Radioprotective properties of polyphenols from Phyllanthus amarus Linn.

Radioprotective activity of pure compounds isolated from the plant Phyllanthus amarus was studied using rat liver mitochondria and pBR322 plasmid DNA as an in vitro model system. These compounds were ellagitannins namely amariin, 1-galloyl-2,3-dehydrohexahydroxydiphenyl (DHHDP)-glucose, repandusinic acid, geraniin, corilagin, phyllanthusiin D, and flavonoids namely rutin, and quercetin 3-O-glucoside. The activity was then correlated with their hydroxyl and superoxide radical scavenging activity. Both ellagitannins and flavonoids effectively prevented lipid peroxidation and protein oxidation in mitochondria. The compounds also prevented radiation induced single strand breaks in pBR322 plasmid DNA. The radioprotective activity of ellagitannins and flavonoids could be due to their ability to scavenge different radicals more or less efficiently, relieving the oxidative stress. Protection conferred by flavonoids, rutin and quercetin 3-O-glucoside to rat liver mitochondria and plasmid pBR322 DNA from radiation induced damage was due to their strong hydroxyl radical scavenging activity. The inhibitory effect of ellagitannins on lipid peroxidation in liver mitochondria was due to their efficient superoxide radical scavenging ability. This is the first report about the radioprotective activity of pure ellagitannins from Phyllanthus amarus.

Formulated antidiabetic preparation Syndrex has a strong antioxidant activity.

Syndrex is a formulated herbal antidiabetic preparation containing powder of germinated fenugreek seeds. We have assessed the antioxidant potential of this drug. Syndrex was fractionated by Soxhlet apparatus and fractions were used to determine their antioxidant potential at different levels. In vitro activity was assessed by ferric reducing antioxidant power assay, radical scavenging by 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferrylmyoglobin/2,2'-azobis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and pulse radiolysis. Inhibition of lipid peroxidation by Syndrex in mitochondrial preparations from rat liver was checked. Methanolic fraction of Syndrex exhibited the highest antioxidant activity as compared to other fractions. This fraction showed maximum phenolic and flavonoid contents. Isolated mouse pancreatic islets also employed to assess antioxidant activity of Syndrex. Islets were treated with streptozotocin, a diabetogen known to damage islet cells by inducing generation of free radicals. Syndrex treated islets were protected from streptozotocin insult as these islets survived better and remained functional as compared to streptozotocin treated islets.

DNA protective properties of vanillin against gamma-radiation under different conditions: possible mechanisms.

Ionizing radiation is an important genotoxic agent. Protecting against this form of toxicant, especially by a dietary component, has several potential applications. In the present study, we have examined the ability of vanillin (4-hydroxy-3-methoxybenzaldehyde), a naturally occurring food flavouring agent, to inhibit radiation-induced DNA damage measured as strand breaks under in vitro, ex vivo and in vivo conditions besides the possible mechanisms behind the observed protection. Our study showed that there was a concentration-dependent inhibition of the disappearance of super-coiled (ccc) form of plasmid pBR322 (in vitro) upon exposure to 50 Gy of gamma-radiation. Presence of 0.5 mM vanillin has a dose-modifying factor (DMF) of 6.75 for 50% inactivation of ccc form. Exposure of human peripheral blood leucocytes (ex vivo) to gamma-radiation causes strand breaks in the cellular DNA, as assessed by comet assay. When leucocytes were exposed to 2 Gy of gamma-radiation there was an increase in parameters of comet assay such as %DNA in tail, tail length, 'tail moment' and 'Olive tail moment'. The presence of 0.5 mM vanillin during irradiation significantly reduced these parameters. Damage to DNA in mouse peripheral blood leucocytes after whole-body exposure of mice (in vivo) to gamma-radiation was studied at 1 and 2 h post-irradiation. There was recovery of DNA damage in terms of the above-mentioned parameters at 2 h post-irradiation. This was more than that observed at 1 h. The recovery was more in vanillin treated mice. Hence our studies showed that vanillin offers protection to DNA against radiation-induced damage possibly imparting a role other than modulation of DNA repair. To examine the possible mechanisms of radioprotection, in terms of radiation-derived radicals, we carried out the reaction of vanillin with ABTS*(+) radical spectrophotometrically besides with DNA peroxyl and carbonyl radicals by using pulse radiolysis. Our present investigations show that vanillin has ability to protect against DNA damage in plasmid pBR322, human and mouse peripheral blood leucocytes and splenic lymphocytes besides enhancing survival in splenic lymphocytes against gamma-radiation, and that the possible mechanism may involve scavenging of radicals generated during radiation, apart from modulation of DNA repair observed earlier.

