Review J. Clin. Biochem. Nutr., 38, 9–18, January 2006

Cardioprotective and Other Beneficial Effects of Some Indian Medicinal

Jai Ashish Tilak-Jain and Thomas Paul Asir Devasagayam*

Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400-085,

Received 29 September, 2005; Accepted 31 October 2005

Summary The distinguishing feature of the traditional Indian medicinal system, Ayurveda, is its emphasis on the maintenance of positive health. As per Ayurveda, Indian medicinal plants are rich sources of substances that have several therapeutic properties including cardiopro- tection. Globally, cardiovascular diseases are a leading cause of mortality. Several epidemio- logical studies suggest a correlation between antioxidant intake and occurrence of various cardiovascular diseases. This review deals with medicinal plants which possess cardiotonic, cardioprotective and antioxidant effects. It also includes our studies on the antioxidant prop- erties of some of these plants such as Terminalia arjuna (arjun), Trigonella foenum-graecum (), Curcuma longa (), indica (kokum) and Vitis vinifera (grapes). Among these, Terminalia arjuna possesses the highest antioxidant potential and inhibit whole plasma oxidation in rats besides showing significant intestinal absorption. Other plants studied also exhibit radical scavenging effects as studied using various biochemical assays. These antioxidant effects may possibly be responsible for their known beneficial therapeutic effects including their use in cardioprotection.

Key Words: Cardiovascular diseases, Indian medicinal plants, antioxidant, extracts, Terminalia arjuna

Role of Ayurveda in Human Health Introduction India is well-known for its rich traditional systems of The World Health Organization (WHO) estimates that medicine, i.e. Ayurvedic, Siddha, Unani and Amchi 80% of the people of developing countries rely on traditional (Tibetan) besides a vast reservoir of living traditions of eth- medicines, mostly plant-derived drugs, for their primary nomedicine. Many rural households in India, with limited health needs. Medicinal plants are commonly used in access to organized health services practice home remedies, treating and preventing specific ailments and are considered the recipes and formulae of which have been handed down to play a significant role in health care. Traditional medicinal from generation to generation. In Indian systems of medi- systems use plants as indispensable sources of medicinal cine, generally the medicines of plant origin are preferred preparations. Hundreds of species are recognized as having over the medicines of animal origin, due to presence of medicinal value. Indeed, ‘phytomedicines’ are beginning to abundant natural flora. The basic concept of disease preven- link traditional and modern medicines. tion, has existed in the ancient Vedic scripture and has been practiced in Indian traditional medicine, the Ayurveda, for *To whom correspondence should be addressed many centuries. The emphasis on the maintenance of posi- Tel: +91-22-25593948 Fax: +91-22-25505151/25560750 tive health or Swastha Vrutta, is a distinguishing feature of E-mail: [email protected]; or [email protected] Ayurveda [1]. In Ayurveda, it is clearly mentioned that any

9 10 J.A. Tilak-Jain and T.P.A. Devasagayam patient can be cured with the help of present in the sur- potential to attenuate atherosclerosis, which is a leading roundings. The two main approaches to illness in Ayurveda cause of mortality in several human populations. There are are preventive and curative. [2]. A harmonious balance be- also epidemiological evidences and interventional studies to tween three humors of the body viz. ‘Vayu’, ‘Pitta’ and correlate higher level of antioxidant-rich food uptake with ‘Kafa’ is needed for positive health; imbalance of these may lower incidence of CHD [12, 13]. Contrary to popular belief, cause disease(s). A significant part of Ayurvedic therapeu- CHD is indeed common in the Indian subcontinent. The tics aims to promote positive health. The prescribed proce- prevalence of CHD increased from 1% to over 8% in urban dures include drugs along with daily routine including population [14]. Indians have among the highest prevalence exercise, diet and nutirtion besides mental attitude and of CHD and have rather unusual risk factors characterized discipline. This is achieved by using extracts of various plant by high triglycerides, low High Density Lipoproteins materials, the rasayanas. These enhance body’s resistance (HDL), glucose intolerance, insulin resistance, abdominal and by using them, one obtains longevity, regains youth, gets obesity and increased lipoprotein (a) levels [15]. Hence there sharp memory and intellect besides curing diseases [3, 4]. is an urgent need to explore various strategies to combat the Indian medicinal plants are rich sources of substances that increasing risk of CVDs in the Indian subcontinent. Medic- have several therapeutic properties like cardioprotective, inal plants with cardioprotective effects can play a major chemopreventive and other effects. role in this aspect.

Cardiovascular Diseases Oxidative Stress, Antioxidants and CVD

Globally, cardiovascular diseases (CVD) constitute a Evidence for the involvement of free radicals in the eti- leading cause of mortality. Developing countries like India ology of CHD comes from several studies that suggest a are also struggling to manage the impact of CVD along with correlation between antioxidant intake and various CVDs the growing burden of obesity, Type II diabetes and hyper- [16, 17]. Antioxidant defenses also appear to be a distin- tension [5]. Heart disease in India occurs 10 to 15 years guishing factor between normal subjects and those with earlier than in the West. One fifth of the deaths in India are symptomatic CHD. Antioxidants remain higher in normal from coronary heart disease (CHD). By the year 2020, it will subjects and keep lipid peroxidation (LP) under control. In account for one third of the deaths. Current projections symptomatic CHD, antioxidant levels are significantly low- suggest that by the year 2020, India will have the largest ered [18]. Hence, increased intake of antioxidants, espe- CVD burden in the world [6]. The prevalence of these dis- cially lipid-soluble and chain-breaking antioxidants that eases is more in urban than in rural areas [7]. Lower vitamin accumulate in lipoproteins, might be expected to have C and selenium in Indians as compared to other ethnic beneficial effects [19]. A large number of studies in experi- groups, particularly in combination, could play a part in their mental animals have shown that hypercholesterolemia, dia- increased risk of CHD. Lower vitamin C in Indians is betes, hypertension, smoking, ageing and nitrate intolerance probably because of its destruction by prolonged cooking are the common risk factors for atherosclerosis. They in- [8]. There are epidemiological correlations between poor crease production of free radicals not only by endothelial plasma levels of essential antioxidants and the risk of cells but also by vascular smooth muscle cells and ad- coronary heart disease [9]. ventitial cells. However, the defense system in terms of Epidemiological studies have revealed many important exogenous antioxidants, i.e. natural compounds such as cur- risk factors of environmental and genetic origin that are , baicalein and resveratrol prevent atherosclerosis for- associated with atherosclerosis. The most important clinical mation by exhibiting radical scavenging effects, as shown in complication is an acute occlusion due to blood clot forma- the schematic Figure 1. tion during rupture of the lesion, resulting in myocardial infarction [10]. One of the major initiating event in athero- Medicinal Plants with Cardioprotective Properties sclerosis is oxidative damage to the cholesterol component of the LDL known as LDL oxidation. Oxidation of LDL In recent times, there is a lot of interest in ‘phytonutrients’ contributes to atherogenesis in various ways [11]. An from plants with potential benefits. While these phyton- appropriate balance between processes that stimulate or in- utrients are not essential by traditional definitions, they hibit oxidative stress, LDL oxidation, and additional LDL apparently reduce risks of diseases. A large number of atherogenic modifications determines the progression of epidemiological studies show that diets rich in fruits and atherogenesis. vegetables, i.e. foods rich in antioxidants, are associated LDL oxidation and atherogenesis can be inhibited by with lower incidence of CVDs [13]. Apart from these dietary antioxidants. Elevation in the activity of nutritional antioxi- sources, Indian medicinal plants are also known for their dants over the damaging effects of prooxidants has the cardioprotective properties and are rich sources of antioxi-

