Antioxidant and anti-inflammatory related activities of certain botanicals frequently used in Ayurveda and other indigenous systems of medication.

Abstract: Crude extracts (in 50% v/v, ethanol) of Wax negundo L. (Verbenaceae), Argemone maxicana L. (Papaveraceae), Cuminum cyminum L. (Apiaceae), Moringa oleifera Lamk (Moringaceae), Solanum virginianum L. (Solanaceae), Datura metal L. (Solanaceae) and Zingiber officinale Rosc. (Zingiberaceae) were screened for antioxidant and anti-inflammatory related activities. The antioxidant potentials were determined by performing reducing ability assay, 2, 2-diphenyl-l-picryl hydrazine (DPPH) radical and OH radical scavenging activity along with inhibition of polyphenol oxidase: a metal containing model oxidizing enzyme. The amount of major antioxidative substances like phenolics and vitamin C were estimated. The anti-inflammatory related activities such as inhibition of trypsin, [beta]-glucuronidase and conjugated then formation were carried out to assess the possible anti-inflammatory potential of the selected botanicals. The result of the present investigations show that the plant samples under study were found to be effective reducing agents along with considerable DPPH radical scavenging activity. With few exceptions all the plant samples showed moderate OH radical scavenging potentials and significantly inhibited the generation of hydroperoxides. All the tested plants showed moderate to poor profiles towards inhibition of trypsin, [beta]-glucuronidase and PPO. In the present growing herbal market worldwide the standardisation, authentication and quality control of medicinal plants has remained a major and critical issue in the focus of converting botanical materials into medicines. The results of the present findings may be useful in this context.

Keywords: Traditional medicine, medicinal plants, antioxidants, anti-inflammatory, enzyme inhibition, Ayurveda.

Introduction

A cursory look at the clinical and epidemiological data accumulated in the recent past clearly advocates the health supporting and therapeutic attributes of natural antioxidants. An antioxidant is any substance in low concentration capable of delaying or preventing the oxidation of cell macromolecules such as proteins, lipids, carbohydrates and nucleic acids. Broadly antioxidant is a group of heterogeneous compounds comprising enzymes (superoxide dismutase, catalase, glutathione peroxidase etc.), high molecular weight compounds (albumin, transferin, ceruplasmin etc.), low molecular weight compounds such as tocopherol, quinines, bilirubin and some ployphenols (flavones, flavonoides and phenolic acids), minerals including selenium, manganese and zinc and some vitamins such as Vitamin A, C and E (Gupta 2006).

The main functional target of antioxidants is to neutralise free radicals (atoms or molecules with one or more unpaired electrons) which are generated as a byproduct of daily episodes of normal metabolism. Reactive oxygen species (ROS) such as superoxide anion ([O.sub.2.sup..-] ), hydroxyl radicals (O[H.sup..]), [H.sub.2][O.sub.2] and singled oxygen ([sup.1][O.sub.2]) is a special category of free radicals mediated by activated oxygen (Gulcin 2002). Other free radicals of physiological concern includes thiyl (RS , a sulphur centred radical), trichloromethyl (C[Cl.sub.3], a carbon centred radical) or nitric oxide (N[O.sup..]) are continuously generated during several oxidation reactions (Tiwari 2004). Every living organism is equipped with radical scavenging devices which suppress, scavenge or clear ROS or alternatively induce the synthesis of antioxidant proteins/ enzymes and protect the cells from indiscriminate and hyper reactivity of free radicals (Noguchi 2000).

Overload of free radicals results in the accumulation of stress wherein the available antioxidant agents fail to circumvent the extra burden of free radical generation resulting in build up of oxidative stress (OS): an imbalance between oxidants and antioxidants in the body (Gutterigde 1995). The OS generated deregulates the normal physiological cell functions and initiates the recruitment of a variety of path ophysiological disorders such as ageing, arthritis, inflammation, diabetes, cataract, cancer, asthma, bronchopulmonary dysplasia, cardiovascular dysfunction, gastroduodenal pathogenesis, atherosclerosis, autoimmune diseases, ischemia perfusion injury, liver disorders, muscular dystrophy, amyotropic lateral sclerosis, pulmonary fibrosis, radiation damage, retinopathy, rheumatism, skin disease porphyria, senile dementia stroke and neurodegenerative diseases such as Parkinson's dementia and Alzheimer's disease (Magi 1987, Maxwell 1997, Thomas 1997, Scandalios 1997, Jose 2000, Tiwari 2001, Camria Borek 2004, Senevirathne 2006).

The physiological implications of ROS are alarming and can be a serious threat to human health if unchecked. Antioxidants can minimise the risk of above human ailments because of their involvement in regulation of various oxidative reactions in the human body. More specifically they can terminate or retard the oxidation process by scavenging free radicals, chelating free catalytic metals and acting as electron donors thereby stabilising the free radicals (Noguchi 2000). It is this reputation of the antioxidants in the mainstream of pharmaceutical research that has attracted the scientific community and increased the popularity as an alternative new generation therapeutic approach for the management of diverse polygenic disorders of humans (Tiwari 2004).