Current status of herbal drugs in India: an overview.

Herbal drugs constitute a major share of all the officially recognised systems of health in India viz. Ayurveda, Yoga, Unani, Siddha, Homeopathy and Naturopathy, except Allopathy. More than 70% of India's 1.1 billion population still use these non-allopathic systems of medicine. Currently, there is no separate category of herbal drugs or dietary supplements, as per the Indian Drugs Act. However, there is a vast experiential-evidence base for many of the natural drugs. This offers immense opportunities for Observational Therapeutics and Reverse Pharmacology. Evidence-based herbals are widely used in the diverse systems and manufactured, as per the pharmacopoeial guidelines, by a well-organised industry. Significant basic and clinical research has been carried out on the medicinal plants and their formulations, with the state-of-the-art methods in a number of Institutes/Universities. There are some good examples. Indian medicinal plants also provide a rich source for antioxidants that are known to prevent/delay different diseased states. The antioxidant protection is observed at different levels. The medicinal plants also contain other beneficial compounds like ingredients for functional foods. Hence, the global knowledge about Ayurveda and Indian herbals will hopefully be enhanced by information on the evidence-base of these plants. This will yield rich dividends in the coming years.

Some novel approaches for radioprotection and the beneficial effect of natural products.

Due to the increased use of ionizing radiation in various aspects of human life especially in areas pertaining to radiotherapy of cancer, food preservation, agriculture, industry and power generation, there is a need to develop an effective and non-toxic radioprotector. The currently available ones have many drawbacks including high cost, side effects and toxicity. Several novel approaches are on to locate a potent radioprotector. These include mimics of antioxidant enzymes, nitroxides, melatonin, growth factors, gene therapy, hyperthermia apart from natural products. The latter has several advantages since they are non-toxic with proven therapeutic benefits. These can be classified as natural compounds and plant extracts; polyherbal formulations; besides natural and semi-natural compounds of plant origin. A review of the above agents, their efficacy in radioprotection and possible mechanisms responsible has been carried out. As India and many Eastern countries have an enormous heritage of vast natural dietary and time tested medicinal resources it is worth exploring the possibility of developing efficient, economically viable and clinically acceptable radioprotectors for human application from these resources.

A modified, economic, sensitive method for measuring total antioxidant capacities of human plasma and natural compounds using Indian saffron (Crocus sativus).

BACKGROUND: Free radicals are involved in various human diseases that can possibly be prevented by antioxidants. There are many but rather expensive methods to determine total antioxidant capacity of human plasma (for endogenous antioxidant levels) or plant extracts/natural compounds (for antioxidant potential in terms of radical inhibiting or scavenging properties). We describe a simple, fast and economical 'crocin assay' using the Indian spice saffron. METHODS: In crocin assay, the extent of bleaching of crocin, a carotenoid from saffron, by peroxyl radicals generated by thermal decomposition of azo-initiator was measured. We examined its applicability to clinical samples and plant extracts. RESULTS: The cost of Indian saffron is almost 38 times less per unit dry weight compared to the 'Sigma' saffron. Yet, it gives 26 times better yield of crocin than that from 'Sigma' saffron. It was also shown that Indian saffron is rich in crocin. The total antioxidant capacity (TAC) values of human plasma from normal, healthy individuals, using Sigma as well as Indian crocin, expressed in terms of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) equivalent antioxidant capacity (TEAC), were comparable. We have also demonstrated that crocin assay can be used for clinical samples such as plasmas from healthy and diabetic individuals. The antioxidant potentials, TEAC, of plant extracts and pure natural compounds by Indian and Sigma crocin assays were similar. Addition of uric acid to plasma induced a concentration-dependent response. The assay was compared to standard radical scavenging 1,1'-diphenyl-2-picrylhydrazyl (DPPH) assay and was found to match well, showing better sensitivity and hence validates this assay for natural compounds and clinical samples. CONCLUSIONS: Development of crocin assay using the Indian saffron is economical and sensitive method for measurement of total antioxidant capacities from human plasma as well as natural compounds and plant extracts.