J. Clin. Biochem. Nutr. Cardioprotective Indian Medicinal Plants 11

Fig. 1. Role of reactive oxygen species (ROS) in cardiovascular disease and its prevention by natural antioxidants. dants [5, 20, 21]. Many plants have been used for cardiopro- officinalis (). These plants exhibit potent antioxidant tection in the traditional Indian medicinal system. A review effects, which might be the mechanism behind their bene- of such plants with cardioprotective/antioxidant effects was ficial therapeutic properties. Details of Indian medicinal carried out. Several herbs and herbal products have been plants having cardioprotective, therapeutic and antioxidant recommended to promote a healthy heart. These include properties are given in Table 1 [23–52]. , guggulipid, tocotrienols derived from palm oil, soy To elucidate the possible positive correlation between the protein isoflavones, and Chinese red yeast rice. They have antioxidant and cardioprotective effects, we studied some been shown to lower cholesterol levels by different mecha- medicinal plants in detail. These include Terminalia arjuna nisms. Other antioxidant-rich and antiangiogenic herbs such (arjun), Trigonella foenum-graecum (fenugreek), Curcuma as green tea, black tea, and red wine have the potential to longa (turmeric), Garcinia indica (kokum) and Vitis vinifera reduce the progression of atherosclerosis [22]. (grapes). A summary of our studies are given below. Terminalia arjuna (Arjun) is an Indian medicinal plant Indian Medicinal Plants and very popular cardiac tonic. It improves cardiac muscle function and subsequently enhances pumping activity of the Ayurveda has identified many plants which possess heart. It has been credited with many beneficial therapeutic cardiotonic and cardioprotective effects. Some of them are properties including cardioprotective effects. It is used as a Allium sativum (garlic), Allium cepa (onion), Asparagus cure for various cardiovascular disorders such as congestive racemosus (shatavari), Caesalpinia bonducella, Cassia heart failure, coronary artery disease, myocardial necrosis, fistula, Curcuma longa (turmeric), Emblica officinalis angina, atherosclerosis and ischemia-reperfusion injury be- (Amla), Garcinia indica (kokum), Hemidesmus indicus sides having hypolipidaemic activity [47, 53]. Its bark is (anantmul), Ocimum sanctum (tulsi), Phyllanthus amarus, mainly used in the ayurvedic medicinal preparations. A Picrorrhiza kurroa (katuka), Terminalia arjuna (arjun), decoction of the bark and minerals was used in the treatment Trigonella foenum-graecum (fenugreek), Vitis vinifera of heart disease of microbial origin. The other beneficial (grapes), Withania somnifera (ashwagandha) and Zingiber properties include anti-hemorrhagic, anti-pyretic, anti-al-

