapy with a prokinetic agent, fibre or a laxative. Thus, there is an overall dissatisfaction with traditional treatment options from patients and physicians.

Clinical studies with herbals in functional gastrointestinal disorders

In the recent past, a few controlled clinical studies were carried out with phytotherapeutic combinations (i.e. combinations of various plant/herbal extracts with a number of different active ingredients) which showed superiority over the placebo treatments (Table: Clinical studies with herbal medicinals in gastrointestinal functional disorders). However, randomised controlled trial (RCTs) data supporting the efficacy of these treatments in patients with functional gastrointestinal disorders (FGIDs) are still lacking. RCTs indicated that peppermint oil could be efficacious for symptom relief in irritable bowel syndrome and meta-analysis confirmed this. Placebo-controlled RCTs demonstrated the clinical efficacy and safety of a polyherbal preparation, STW 5 (Iberogast, Enzymatic Therapy Inc.) for the treatment of both functional dyspepsia and patients with irritable bowel syndrome. The pharmacological effects, as well as the therapeutic effectiveness, tolerability, and toxicity, of Iberogast were experimentally and clinically recorded and documented. These studies indicated that Iberogast promotes gastric relaxation and stimulates antral motility. Tong-xie-ning, a standard Chinese traditional herbal formula, and Padma Lax, a Tibetan herbal medicine, showed significant improvement of global symptoms in FGID, when compared with placebo treatment. An RCT assessing artichoke leaf extract in 247 patients with functional dyspepsia demonstrated a significant improvement in both overall symptoms and disease-specific quality of life compared with placebo.

Table: Clinical studies with herbal medicinals in gastrointestinal functional disorders

Name	Part used	Use	Constituents	Study type	Results
STW 5 lberogast® (herbal formula)	Ethanolic extracts of 9 plants	Functional dyspepsia and IBS	Methionine like sulphur-compounds	Placebo-controlled RCT	Improved gastrointestinal symptom-severity score
Carmint	Total extracts of 3 plants	Irritable bowel syndrome (IBS)	(E)-2-dodecenal, eugenol, menthol and L-carvone	Pilot clinical study	Reduced the severity and frequency of abdominal pain/ discomfort
Tong-xie-ning (TCM-herbal formula)	Dried extract of four herbs	Diarrhoea-predominant IBS	Complex compound containing paeoniflorin	Placebo-controlled RCT	Reduced symptom severity
Hange-koboku-to (HKT, Kampoo medicine)	Dried mixture of 5 crude herbs	Functional dyspepsia	Magnorol, konokiol, perillaldehyde, 6-gingerol, 6-shogaol	Open clinical study	Improves delayed gastric emptying (prokinetic effect)
Padma Lax (Tibetan herbal medicine)	Dried extract from 10 plants	Constipation-predominant IBS	Multicomponent	Double-blind randomised pilot study	Reduced symptom severity
Artichoke (Cynara scolymus L.)	Leaf extract	Dyspepsia and IBS	Caffeoylquinic acids and flavonoids	Open clinical study	Amelioration of global symptoms and improvement in QOL score
Peppermint (Mentha piperita L.)	Leaf essential oil	Irritable bowel syndrome (IBS)	Menthol and menthonin	Double-blind RCT	Smooth muscle relaxation; analgesia
Turmeric (Curcuma longa L.)	Rhizome extract	IBS; ulcer and non-ulcer dyspepsia	Curcumin (diferuloyl methane)	Placebo-controlled RCT	Improved healing of peptic ulcer and of non-ulcer dyspepsia but not of IBS
Red pepper (Capsicum annuum ssp.)	Powder	Functional dyspepsia	Capsaicin	Placebo-controlled RCT	60% Reduction of symptom score
Mangava-brava (Lafoensiapacari St Hil.)	Methanolic extract	Used in gastric ulcer and inflammatory conditions	Ellagic acid	Double-blind RCT against H. pylori	Well tolerated and patients were symptom-free in an 8-week trial

STW 5 is composed of ethanolic extracts from nine plants (Ibera AmaraTotalis, Angelicae Radix, Cardui Mariae Fructus, Carvi Fructus, Chelidonii Herba, Liquiritae Radix, Marticariae Flos, Melissae Folium, Menthae Piperitae Folium).

Carmint contains total extracts from three plants (Melissa officinalis, Mentha spiccata and Coriandrum sativum).

Tong-xie-ning consists of dried substances of Paeonia lactiflora Pali, (root), Atractylodes macrocephala Koidz. (rhizome), Citrus reticulata Blanco, (unripe exocarp) and Allium macrostemon Bge. (bulb).