Antioxidant properties of Plumbago zeylanica, an Indian medicinal plant and its active ingredient, plumbagin.

Plumbago zeylanica (known as "Chitrak") is a useful Indian medicinal plant. The root of the plant and its constituents are credited with potential therapeutic properties including anti-atherogenic, cardiotonic, hepatoprotective and neuroprotective properties. To examine possible mechanisms of action of P. zeylanica (Chitrak), in relation to its reported beneficial properties, antioxidant effects of the aqueous/alcoholic extracts of root, corresponding to medicinal preparations, and the active ingredient, plumbagin, were studied. Methods used included: ferric reducing/antioxidant power (FRAP), radical scavenging of 1,1-diphenyl-2-picryl hydrazyl (DPPH) and 2,2'-azobis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS), lipid peroxidation in rat liver mitochondria induced by different agents, and estimating phenolic and flavonoid content. In FRAP/DPPH assays, boiled ethanolic extracts were the most effective, while in the ABTS assay boiled aqueous extracts were the most efficient. These extracts also significantly inhibited lipid peroxidation induced by cumene hydroperoxide, ascorbate-Fe(2+) and peroxynitrite and contained high amounts of polyphenols and flavonoids. To examine the mechanisms of action in detail, antioxidant and pulse radiolysis studies with plumbagin were conducted. The hydroxyl (.OH), alkyl peroxyl (CCl(3)OO.), linoleic acid peroxyl (LOO.), and glutathiyl (GS.) radicals generate a phenoxyl radical upon reaction with plumbagin. The bimolecular rate constants were: .OH, 2.03 x 10(9) dm(3)mol(-1)s(-1); CCl(3)OO., 1.1 x 10(9) dm(3)mol(-1)s(-1); LOO., 6.7 x 10(7) dm(3)mol(-1)s(-1); and GS., 8.8 x 10(8) dm(3)mol(-1)s(-1). In conclusion, our studies reveal that extracts of P. zeylanica and its active ingredient plumbagin have significant antioxidant abilities that may possibly explain some of the reported therapeutic effects.

Biological significance of singlet oxygen.

The biological significance of singlet oxygen (1O2), an electronically excited species of oxygen, has been realized only in the last two decades. This was mainly due to the lack of proper methodology to generate this reactive oxygen species (ROS) in pure form and its reactions with biological molecules. Recent studies, using newly developed detection methods, show that 1O2 being generated in many biological systems, can significantly and quite often adversely alter several crucial biomolecules including DNA, proteins and lipids with undesirable consequences including cytotoxicity and/or disesase development. The reactions of 1O2 with the biological molecules are rather specific, as compared to other ROS. There are various compounds, mainly derived from natural sources that offer protection against damage induced by 1O2. Among the antioxidants carotenoids are the most effective singlet oxygen quenchers followed by tocopherols and others. The same reactive species if generated specifically in diseased states such as cancer can lead to the cure of the disease, and this principle is utilized in the newly developing modality of cancer treatment namely photodynamic therapy. Singlet oxygen, in low concentrations can also act as signaling molecule with several biological implications. This review clearly brings out the biological significance of 1O2.

Cardioprotective effects of an aminothiazole compound on isoproterenol-induced myocardial injury in mice.