Vol. 38, No. 1, 2006 12 J.A. Tilak-Jain and T.P.A. Devasagayam

Table 1. Indian medicinal plants having antioxidant and cardioprotective effects Indian medicinal Antioxidant effects Cardioprotective effects Other therapeutic effects References plants Allium Oils isolated from garlic inhibit nicotine-induced lipid Cardioprotective, hypolipidaem- Antimicrobial, antiarthritic; anti- [23, 24] sativum peroxidation in rat tissues; Aqueous garlic extract ic, antithrombotic, mild choles- tumor, hypoglycemichepatopro- (garlic) (250 mg/kg) decreases lipid peroxidation, increases terol lowering effect and reduces tective and anticarcinogenic GPX, GST and GSH during buccal pouch blood clotting carcinogenesis in Syrian male hamsters Allium cepa Oils isolated from onion inhibit nicotine-induced lipid Cardioprotective, hypolipidaem- Antimicrobial, antitumor, [23] (onion) peroxidation in rat tissues ic, antithrombotic hypoglycemic, antiarthritic Asparagus Purified polysaccharide fraction inhibited Ayurvedic preparation using this, Immunomodulator, anti-stress [25, 26] racemosus γ-radiation-induced lipid peroxidation in rat liver abana, gives cardioprotection agent, antihepatotoxic (shatavari) mitochondria Caesalpinia Leaves decreased lipid peroxidation and increased GSH, Decreases blood glucose levels Anti-diabetic, anti-pyretic, [27, 28] bonducella SOD, and CAT significantly analgesic, antitumor Cassia fistula Aqueous extract of flowers decreased levels of CD, Used for treatment of cardiovas- Hepatoprotective, antidiabetic, [29] LOOH, TBARS and restored GSH, SOD, CAT, GPX, cular diseases antitumor GR in diabetic rat heart tissues Curcuma Natural curcuminoids act as antioxidants; turmeric Decreases proliferation of Anticarcinogenic, anti-inflam- [30–33] longa extracts have antioxidant effects, curcumin inhibits smooth muscles in blood vessels, matory, radioprotective, hepato- 1 (turmeric) O2-induced DNA damage; inhibits γ-radiation-induced protects from blockage of protective and anticlastogenic lipid peroxidation in rat liver microsomes arteries Emblica Inhibits TBARS formation and increases SOD, CAT, Hypolipidemic, decreases Anti-inflammatory, hepatopro- [34, 35] officinalis GPX during oxidative stress in rat brain ischemia-reperfusion-induced tective, anticandidal, cytoprotec- (Amla) oxidative stress in rat heart tive, anticlastogenic, anti-ulcer, anti-pyretic, anti-tumor and analgesic Garcinia Garcinol is a good antioxidant and inhibits iNOS Cardiotonic Neuroprotective, anti-inflamma- [36–38] indica (kokum) induction in astrocytic cells tory, anti-tumor Hemidesmus Inhibition of free radical formation, inhibits LP, Caps HT2, a herbal Ayurvedic Anti snake venom activity, [39, 40] indicus increases SOD medicine is antiatherogenic anti-pyretic, anti-inflammatory, (anantmul) antipyretic Ocimum Orientin and vicenin inhibited radiation induced lipid Hypoglycaemic in diabetic rats, Chemoprotectant, anti-thyroid, [40–42] sanctum (tulsi) peroxidation in vivo; Ocimum seed oil increased SOD, cardiac protection in isoprotere- radioprotective, neuroprotective, CAT, GST, GSH and decreased lipid peroxidation in nol induced myocardial infarc- anti-ulcerogenic mice tion in rats Phyllanthus Antioxidant, increases SOD, GST, GPX Significantly reduces blood Anti-diabetic, antiinflammatory, [43, 44] amarus glucose anti-mutagenic, antidiarrhoeal radioprotective Picrorrhiza Alcoholic extract prevented lipid peroxidation and Cardioprotection against isoprot- Hepatoprotective, anti-allergic, [45] kurroa increases activities of SOD, CAT during erenol-induced myocardial stress antidiabetic, anti-ulcerogenic, (katuka) D-galactosamine induced hepatitis in rats in rats, hypolipidaemic antileishmanial Terminalia Potent free radical scavenger, inhibits oxidative damage Cardiotonic, cardioprotective, Hepatoprotective, antimutagen- [46, 47] arjuna (arjun) to lipids, proteins and DNA in rats hypocholesterolaemic, anti-angi- ic, anticarcinogenic, anti- nal, anti-ischemic, hypolipidem- haemorragic, induces union of ic, anti-atherosclerotic fractures Trigonella foe- Antioxidant in diabetic rats Decreased blood glucose levels, Antidiabetic activity [48] num-graecum lowers cholesterol, LDL, VLDL, (fenugreek) triglycerides Vitis vinifera Antioxidant activity due to isoflavons Cardioprotective Antiinflammatory, chemopre- [49–50] (grapes) ventive; anti- mutagenic Withania Sitoindosides VII-X and withaferin A increased SOD, Cardioprotective and Immunomodulatory, anti-inflam- [51] somnifera CAT, GPX in rat frontal cortex and striatum anticoagulant matory, anti-aging, anti-stressor, (ashwagandha) anticarcinogenic and thyroid stimulatory Zingiber Lowers lipid peroxidation and maintains SOD, CAT, Cardiotonic Carminative, antibacterial, [36, 52] officinalis GPX and increased blood GSH levels in rats aphrodisiac, general tonic, blood (ginger). purifier, anti-inflammatory Abbreviations: CAT- catalase; CD- conjugated dienes; DNA- deoxyribose nucleic acid; GPX- glutathione peroxidase; GR- glutathione reductase; GSH- reduced glutathione; GST- glutathione transferase; iNOS- inducible nitric oxide synthase; LDL- low density lipoprotein; LOOH- lipid hydroperoxide; LP- lipid peroxidation, 1 O2- singlet oxygen; SOD, superoxide dismutase; TBARS- thiobarbituric acid reactive substances; VLDL- very low density lipoprotein.