HKT is a composite of five crude herbs: Pinelliae Tuber, Hoelen, Magnoliae Cortex, Perillae Herba and Zingiberis Rhizoma.

Padma Lax is composed of Aloes, Calumba, Cascara, Myrobalan, Condurango, Elecampane, Frangula, Gentian, Pepper and Nux vomica.

RCT = randomised, controlled trial; QOL = quality of life.

Studies on curcumin (diferuoylmethane), an ingredient of turmeric (Curcuma longa) demonstrated improvement in healing of peptic ulcer and symptoms in non-ulcer dyspepsia but not in irritable bowel syndrome. Capsaicin, the active ingredient of another spice, red chilli pepper, has been evaluated in small RCTs, which yielded conflicting results in patients with functional dyspepsia. While one study reported significant improvement in overall symptoms, epigastric pain, fullness and nausea compared with placebo, an earlier placebo-controlled crossover trial was unable to show significant improvements in postprandial dyspepsia scores with capsaicin.

The combination of extracts of different plants appears to be advantageous to treat functional gastrointestinal disorders (FGIDs) compared with conventional chemically well-defined drugs. Nevertheless, several issues regarding herbal products deserve mention. Based on current evidence, the scientific validity of the use of many of these commercial natural products in FGIDs is severely limited, with quality control and regulatory issues continuing to be a concern. The available trials almost all suffer from significant methodological flaws making the results difficult to interpret. Although the short-term use appears relatively safe, the long-term safety of these agents has not been established. Further, because these natural products are not regulated as pharmaceuticals, questions regarding agent purity and potency could be raised.

Safety and efficacy issues

The quality of herb used and its chemical constitution is fundamental to understanding the product’s intended use and factors affecting its safety. It is the chemical constituents that are the basis for the pharmacological activity of a herbal product, and consequently for the therapeutic efficacy. Use of validated, reliable, and relevant methods for efficacy/toxicity studies with regulatory strategies are essential to create a stronger evidence base on the safety, efficacy and quality of the herbal products for functional gastrointestinal disorders.

Evaluation of herbal medicines: preclinical studies

Preclinical assays are essential to guarantee the safety and efficacy of natural products in functional gastrointestinal disorders (FGIDs). The pathophysiology of FGID is not firmly established, and is characterised by recurrent episodes of gastrointestinal symptoms with no structural alterations. Visceral hypersensitivity, disordered gastrointestinal motility and secretion are presently considered key mechanisms underlying FGID symptoms. Therefore, pharmacological studies with new herbal products for efficacy and safety assessment are carried out using in-vitro and in-vivo experimental models that helped define basic mechanisms of functional dyspepsia and IBS symptoms. Animal testing should follow the ethical considerations and the approval of the Institutional Committee on the Use of Animals for experimentation.

In-vitro and animal testing

The emerging therapies for functional gastrointestinal disorders are largely aimed at normalising pain perception and gastrointestinal motor and reflux function. Helicobacter pylori infection, NSAID consumption and severe physical stress are associated with FGID. For this reason, the most common pharmacological properties determined in the evaluation of drugs effective for FGID are the anti-Helicobacter, cytoprotection, visceral analgesic, spasmolytic, antisecretory, antidiarrheal and prokinetic effects. The requirement for each one of these studies include experimental animals (two species, one rodent and another non-rodent, 6-8 per group), appropriate controls (negative and positive), and product evaluation at three dose levels.

H. pylori may induce gastritis and chronic dyspepsia (functional non-ulcer dyspepsia or gastro-duodenal ulcer) in humans. Mongolian gerbils or BALB/c mice infected with H. pylori are often used as animal models to screen test compounds for their efficacy in reducing the extent of gastric ulceration or inflammation. Although these animals demonstrate gastric ulceration, virtually no gastritis is seen in the antrum of infected animals, but this is, however, the hallmark of human infection. The anti-H. pylori activity of a test drug can be assessed through in-vitro studies employing rat gastric epithelial cell cultures and H. pylori isolates from gastric mucosal biopsy patients. The minimum inhibitory concentration value of test drug is first established against H. pylori and then H. pylori is co-cultivated with rat gastric epithelial cells in the presence/absence of test drug at its minimum inhibitory concentration. A reduction in the activity of urease, a normal appearance of the epithelial cells on electron microscopic examination, a decrease in lipid peroxidation and lactate dehydrogenase suggests the possible anti-H. pylori activity of PS.