Dendrodoine analogue (DA), an aminothiazole compound derived from dendrodoine, present in a marine tunicate, has been shown to possess many beneficial properties. This study was aimed to evaluate its cardioprotective effect against isoproterenol (ISO)-induced myocardial damage in mice. Swiss mice were pretreated with DA for 7 days and then treated with ISO (5 mg/kg BW, for 2 consecutive days). Biochemical assessment of myocardial injury was carried out by measuring marker enzymes, antioxidant enzymes and levels of lipid peroxidation. Histological studies of hearts were also carried out. ISO administration increased the activities of creatine kinase-MB, lactate dehydrogenase and aspartate aminotranferase (AST) in serum. Prior administration of DA restored the levels of these enzymes and the heart coefficient close to normal levels. DA at a concentration of 5 mg/kg BW was most effective in reducing AST, and this concentration was used for further studies. DA also gave significant protection against lipid peroxidation in the heart besides restoring histopathological alterations. DA showed significant reactivity towards superoxide radicals. In conclusion our study indicates that DA can protect mouse myocardium against damage and one of the possible reasons behind this protective effect can be attributed to its antioxidant property.

Geraniin and amariin, ellagitannins from Phyllanthus amarus, protect liver cells against ethanol induced cytotoxicity.

Ethanol mediated free radical generation plays an important role in the pathogenesis of liver injuries and alcoholic liver diseases. In the present study two ellagitannins namely geraniin and amariin isolated from Phyllanthus amarus were examined for their ability to protect mouse liver slices against ethanol induced toxicity and possible mechanism of its protection. Oxidative stress markers such as, lipid peroxidation, protein carbonyl formation, amount of 8-hydroxy-2-deoxyguanosine and antioxidant enzymes levels were measured using specific biochemical assays. Poly (ADP-ribose) polymerase (PARP), Bax and Bcl2 were checked to assess the induction of apoptosis using western blots. The results showed that geraniin and amariin protected mouse liver slices against ethanol induced cytotoxicity. Both compounds inhibited oxidation of lipid, protein and formation of 8-hydroxy-2-deoxyguanosine, all of which were found to be elevated on exposure to ethanol. These compounds restored the antioxidant enzymes altered on ethanol exposure. Compounds also inhibited the cleavage of PARP and bax and restored Bcl2, induced on exposure to ethanol. In summary, both ellagitannins effectively protected mouse liver slices against ethanol induced cytotoxicity and apoptosis by reducing oxidative damage to biological molecules and modulating Bax/Bcl-2 ratio respectively, thus minimizing liver injury.

Protective effect of an aminothiazole compound against γ-radiation induced oxidative damage.

Ionizing radiation causes its biological effects mainly through oxidative damage induced by reactive oxygen species. During radiotherapy of cancer, one of the undesirable side-effects is toxicity to normal cells. Compounds with antioxidant activities are being tried as 'prophylactic radioprotectants' to overcome this problem. We evaluated the protective effect of an aminothiazole compound, in the form of dendrodoine analogue (DA) originally derived from a marine tunicate, against γ-radiation-induced damage to lipid, protein, and DNA besides its cytotoxicity. Oxidative damage was examined by different biochemcial assays. Our studies reveal that DA gave significant protection, in fairly low concentrations, against damage induced by γ-radiation to rat liver mitochondria, plasmid pBR322 DNA, and mouse splenic lymphocytes in vitro. It also protected against oxidative damage in whole-body irradiated mice exposed to therapeutic dose of radiation (2 Gy) in vivo. Spleen, a major target organ for radiation damage, of the irradiated mice showed significant protection when treated with DA, as examined by histopathology. In conclusion, due to the possible protective effects against normal cells/tissues both in vitro and in vivo, DA shows potential to be a radioprotector for possible use during radiotherapy.

Oxidative stress perturbs cell proliferation in human K562 cells by modulating protein synthesis and cell cycle.