J. Clin. Biochem. Nutr. Cardioprotective Indian Medicinal Plants 13 lergic, antimutagenic, antigenotoxic, antitumor, chemopre- anion production [60, 61]. Some of the beneficial activities ventive and anti-radical effects [54]. There are several of turmeric have been linked to the ’s antioxidant formulations of arjun in Ayurveda/Siddha systems of medi- ability in different systems [4, 32]. However, the availability cine such as Arjunsal, Arjunaristam, Arjunaghrita and Arjun of antioxidant effects of turmeric and its constituents, in re- Siddha Dugdha. Baicalein is a naturally occurring flavonoid lation to the different beneficial properties as is being used (5,6,7-trihydroxy-2-methyl-4H-1-benzopyran-4-one) present in cooking and medicine has not been evaluated earlier. in bark of Terminalia arjuna. It is credited with anti-allergic, Garcinia indica (dried rind known as ‘kokam’) is an anti-proliferative, antitumor and other beneficial properties Indian spice used in many parts of India in making several [55, 56]. vegetarian and non-vegetarian ‘curry’ preparations. It is also Trigonella foenum-graecum (Fenugreek, Methi) is an im- used in preparing healthy soft drink mixes [36]. Kokam’s re- portant spice used in India and various other Asian, African frigerant properties are well documented. It is useful as an and European countries for its health benefits. Its leaves, infusion, or by direct application, in skin ailments such as tender shoots and germinated seeds are being consumed as rashes caused by allergies. Kokam butter is an emollient vegetables. They are also considered as health foods [36]. being helpful in the treatment of burns, scalds and chaffed Fenugreek seeds are mainly used for flavouring pickles and skin [62]. Kokam in food preparations is used as an chutnies. The major use of fenugreek is in . appetizer and good liver tonic. It improves appetite and Fenugreek also is credited with many medicinal properties. alleys thirst. It is a cardiotonic and is used in bleeding, piles, It has antidiabetic effects and is helpful in digestive disor- dysentery, tumours and heart diseases [36]. ders as a tonic besides in the treatment for edema of legs, Grape juice and red wine are considered to confer certain flatulence, dysentery, diarrhoea, dyspepsia, chronic cough degree of protection against cardiovascular ailments [63]. and enlargement of liver and spleen [57]. In Type 1 diabetic Grape is known to have several therapeutic effects. Resvera- patients, supplementation of fenugreek in the diet lowers trol is a natural antioxidant found in grape known to exhibit lipid peroxidation [48], induces hypocholesterolemia and cardioprotective, chemopreventive/anticarcinogenic and anti- hypoglycaemia [58]. Germinated fenugreek seeds are used mutagenic properties. These properties have been linked to in various food preparations and as a natural remedy for dia- its antioxidant activity. The composition and properties of betes. They are shown to reduce blood sugar levels and grapes have been extensively investigated. Reports indicate cholesterol in diabetic patients [58]. Fenugreek significantly that grapes contain large amounts of phenolic compounds decreased the blood lipids (total cholesterol and triglycer- [64], which play an important role in human health, such as ides) without affecting the HDL and also reduced the blood lowering of human low density lipoprotein. Grape juice de- sugar in patients with coronary artery disease (CAD) [59]. layed oxidation of LDL in healthy volunteers and CAD pa- Curcuma longa (turmeric) is a yellow-coloured rhizome tients. It reduced free radical release and DNA damage and known for its use in curries and in different medicinal increased antioxidant capacity [65]. Grape seed proanthocy- preparations. It is used for cooking and therapeutic purposes anin has also been reported to have potent antioxidant prop- [36]. Turmeric has been used in traditional medicine in erties [66]. Grape seed oil has the highest antioxidant activity India, for more than 2000 years. The characteristic yellow among various other oils. The antioxidant abilities of domi- colour of this rhizome is due to the presence of the compo- nant grape varieties, however, are not examined. We studied nent ‘curcumin’. Curcumin is a major colouring component antioxidant ability of some grape varieties of the Asian re- of Curcuma longa and exhibits a variety of biological activ- gion and Indian subcontinent, mainly Thompson Seedless ities including anti-inflammatory, hepatoprotective, antimu- and its clones (Tas-E-Ganesh, Sonaka and Manikchaman), tagenic and anti-neoplastic properties [32]. The beneficial Sharad Seedless, which are considered the ruling grape vari- properties of turmeric include use as deodorizing agent, eties. disinfectant, cold, cough and various types of skin diseases. The mechanisms responsible for the reported therapeutic It is also used in dental diseases, digestive disorders like effects of the above medicinal plants are probably due to dyspepsia and acidity, indigestion, flatulence, upper abdom- their antioxidant and radical scavenging activities. Since inal pain, asthma, gastric and duodenal ulcers. The rhizome antioxidant activities are known to correlate with cardiopro- has carminative, diuretic, antimicrobial, antigenotoxic, anti- tective effects we have examined the antioxidant effects of inflammatory, anticarcinogenic, antiatherogenic and antihel- the extracts from these plants and their pure active ingredi- mintic activities [36]. Curcumin was found to protect rat ents whenever possible, at different levels of antioxidant myocardium against ischaemic insult and rat forebrain protection by using various physicochemical and biochem- against ischemia-reperfusion (I/R) insult and the protective ical assays. effect could be attributed to its antioxidant properties as well as its inhibitory effects on xanthine dehydrogenase/xanthine oxidase (XD/XO) conversion and resultant superoxide