Gastric cytoprotection can be evaluated using rat/mouse/guinea pig models of gastric lesions induced by absolute ethanol, indometacin, histamine and immobilisation stress and, to elucidate the physiological mechanism, effects of herbal products on mucus secretion, acid secretion (pylorus-ligated animals), glutathione reserve, mucosal blood flow and gastric emptying time are analysed. In addition, to understand the pharmacological mechanism underlying the gastroprotection, the role of capsaicin-sensitive fibres, endogenous prostaglandins and nitric oxide may be verified, using capsaicin-desensitisation procedure, and/or analysis of prostaglandins by ELISA and nitrite/nitrate by Griess reaction or indirectly by the use of nitric oxide synthase inhibitors.

Visceral hyperalgesis is a characteristic feature of functional dyspepsia and irritable bowel syndrome. Visceral antihyperalgesic effects of herbals can be examined using animal models of nociception induced by intracolonic administration of mustard or capsaicin. These chemicals evoke both inflammatory and non-inflammatory pain through sensitisation of neurones at the peripheral and/or central sites, involving several neuropeptides and a great variety of inflammatory mediators. They serve as valuable tools in assessing gut pain and to study the possible mechanism.

Spasmolytic and antidiarrheal properties of test compounds can be assessed in well-established models such as the USSING-chamber, a pharmacological model for diarrhea, and the isolated guinea pig ileum, a model for modulatory effects on ileum contraction. The inhibitory effect observed in these models provides ex-vivo evidence for the spasmolytic and antidiarrheal activities of herbal products. Several studies have investigated abnormalities of serotonin signalling in IBS. Decreased postprandial serotonin plasma levels have been reported to occur in constipation-predominant IBS, and increased plasma levels have been reported to occur in diarrhea-predominant irritable bowel syndrome.

To verify the potential usefulness of herbals in diarrhea-prone IBS, in-vivo testing could be carried out in mice on castor oil- or croton oil-induced diarrhea, cholera toxin-induced intestinal secretion and on gastrointestinal transit induced by 5-HTP, using alosetron, an antagonist for 5-HT₃ receptors and a known agent effective for diarrhea-predominant irritable bowel syndrome as a positive control. Further, morphine-induced gastrointestinal delay in mice may be used as a model to test the compound’s likely use in constipation-predominant IBS, using tegaserod, a 5-HT₄ receptor agonist, as a positive control. Tegaserod is a known prokinetic agent that speeds small-bowel transit and right-colon transit in irritable bowel syndrome, reducing symptoms of constipation, pain and bloating.

Safety pharmacology

Clinical assessment of the gastrointestinal tract is often limited to measurements of transit time and observations of vomiting or diarrhea. In-vitro functional human tissue assays can be performed to measure a vast range of toxic effects of drugs under investigation, at the level of the organ, cell or even gene and these assays are considered an important adjunct to routine safety pharmacology tests. The cytotoxicity of new compounds can be determined by the MTT reduction assay using human lung fibroblasts (MRC-5). These models are rapid, less expensive and reveal mechanisms of action. The data obtained serve as signals of potential harmful effects in humans and are often superior to extrapolation from animals. The heart is a frequent site of toxicity of pharmaceutical compounds in humans, and, when developing a new drug, it is critical to conduct a thorough preclinical evaluation of its possible adverse effects on cardiac structure and function. Changes in cardiac morphology such as myocardial necrosis, hypertrophy or valvulopathy are assessed in laboratory animals. The potential proarrhythmic risk of new drugs is a major subject of concern and needs to be fully addressed before treatment of volunteers or patients takes place. The recommended tests to detect most arrythmic drugs were to determine the effects on cardiac ion channels, in particular I(Kr) potassium channel antagonism in vitro and prolongation of the QT interval, assessed in vivo, in telemetred dogs.

In-vivo animal safety data serve as important signal generators and in some cases, may stand alone as indicators of unreasonable risks. These include acute toxicity, and long-term toxicity, reproduction toxicity, genotoxicity and carcinogenicity studies. Knowledge of an ingredient’s pharmacokinetics and in-vivo metabolism will allow most appropriate interpretation of relevancy of the dose/concentration used in the in-vitro tests. Evidence of abnormalities from laboratory animal studies can be indicative of potential harm to humans.