In Ayurveda the antioxidant properties of many rasayanas are well defined (Scartezzini 2000). The awareness and acceptance of traditional or indigenous plant drugs is increasing. There is renewed clinical interest in complementary medicines with hopes of discovering new leads in the present target-rich lead-poor pharmaceutical research. Traditional plant medications coupled with dietary measures are prescribed in Ayurveda and other indigenous systems of medicine. In Australia and the US, a sizable number of populations use at least one form of unconventional therapy including herbal medicine (MacLennan 1996, Eisenberg 1993).

The medicinal plants considered in this study possess multiple therapeutic potentials and are described in traditional and other indigenous systems of medicine. A brief pharmacological overview of the selected medicinal plants is described here highlighting the medicinal importance of the selected plants. Traditionally the whole plant of Vitex negundo L. (Verbenaceae) is described as useful in rheumatism. It is used as an astringent, cephalic, stomachic, promotes growth of hair, is prescribed against asthma, bronchitis, leucoderma and spleen enlargement. The fruits of the plant are used as an emmenagogue, nervine tonic. Seeds are used in cutaneous diseases and leprosy, while flowers are cardioprotective and prescribed in cholera, diarrhea, fever and liver complaints. The leaves of the plant have anti-inflammatory, discutient, anodyne and antiparasitic action. Rheumatic patients have benefited from bathing in water boiled with the leaves. The anti-rheumatic Ayurvedic preparation Vishagarbha Taila is prepared from the leaves and roots of the Vitex negundo (Chatterjee 1995).

Argemone maxicana L. (Papaveraceae) is a native of America which has now run wild all over India. In its homeland the yellow milky latex is used for the treatment of syphilis and skin diseases. In the Concan region (MS) of India the latex with milk is given in leprosy. The seeds and seed oil have been used by European physicians in India with considerable differences of opinion regarding their properties, some considering them as inert and others asserting that the oil in different doses is a valuable remedy for dysentery and other intestinal infections (William 2005).

Traditionally the fruit of Cuminum cyminum L. (Apiaceae) is used as a spice. It has antidiarrheal, antidysenteric, aromatic, astringent, carminative, cooling, galactagogue, diuretic and stimulant activities. The fruit is prescribed for correction of hoarseness in voice; the paste has been applied externally to allay pain and irritation of intestinal worms. The oil is useful in eczema (Chatterjee 1995).

Moringa oleifera Lamk (Moringaceae) commonly known as chum stick in India, is widely reputed for its therapeutic applications in traditional and indigenous systems of medicine. The pods are rich in leucine. All parts of the plant are used in the treatment of ascites, snake bites, rheumatism and as a circulatory stimulant. The leaves are rich in vitamins C and A, useful in management of scurvy. Flowers are used as a tonic, diuretic and cholagogue. The seeds are prescribed as antipyretic and seed oil is applied in rheumatism (Ambasta, 1992).

The whole plant of Solanum virginianum L. (Solanaceae) is described as antiasthmatic, aperient, diuretic, febrifuge, digestive, promotes conception and is used in constipation, dropsy and gonorrhea. The Ayurvedic chug Arkadhi is prescribed as a remedy for bronchitis, dengue and fever with chest affections. Fruit, flowers and stem are bitter, carminative, anti-rheumatic and it is prescribed in conditions associated with vascular and watery eruption. Its root is one of the Dasamula (ten roots), a reputed Ayurvedic decoction prescribed by the physicians for the management of arthritis and as an anti-inflammatory agent (William 2005a).

The seeds and roots of the Datura metel L. (Solanaceae) are useful as antidiarhoeal, antipyretic, antiseptic, antispasmodic, narcotic, anodyne and as a remedy for skin diseases. As a domestic remedy the pounded leaves of Datura metel mixed with turmeric in the form of a paste is applied over inflamed and painful parts. A pill made on the pounded seeds is placed in decayed teeth to relieve toothache. The Ayurvedic preparation Svalpajvaranausa prescribed for fever with catarrhal symptoms contains two parts of seeds of Datum metel with mercury, sulphur, aconite and ginger (William 2005a).

The rhizome of the Zingiber officinale Rose. (Zingiberceae), ginger, is routinely added as a taste and aromatic ingredient in the preparation many Asian dishes. The rhizomes are described as an appetizer, laxative, stomachic, aphrodisiac, carminative and are useful in heart and throat diseases, inflammations, bronchitis, asthma, vomiting and pain. Ginger is antihelmintic, useful in piles, rheumatism, headache and lumbago. Fresh rhizomes are ground and mixed with honey to cure colds and coughs (Bhattacharjee 2005).