Vol. 38, No. 1, 2006 14 J.A. Tilak-Jain and T.P.A. Devasagayam

Antioxidant Potential of Plant Extracts and Natural reaction by spin trapping agents. Since flavonoids and Compounds polyphenols are major phytochemicals in medicinal plants responsible for antioxidant effects, we have also estimated A number of epidemiological studies show that diets high their content in these extracts. in fruits and vegetables, the foods rich in antioxidant com- The methodologies used for determination of antioxidant pounds, are associated with a lower incidence of cardiovas- properties of the compounds have been presented in Table 2. cular disease. Observational data in animals and humans It was clearly established that among the plants examined suggest that greater intake of antioxidant vitamins are asso- namely Terminalia arjuna, Trigonella foenum-graecum, ciated with the reduced risk of atherosclerotic vascular Curcuma longa, Garcinia indica and Vitis vinifera. disease [67]. Antioxidants may at least in part prevent ath- Terminalia arjuna possessed the highest antioxidant poten- erosclerosis and cardiovascular disease [68]. Hence it is tial and inhibit whole plasma oxidation in rats besides pertinent to examine cardioprotective and antioxidant effects showing significant intestinal absorption. In case of primary of plants used in Indian herbal preparations. radical scavenging, as assessed by DPPH method, methan- For ease of study, in biological systems, oxidative stress olic extract possesses the highest Trolox Equivalent Antiox- can be generated using various physical/chemical agents. idant Capacity (TEAC), among the T. arjuna extracts. In Among them, ionizing radiation such as γ-rays is an impor- ferrylmyoglobin/ABTS assay, TEAC of acidic methanolic tant source of reactive oxygen species. The exposure of bio- extracts was the most significant and in FRAP assay, logical systems to radiation results in radiolytic cleavage of methanolic extract had the highest reducing activity. Thus . . − water yielding OH, H, e aq etc. in presence of oxygen even methanolic and acidic methanolic extract as well as baicalein .− 1 O2 , H2O2, O2 etc. are produced. Thermal decomposition of showed significant antioxidant capacities as compared to the an azo-initiator, 2.2'-Azobis (2-amidinopropane) dihydro- standard antioxidant, Trolox. We examined the effect of dif- chloride (AAPH) in presence of oxygen gives rise to a ferent free radical inducers such as γ-radiation, ascorbate- constant source of peroxyl radicals. These free radicals Fe2+, AAPH and peroxynitrite. When we studied the effect especially .OH and LOO. can initiate lipid peroxidation. of baicalein on oxidative stress induced lipid peroxidation in Cumene hydroperoxide, ascorbate-Fe2+ and peroxynitrite are rat heart homogenate, we found that it exerts protective some other free radical generators. effects even at low concentrations such as 10 µM. Baicalein Antioxidants exhibit their effects at different levels. These at 100 µM concentration, inhibited radiation-induced single include ability to bind iron that can prevent radical forma- strand breaks in pBR322 plasmid DNA, almost by 96%. Ba- tion, the scavenging of primary and secondary radicals and icalein also possessed fairly high rate constants, in the range 9 9 8 3 −1 −1 ability to inhibit free radical induced membrane damage. of 3.7 × 10 , 1.3 × 10 and 8.0 × 10 dm mol s for hy- Among the sub-cellular organelles mitochondria are crucial droxyl, azidyl and alkylchloroperoxyl radicals, respectively targets for oxidative damage. In this paper, we have demon- with the biologically relevant radicals as compared to other strated that ROS induce significant lipid peroxidation in the standard antioxidants, as studied by pulse radiolysis. The model system i.e. rat liver mitochondria as measured by IC50 values for baicalein for superoxide and singlet oxygen LOOH, an unstable intermediate, which further breaks down quenching were 18 and 128 µM, respectively, as determined to stable aldehydes and react with thiobarbituric acid (TBA) from ESR. These values are significantly higher than those to form TBARS, the final stable end product. Apart from for catechin, rosmarinic acid etc. enhancing lipid damage to membranes, oxidative damage In case of fenugreek seeds, the petroleum ether fraction leads to protein oxidation resulting in the formation of pro- showed highest peroxyl radical scavenging activity with tein hydroperoxide, protein carbonyls besides inactivation of ORAC value of 32.29 µM of Trolox. In case of Soxhlet antioxidant enzymes such as superoxide dismutase (SOD), fractions of fenugreek, petroleum ether fraction showed glutathione peroxidase (GPX) and glutathione reductase highest protection of 74.53% followed by Chloroform (GR). To examine the mechanisms underlying the observed extract (63.19%), Aqueous extract (59.77%) and then meth- protection by the plant extracts and the pure compounds, and anolic extract showing 44.32% protection against damage to evaluate radical scavenging abilities, we have utilized induced by Asc-Fe2+. biochemical assays such as Ferric Reducing Antioxidant In another set of experiments, the results obtained showed Power (FRAP), DPPH (1,1'-diphenyl-2-picrylhydrazyl) and that the ORAC values of turmeric extract ranging from 10 to ferrylmyoglobin/ABTS assay. The Oxygen Radical Absor- 25 µmoles of Trolox equivalent per gm fresh weight, are bance Capacity (ORAC) values are used as ‘standard’ similar to or higher than the values observed for some fruits measures for comparing antioxidant activity of food mate- and vegetables including those for garlic (19.4), spinach rials. We have also used pulse radiolysis to determine the (12.6) and onion (4.5) [69]. Ethanolic preparations of both rate constants of the compound with the reactive species, raw and processed turmeric are effective in giving antioxi- and electron spin resonance (ESR) to study the radical dant protection at various levels [33].

J. Clin. Biochem. Nutr. Cardioprotective Indian Medicinal Plants 15

Table 2. Methods used for studying antioxidant actions in relation to health implications of some Indian medicinal plants. Results are given in text. Antioxidant actions Methodology used Hydroxyl radicals Scavenging hydroxyl radicals generated by irradiation with γ-ray or Fenton reaction .OH generated by linear accelerator and detected spectrophotometrically Superoxide anion Scavenging superoxide anion induced by xanthine-xanthine oxidase as studied by ESR by using spin trap, 5,5-dimethyl-1-pyroline-N-oxide (DMPO) .− O2 generated by linear accelerator and detected spectrophotometrically Protection of iron-containing superoxide dismutase (SOD) Peroxyl radicals Scavenging peroxyl radicals induced by thermolysis of 2.2'-Azobis (2-amidinopropane) dihydrochloride (AAPH) by pulse radiolysis Scavenging of AAPH induced peroxyl radicals by using Oxygen Radical Absorbance Capacity (ORAC) assay . CCl3O2 generated by linear accelerator and detected spectrophotometrically Thiyl radicals Scavenging thiyl radicals by pulse radiolysis Singlet oxygen Scavenging singlet oxygen induced by photosensitization of Rose Bengal by ESR using spin trap, 2,2,6,6-tetramethyl-4-hydroxypiperidine (4-OH TEMP) Total ROS Scavenging of ROS in NIH3T3 cells induced by γ-ray by using DCFDA fluorescence assay Lipid peroxidation Inhibition of lipid peroxidation in rat liver mitochondria induced by biologically relevant γ-ray, ascorbate-Fe2+, peroxynitrite, AAPH, cumene hydroperoxide, photosensitization of Rose Bengal, xanthine-xanthine oxidase systems, in vitro and in vivo. Antioxidant enzymes Protection of Superoxide dismutase (SOD), Glutathione peroxidase (GPx), Glutathione reductase (GR) activities in rat liver mitochondria in vitro. DNA damage Inhibition of γ-radiation induced single strand breaks in pBR322 plasmid DNA Biological applications- Inhibition of whole plasma oxidation induced by thermolysis of 2.2'-Azobis (2-amidinopropane) Cardioprotection in rats dihydrochloride (AAPH) in blood plasma of rat

