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 Rg₃ reduced 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.

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

Table: In vitro and in vivo angiogenesis assays

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.