Acute toxicity testing involves the study of toxic effects after a single (oral/parenteral) administration of the test compound with the objective to classify the substance associated on the basis of acute toxicity, identification of target involved in the acute toxic effect (signs: time of appearance, progression and reversibility), and to establish the dose intervals that may be of relevance to other toxicological studies. A long-term toxicity study (4-12 weeks) involves the repeated doses of the test substance, aimed to determine the maximum tolerable dose, the highest dose that does not result in toxic effects and the mechanism underlying the toxic effects in vivo by means of biochemical, behavioural and histopathological studies. Genotoxicity and carcinogenesis studies are important because several medicinal plants contain substances such as pyrrolizidine alkaloids, flavonoids, phorbol esters, etc., which can cause mutagenic effects. The Ames test using Salmonella typhimurium strains and the CHO (Chinese hamster ovary) chromosomal aberration test are the two most commonly used methods for the evaluation of genetic mutation and chromosome damage.

Evaluation of herbal medicines: controlled clinical trials

The choice of primary endpoint for a clinical trial is to demonstrate the efficacy of a therapeutic agent.

The Rome III Committee recommends two types of measures to assess the efficacy of new treatments for irritable bowel syndrome and functional dyspepsia:

• binary endpoints addressing the construct of relief (that is, adequate relief and satisfactory relief)

• an integrative symptom questionnaire that addresses the change in severity of a representative group of symptoms of irritable bowel syndrome (that is, the IBS Severity Scale).

The current evidence suggests that at present, adequate relief should be recognised by regulatory authorities as an acceptable primary endpoint in clinical trials. This analysis also suggests that data from individual clinical trials should be pooled and undergo meta-analysis, and that prospective studies should be considered to further characterise the performance of available endpoints as outcome measures in pharmacotherapeutic trials. Literature search reveals that few well-controlled, double-blind (placebo-controlled) trials have been carried out with herbal medicines (see Table: Clinical studies with herbal medicinals in gastrointestinal functional disorders). Meta-analyses of reviews published reveal several discrepancies, and these are mostly due to:

• lack of standardisation and quality control of the herbal drugs used in clinical trials

• use of different dosages of herbal medicines

• inadequate randomisation in most studies, and patients not properly selected

• numbers of patients in most trials insufficient for the attainment of statistical significance

• difficulties in establishing appropriate placebos because of the tastes, aromas, etc.

• wide variations in the duration of treatments using herbal medicines.

A few herbal products, e.g. STW 5 (Iberogast), Artichoke, Carmint, Tong-xie-ning (Chinese herbal formula), Hange-koboku-to (Kampoo medicine), and Padma Lax (Tibetan herbal medicine) have been evaluated in clinical trials, but they still have the difficulties mentioned above and need additional, well-controlled and appropriate randomised clinical trials to prove their efficacy.

To assess the efficacy of new herbals in functional gastrointestinal disorders, a double-blind, randomised, placebo-controlled, parallel group trial remains the preferred design. Investigators should include as broad a spectrum of patients as possible and should report recruitment strategies, inclusion/exclusion criteria, and attrition data. The primary analysis should be based on the proportion of patients in each treatment arm who satisfy a prespecified clinically meaningful change in a patient-reported symptom-improvement measure. Such measures of improvement are psychometrically validated subjective global assessments or a change from baseline in validated symptom guidelines and include an analysis of harms data and secondary outcome measures to support severity questionnaire. Data analysis should address all patients enrolled, using an intention-to-treat principle. Reporting of results should follow the Consolidated Standards for Reporting Trials or explain the primary outcome. Trials should be registered in a public location, prior to initiation, and should be reported even if the results are negative or inconclusive.

Conclusions

No standard therapy is currently available for functional gastrointestinal disorders such as functional dyspepsia and irritable bowel syndrome, which are characterised by multiple symptoms associated with disordered gut function. There is limited evidence for the efficacy, safety and tolerability of currently available conventional therapies and it may be that, with traditional herbal therapies, patients may respond better in symptom improvement because of their multicomponent nature with different active constituents having pleotropic actions. However, herbal medicinals should undergo the same procedures as conventional drugs and should not be considered differently because they are of natural origin. Preclinical studies on very many herbal extracts and their active constituents demonstrated interesting pharmacological properties relevant to functional dyspepsia and irritable bowel syndrome, but only few of them were taken to clinical trials with limited success. Future studies should address their effectiveness and safety in patients with FGIDs by the double-blind, randomised, placebo-controlled, parallel-group trial study design. The safety and their quality should be ensured through greater pharmacovigilance studies and by governmental regulatory mechanisms. To provide uniform quality of raw material, emphasis must be laid on domestication, production and biotechnological studies and genetic improvement of medicinal plants to provide uniform and high-quality raw material.