In case of G. indica extracts, in the DPPH assay, fractions protective effects. T. arjuna extracts prevented in vivo from chloroform and methanol-HCl extracts were the most ischemic-reperfusion injury induced oxidative stress, tissue effective while in the ABTS assay, fraction from acetone injury of heart and haemodynamic effects in rabbit hearts was the most potent. Studies on ascorbate-Fe2+ induced lipid [47, 70]. Curcumin was found to protect rat against is- peroxidation showed that chloroform and benzene extracts chemia-reperfusion (I/R) insult due to its antioxidant were very effective. Kokam syrup and the boiled aqueous properties as well as its inhibitory effects on xanthine extract of kokam also had significant antioxidant effects in dehydrogenase/xanthine oxidase (XD/XO) conversion and the above assays. Aqueous and boiled aqueous extracts of resultant superoxide anion production [60, 61]. Resveratrol kokam besides the commercial kokam syrup have high from grapes and curcumin from turmeric also appeared to be ORAC values (28.5, 23.8 and 20.4 respectively) comparable interesting for atherosclerosis treatment [71]. Resveratrol to that of other . mediates cardioprotection and neovascularization which Our studies also revealed that ethanolic extracts of black may be attributed to its ability to stimulate nitric oxide and red grapes possess higher levels of antioxidant abilities production and its free radical scavenging activity [72]. Our as compared to the green varieties. This could perhaps be results also showed that extracts of arjun, turmeric, due to high polyphenolics present in the extracts. In the fenugreek, kokum and grapes possessed significant antioxi- radical scavenging assays, our study indicates that cv. dant activities. Various extracts of T. arjuna as well as baica- Mango is the most potent followed by cv. Sharad seedless lein showed significant activities in the different assays and cv. Flame Seedless. In the lipid peroxidation assay, used. Other plants studied also exhibit significant radical ethanolic extracts of cv. Sharad seedless and cv. Mango scavenging effects as studied using various biochemical as- could significantly inhibit peroxidation in rat liver mito- says. These observed antioxidant effects may possibly be chondria by 74% and 70% respectively, as compared to responsible for their known beneficial therapeutic effects of aqueous extracts with 71% and 60% protection respectively. the above mentioned plants including in cardioprotection. It has been shown earlier that the antioxidant ability of the plant extracts was possibly responsible for their cardio-

Vol. 38, No. 1, 2006 16 J.A. Tilak-Jain and T.P.A. Devasagayam

References [19] Noguchi, N. and Niki, E.: Phenolic antioxidants: a rationale for design and evaluation of novel antioxidant drug for [1] Lele, R.D.: Ayurveda and Modern Medicine. Bharatiya Vidya atherosclerosis. Free Rad. Biol. Med., 28, 1538–1546, 2000. Bhavan, Mumbai, 2001. [20] Scartezzini, P. and Speroni, E.: Review on some plants of [2] Upadhyay, S.N.: Immunomodulation. Narosa Publishing Indian traditional medicine with antioxidant activity. J. House, New Delhi, 1997. Ethnopharmacol., 71, 23–43, 2000. [3] Agarwal, S.S. and Singh, V.K.: Immunomodulators: A re- [21] Jadhav, H.R. and Bhutani, K.K.: Antioxidant properties of view of studies on Indian medicinal plants and synthetic Indian medicinal plants. Phytother. Res., 16, 771–773, 2002. peptides. Part I: Medicinal Plants. Proc. Indian Natl. Sci. [22] Heber, D.: Herbs and atherosclerosis. Curr. Atheroscler. Rep., Acad. B., 65, 179–204, 1999. 3, 93–96, 2001. [4] Devasagayam, T.P.A. and Sainis, K.B.: Immune system and [23] Helen, A., Rajasree, C.R., Krishnakumar, K., Augusti, K.T., antioxidants, especially those derived from Indian medicinal and Vijayammal, P.L.: Antioxidant role of oils isolated from plants. Indian J. Expt. Biol., 40, 639–655, 2001. garlic (Allium sativum Linn) and onion (Allium cepa Linn) on [5] Devasagayam, T.P.A., Tilak, J.C., Boloor, K.K., Sane, K.S., nicotine-induced lipid peroxidation. Vet. Hum. Toxicol., 41, Ghaskadbi, S., and Lele, R.D.: Free radicals and antioxidants 316–319, 1999. in human health: current status and future prospects. J. Asso. [24] Balasenthil, S., Arivazhagan, S., and