Oxidative stress leads to perturbation of a variety of cellular processes resulting in inhibition of cell proliferation. This study has determined the effect of oxidative stress on protein synthesis in human K562 cells using a hydrophilic peroxyl radical initiator, AAPH and H(2)O(2). The results indicated that oxidative stress leads to a significant decrease in the rate of protein synthesis caused due to induced activation as well as expression of the erythroid cell-specific eIF-2alpha kinase, called the Heme Regulated Inhibitor (HRI). Elevated levels of HRI expression and activity were accompanied by increased lipid peroxidation and decreased cell proliferation. Further, oxidative stress also caused inactivation of p34(cdc2) kinase, thereby arresting cell division leading to apoptosis. Thus, the data provides the mechanism of inhibition of protein synthesis and perturbation of a cell cycle regulatory protein leading to inhibition of cell proliferation in K562 cells during oxidative stress.

Antioxidant activity of some polyphenol constituents of the medicinal plant Phyllanthus amarus Linn.

Phyllanthus amarus Linn is a widely distributed tropical medicinal plant highly valued for its therapeutic properties. The antioxidant activity of some of its principal constituents, namely amariin, 1-galloyl-2,3-dehydrohexahydroxydiphenyl (DHHDP)-glucose, repandusinic acid, geraniin, corilagin, phyllanthusiin D, rutin and quercetin 3-O-glucoside were examined for their ability to scavenge free radicals in a range of systems including 2,2-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azobis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS)/ferrylmyoglobin, ferric reducing antioxidant power (FRAP) and pulse radiolysis. In addition, their ability to protect rat liver mitochondria against oxidative damage was determined by measuring the ROO* radical induced damage to proteins and lipids and *OH radical induced damage to plasmid DNA. The compounds showed significant antioxidant activities with differing efficacy depending on the assays employed. Amariin, repandusinic acid and phyllanthusiin D showed higher antioxidant activity among the ellagitannins and were comparable to the flavonoids, rutin and quercetin 3-O-glucoside.

A study of antioxidant properties of some varieties of grapes (Vitis vinifera L.).

Grapes (Vitis vinifera L.) are a major fruit crop in the world. Grapes seem to confer health benefits due to their antioxidant activity. We have evaluated the antioxidant potential of 11 grapes varieties from India and nearby Asian countries. The assays employed involve different levels of antioxidant action like ferric reducing antioxidant power, radical scavenging by 1,1-diphenyl-2-picrylhydrazyl, ferrylmyoglobin/2,2'-azobis-3-ethylbenzthiazoline-6-sulfonic acid, oxygen radical absorbance capacity (ORAC), and inhibition of lipid peroxidation. The total phenolic and flavonoids contents were also estimated. Our study indicates that cv. Mango is the most potent followed by Sharad Seedless. Ethanolic extracts were found to be more effective than aqueous extracts. Cv. Sharad Seedless, Mango, and Manikchaman also had high ORAC values. Their HPLC analysis showed the presence of various antioxidant polyphenols. In conclusion our studies identified some varieties of grapes with high antioxidant activities and showed that their high antioxidant potential may be due to their phenolic and flavonoid contents.

Antioxidant properties of germinated fenugreek seeds.

Fenugreek (Trigonella foenum-graecum) is used as a spice, vegetable and a medicinal plant. Since antioxidant properties have been linked to health benefits of natural products, such properties were studied in germinated fenugreek seeds which are considered to be more beneficial than dried seeds. Different fractions of the germinated seeds were used to determine their antioxidant potential at different levels. The assays employed were ferric reducing antioxidant power, radical scavenging by 1,1-diphenyl-2-picrylhydrazyl, ferrylmyoglobin/2,2'-azobis-3-ethylbenzthiazoline-6-sulfonic acid, pulse radiolysis, oxygen radical absorbance capacity and inhibition of lipid peroxidation in mitochondrial preparations from rat liver. An aqueous fraction of fenugreek exhibited the highest antioxidant activity compared with other fractions. As the quantity of phenolic and flavonoid compounds can be related to antioxidant activity, the contents from these extracts were measured. HPLC analysis was carried out to detect polyphenols, flavonoids and other components. This study reveals significant antioxidant activity in germinated fenugreek seeds which may be due partly to the presence of flavonoids and polyphenols.