Nagini, S.: Garlic Physi. India, Oct., 794–804, 2004. enhances circulatory antioxidants during 7,12-dimethyl- [6] www.healthinitiative.org benz(a)anthracene-induced hamster buccal pouch carcino- [7] www.csiindia.org genesis. J. Ethnopharmacol., 72, 429–433, 2000. [8] Hughes, K. and Ong, C.N.: Vitamins, selenium, iron and [25] Kamat, J.P., Boloor, K.K., Devasagayam, T.P.A., and coronary heart disease risk in Indians, Malays and Chinese in Venkatachalam, S.R.: Antioxidant properties of Asparagus Singapore. J. Epidemiol. Comm. Health, 52, 181–185, 1998. racemosus against damage induced by γ-radiation in rat [9] Ong, A.S.H. and Packer, L.: Lipid-soluble Antioxidants: liver mitochondria. J. Ethnopharmacol., 71, 425–435, 2000. Biochemistry and Clinical Applications. Basel: Birkhauser [26] Goyal, R.K., Singh, J., and Lal, H.: Asparagus racemosus-an Verlag, 1992. update. Indian J. Med. Sci., 57, 408–414, 2003. [10] Lusis, A.J.: Atherosclerosis. Nature, 407, 233–241, 2000. [27] Chakrabarti, S., Biswas, T.K., Seal, T., Rokeya, B., Ali, L., [11] Stocker, R.: The ambivalence of vitamin E in atherogenesis. Khan, A.K., Nahar, N., Mosihuzzaman, M., and Mukherjee, Trends Biochem. Sci., 24, 219–223, 1999. B.: Antidiabetic activity of Caesalpinia bonducella F. in [12] Packer, L. and Ong, A.S.H.: Biological Oxidants and Anti- chronic type 2 diabetic model in Long-Evans rats and oxidants: Molecular Mechanisms and Health Effects. Cham- evaluation of insulin secretagogue property of its fractions on paign: AOCS Press, 1998. isolated islets. J. Ethnopharmacol., 97, 117–122, 2005. [13] Chopra, M.U., McLoone, M., O’Neill, N., and Williams, [28] Archana, P., Tandan, S.K., Chandra, S., and Lal, J.: Anti- D.I.: Fruit and vegetable supplementation-effect on ex-vivo pyretic and analgesic activities of Caesalpinia bonducella LDL oxidation in humans, in Natural Antioxidant and Food seed kernel extract. Phytother. Res., 19, 376–381, 2005. Quality in Atherosclerosis and Cancer Prevention, eds. By [29] Manonmani, G., Bhavapriya, V., Kalpana, S., Govindasamy, Kumpulaine J.T. and Salonel J.P., The Royal Society of S., and Apparanantham, T.: Antioxidant activity of Cassia Chemistry, Cambridge, pp. 151–155, 1996. fistula (Linn.) flowers in alloxan induced diabetic rats. J. [14] Gupta, R. and Gupta, V.P.: Meta-analysis of coronary heart Ethnopharmacol., 97, 39–42, 2005. disease prevalence in India. Indian Heart J., 48, 241–245, [30] Subramanian, M., Sreejayan, N., Rao, M.N.A., Devasagayam, 1996. T.P.A., and Singh, B.B.: Diminution of singlet oxygen in- [15] Bhatnagar, D.: The metabolic basis of increased coronary duced DNA damage by curcumin and related antioxidants. risk attributed to people from Indian subcontinent. Curr. Sci., Mutat. Res., 311, 249–255, 1994. 74, 1087–1094, 1998. [31] Sreejayan, N., Rao, M.N.A., Priyadarsini, K.I., and [16] Fruchart, J.C. and Duriez, P.: Free radicals and athero- Devasagayam, T.P.A.: Inhibiton of radiation induced lipid sclerosis. in ‘Free radical damage and its control’, eds. By peroxidation by curcumin. Int. J. Pharm., 151, 127–130, Rice-Evans, C.A. and Burdon, R.H., Elsevier Science B.V., 1997. 1994. [32] Devasagayam, T.P.A., Kamat, J.P., and Sreejayan, N.: Anti- [17] Harrison, D., Griendling, K.K., Landmesser, U., Hornig, B., oxidant action of curcumin. in Micronutrients and Health: and Drexler, H.: Role of oxidative stress in atherosclerosis. Molecular Biological Mechanisms, eds. By Nesaretnam, K. Am. J. Cardiol., 91, 7A–11A, 2003. and Packer, L., AOCS Press, USA, pp. 42–59, 2001. [18] Shanmugasundaram, K.R., Srinivas, K., Sundaram, P., [33] Tilak, J.C., Banerjee, M., Hari Mohan, and Devasagayam, Satyaraj, D., Muthu, R., Padma, P., Maheswari, R.U., T.P.A.: Antioxidant availability of turmeric in relation to its Veeraraghavan, V., Sharada, V., and Amudha, B.: Lipid medicinal and culinary uses. Phytother. Res., 18, 798–804, peroxidation, antioxidant defenses and rod-cell membrane 2004. changes in relation to coronary risk index and symptomatic [34] Bhattacharya, A., Chatterjee, A., Ghosal, S., and Bhattacharya, coronary heart disease. J. Cardiovasc. Risk, 2, 551–561, S.K.: Antioxidant activity of active tannoid principles of 1996. Emblica officinalis (amla). Indian J. Exp. Biol., 37, 676–680,

J. Clin. Biochem. Nutr. Cardioprotective Indian Medicinal Plants 17

1999. chemopreventive activity of resveratrol, a natural product de- [35] Devasagayam, T.P.A., Subramanian, M., Singh, B.B., rived from grapes. Science, 275, 218–220, 1997. Ramanathan, R., and Das, N.P.: Protection of plasmid [50] Hung, L.M., Chen, J.K., Huang, S.S., Lee, R.S., and Su, pBR322 DNA by flavonoids against single-strand breaks in- M.J.: Cardioprotective effect of resveratrol, a natural antioxi- duced by singlet molecular oxygen. J. Photochem. Photobiol. dant derived from grapes. Cardiovasc. Res., 47, 549–555, B: Biol., 30, 97–103, 1995. 2000. [36] Kirtikar, K.R. and Basu, B.D.: Indian Medicinal Plants, Vol. [51] Dhuley, N.: Adaptogenic and cardioprotective action of ash- I. Blatter, E., Cauis, J.R., Mhaskar, K.S. and Basu, L.M., wagandha in rats and frogs. J. Ethnopharmacol., 70, 57–63, (Eds.) Allahabad, India, 1984. 2000. [37] Yamaguchi, F., Saito, M., Ariga, T., Yoshimura, Y., and [52] Ahmed, R.S., Seth, V., and Banerjee, B.D.: Influence of Nakazawa, H.: Free radical scavenging activity and antiulcer dietary ginger (Zingiber officinalis Rosc) on antioxidant de- activity of garcinol from Garcinia indica fruit rind. J. Agri. fense system in rat: Comparison with ascorbic acid. Indian J. Food Chem., 48, 2320–2325, 2000. Exp. Biol., 38, 604–606, 1997. [38] Liao, C-H., Ho, C-T., and Lin, J-K.: Effects of garcinol on [53] Dwivedi, S. and Agarwal, P.: Antianginal and cardioprotec- free radical generation and NO production in embryonic rat tive effects of Terminalia arjuna, an indigenous drug, in coro- cortical neurons and astrocytes. Biochem. Biophys. Res. nary artery disease. J. Assoc. Physicians India, 42, 287–289, Comm., 329, 1306–1314, 2005. 1994. [39] Alam, M.I. and Gomes, A.: Viper venom-induced inflamma- [54] Gupta, R., Singhal, S., Goyle, A., and Sharma, V.N.: Antiox- tion and inhibition of free radical formation by pure com- idant and hypocholesterolemic effects of Terminalia arjuna pound (2-hydroxy-4-methoxy benzoic acid) isolated and pu- bark powder: a randomized placebo-controlled trial. J. rified from anantamul (Hemidesmus indicus R. BR) root ex- Asso. Physicians India, 49, 231–235, 2001. tract. Toxicon., 36, 207–215, 1998. [55] Gao, D., Tawa, R., Masaki, H., Okano, Y., and Sakurai, H.: [40] Mary, N.K., Babu, B.H., and Padikkala, J.: Antiatherogenic Protective effects of baicalein against cell damage by reac- effect of Caps HT2, a herbal Ayurvedic medicine formula- tive oxygen species. Chem. Pharm. Bull. Tokyo, 46, 1383– tion. Phytomed., 10, 474–482, 2003. 1387, 1998. [41] Grover, J.K.: Hepato and cardio protective action of tulsi [56] Shieh, D.E., Liu, L.T., and Lin, C.C.: Antioxidant and free (Ocimum sanctum linn). Eur. J. Pharmacol., 183, 872, 1990. radical scavenging effects of baicalein, baicalin and wogo- [42] Gupta, S.K., Sharma, M., Kishore, K., and Arya, D.S.: nin. Anticancer Res., 20, 2861–2866, 2000. Ocimum sanctum provides cardiac protection in isoproterenol [57] Bach, E.: Therapeutic applications of fenugreek. Alt. Med. induced myocardial infirction in rats. J. Mol. Cell. Cardiol., Rev., 8, 20–27, 2003. 33, A42, 2001. [58] Sharma, R.D., Raghuram, T.C., and Rao, N.S.: Effect of [43] Raphael, K.R. and Kuttan, R.: Inhibition of experimental fenugreek seeds on blood glucose and serum lipids in type I gastrict lesion and inflammation by Phyllanthus amarus ex- diabetes. Eur. J. Clin. Nutr., 44, 301–306, 1990. tract. J. Ethanofarmacol., 87, 193–197, 2003. [59] Bordia, A., Verma, S.K., and Srivastava, K.C.: Effect of gin- [44] Kumar, K.B. and Kuttan, R.: Protective effect of an extract of ger (Zingiber officinale Rosc.) and fenugreek (Trigonella Phyllanthus amarus against radiation induced damage in foenumgraecum L.) on blood lipids, blood sugar and platelet mice. J. Radiat. Res. (Tokyo), 45, 133–139, 2004. aggregation in patients with coronary artery disease. Prostag- [45] Subramaniam, H., Kumar, S., Anandan, R., Devaki, T., and lan. Leukotrien. Essn. Fatty Acids, 56, 379–384, 1997. Santhosh Kumar, M.: Cardioprotective effects of Picrorrhiza [60] Ghoneim, A.I., Abdel-naim, A.B., Khalifa, A.E., and kurroa against isoproterenol-induced myocardial stress in El-denshary, E.S.: Protective Effects of Curcumin Against rats. Fitoterapia, 72, 402–405, 2001. Ischaemia/Reperfusion Insult in Rat Forebrain. Pharmacol. [46] Bharani A., Ganguli, A., Mathur, L.K., Jamra, Y., and Res., 46, 273–279, 2002. Raman, P.G.: Efficacy of Terminalia arjuna in chronic stable [61] Manikandan, P., Sumitra, M., Aishwarya, S., Murali angina: a double-blind, placebo-controlled, crossover study Manohar, B., Lokanadam, B., and Puvanakrishnan, R.: Cur- comparing Terminalia arjuna with isosorbide mononitrate. cumin modulates free radical quenching in myocardial is- Indian Heart J. 54: 170–175, 2002. chaemia in rats. Int. J. Biochem. Cell Biol., 36, 1967–1980, [47] Gauthaman, K., Banerjee, S.K., Dinda, A.K., Ghosh, C.C., 2004. and Maulik, S.K.: Terminalia arjuna (Roxb.) protects rabbit [62] Drury, H.: The Useful Plants of India. Allied Book Centre: heart against ischemic-reperfusion injury: role of antioxidant Dehradun, 1991. enzymes and heat shock protein. J. Ethnopharmacol., 96, [63] Stein, J.H., Keevil, J.G., Wiebe, D.A., Aeschlimann, S., and 403–409, 2005. Folts, J.D.: Purple grape juice improves endothelial function [48] Ravilumar, P. and Anuradha, C.V.: Effect of fenugreek seeds and reduces the susceptibility of LDL cholesterol to oxida- on blood lipid peroxidation and antioxidants in diabetic rats. tion in patients with coronary artery disease. Circulation. Phytother. Res., 13, 197–201, 1999. 100, 1050–1055, 1999. [49] Jang, M., Cai, L., Udeani, G.O., Slowing, K.V., Thomas, C.F., [64] Oszmianski, J. and Lee, C.Y.: Isolation and HPLC determina- Beecher, C.W., Fong, H.H., Farnsworth, N.R., Kinghorn, tion of phenolic compounds in red grapes. Am. J. Enol. Vitic., A.D., Mehta, R.G., Moon, R.C., and Pezzuto, J.M.: Cancer 39, 259–262, 1990.

Vol. 38, No. 1, 2006 18 J.A. Tilak-Jain and T.P.A. Devasagayam

[65] Park, Y.K., Park, E., Kim, J.S., and Kang, M.H.: Daily grape tor of bone mineral density. Nutr. Res., 22, 1389–1399, 2002. juice consumption reduces oxidative DNA damage and plas- [70] Karthikeyan, K., Sarala Bai, B.R., Gauthaman, K., Sathish, ma free radical levels in healthy Koreans. Mutation Res., K.S., and Niranjali Devaraj, S.: Cardioprotective effect of the 529, 77–86, 2003. alcoholic extract of Terminalia arjuna bark in an in vivo [66] Sen, C.K. and Bagachi, D.: Regulation of inducible adhesion model of myocardial ischemic reperfusion injury. Life Sci., molecule expression in human endothelial cells by grape 73, 2727–2739, 2003. seed proantocyanidin extract. Mol. Cell. Biochem., 216, 1–7, [71] Deby-Dupont, G., Mouithys-Mickalad, A., Serteyn, D., 2001. Lamy, M., and Deby, C.: Resveratrol and curcumin reduce [67] Buring, J.E. and Hennekens, C.H.: Antioxidant vitamins and the respiratory burst of Chlamydia-primed THP-1 cells. Bio- cardiovascular disease. Nutr. Rev., 55, S53–S60, 1997. chem. Biophys. Res. Comm., 333, 21–27, 2005. [68] Diaz, M.N., Frei, B., Vita, J.A., and Keaney, J.F.: Antioxi- [72] Kaga, S., Zhan, L., Matsumoto, M., and Maulik, N.: Resvera- dants and atherosclerotic heart disease. N. Eng. J. Med., 337, trol enhances neovascularization in the infarcted rat myocar- 408–416, 1999. dium through the induction of thioredoxin-1, heme oxygena- [69] Lachnicht, D., Brevard, P.B., Wagner, T.L., and DeMars, se-1 and vascular endothelial growth factor. J. Mol. Cell. C.E.: Dietary oxygen radical absorbance capacity as a predic- Cardiol. In Press, Corrected Proof, 2005.

J. Clin. Biochem. Nutr.