Materials and methods

The selected medicinal plants Vitex negundo (leaves), Argemone maxicana (roots), Moringa oleifera (bark), Solanum virgirnianum fruit) and Datura metel (roots) were collected in the month of November 2006 from a field near Nanded City (MS), while the Cuminum cyminum (seeds) and Zingiber officinale (rhizome) were purchased from the local market at Nanded city (MS). The plants were identified and authenticated by the Taxonomist from the Department of Botany, School of Life Sciences and the voucher specimens were deposited in the herbarium of the School of Life Sciences of the host institute. The collected samples were dried at room temperature and were made into fine powder for extraction process. DPPH (2, 2-diphenyl-l-picryl hydrazine) and [beta]-glucuronidase (EC 3.2.1.31, 25,000 units, source: E. coli), were obtained from Sigma-Aldrich Co. (St Louis MO, USA), Trypsin was obtained form SISCO Research Lab. Ltd. Mumbai, p-nitrophenyl-[beta]-D-glucopyranosiduric acid was obtained from Calbiochem (EMD Biosciences Inc. La Jolla CA), L-DOPA (3,4 dihydroxy phenyl L-alanine) and glutathione (reduced form) were purchased from s. d. Fine Chemicals Ltd. Mumbai. Blood samples (containing 2 mg/ml EDTA) were collected from the local slaughterhouse at Nanded City (MS) for the preparation of RBC membrane solution. Apples (for extraction of PPO) were purchased from the local market at Nanded City. All other reagents and solvents were obtained from commercial sources and were of analytical grade

Extraction of plant samples

Approximately 10 g of individual plant sample was placed in Soxhlets extraction apparatus containing 50% (v/v) ethanol. The process of extraction was up to 6 hours. The final extracts were air dried and maintained at 4[degrees]C for further investigations.

Reducing activity assay

The reducing activity assay was performed as described by Sasaki (1991). The reduction of [Fe.sup.3+], of [K.sub.3]Fe[(CN).sub.e], to [Fe.sup.2+] by reducing agents results in a fall in extinction at 420 nm. The reaction mixture contained 500 [micro]L solution of individual plant extract in 3 mL of 1 mM potassium ferricynide solution; after 10 min the absorbance was recorded at 420 nm. The OD of the control sample (without plant extract) was considered as 100% potassium ferricynide, the amount of plant samples required to reduce 50% potassium fenicynide to [Fe.sup.2+] was calculated as [IC.sub.50] value. Ascorbic acid (1 mM, [IC.sub.50] = 0.049 mg/mL) was used as a standard reducing agent

DPPH radical scavenging assay

The DPPH radical scavenging assay was performed as described by Bartolome (2004). The reaction mixture contained different concentrations of individual plant extract (in absolute ethanol) and DPPH radical (1 mM in absolute ethanol) solution. The contents of the reaction mixture were observed spectrophotometrically at 517 non after 20 min. The OD of the control was considered as a 100% unreduced DPPH, while the amount of plant sample required to reduce 50% of DPPH was calculated as [IC.sub.50] value. Reduced form of glutathione (1mM, [IC.sub.50] = 0.52 mg/ml) was used as a reference compound.

Determination of OH radical scavenging activity

A method described by Christos and Dimitra (2003) was used for the determination of OH radical scavenging activity. OH radicals were generated by using [Fe.sup.3+]/ascorbic acid. The formaldehyde generated from the oxidation of dimetlryl sulfoxide (DMSO) was measured for the detection of OH radicals generated in the [Fe.sup.3+]/ascorbic acid system.

The reaction mixture contained 0.1 mM EDTA, 167 [micro]M [Fe.sup.3+], 33 mM DMSO in phosphate buffer of 50 mM pH 7.4. 0.1 mL different concentrations of individual plant extract and 150 pl of ascorbic acid (10 mM in phosphate buffer) were added to start the reaction. Trichloroacetic acid (17%y w/v) was used to terminate the reaction after 30 min. The formaldehyde produced was detected spectrophotometrically at 412 mn.

The [IC.sub.50] values were determined as the concentration of the individual plant sample required to achieve 50% of formaldehyde production as compared to control (without sample). Coumarin (1 mM, [IC.sub.50] = 0.95 mg/ml) was used as a standard compound for comparative study.

PPO inhibition assay

A semi pure preparation of PPO (polyphenol oxidase) extracted from apple was used to study the effect of individual plant extract on activity of PPO. The extraction of PPO was carried out as the experimental protocol described by Gacche (2004). The PPO assay was carried out as described by Pathak (1992). The reaction mixture contained L-DOPA (1 mL, 2 mM), 0.5 ml enzyme, 1 ml, individual concentration of plant extract and citrate buffer (0.5 ml, pH 4.8, 0.1 M). After 5 min the contents of the reaction mixture were observed spectrophotometrically at 470 nm. The OD of the control was considered as 100% activity of PPO and the concentration of plant samples required to achieve 50% PPO activity were calculated as [IC.sub.50] values. L-cysteine (1 mM, [IC.sub.50] = 0.91 mg/ml) was used as a standard PPO inhibitor.

Phenolic content

A method described by Bray and Thorpe (1954) was adapted for the estimation of total polyphenol contents from the ethanol extracts of the selected plant samples. The phenolic compound undergoes reaction with an oxidizing agent phosphomolybdate present in the Folin-Ciocalteau reagent, the resultant reaction product is a blue coloured complex having maximum extinction at 660 non. The amount of phenolics was calculated by using a standard curve constructed using serial dilutions of catechol (500 [micro]g/ml). The total amount of polyphenol was estimated as mg/g of samples.

Vitamin C content

A dye reduction method described by Sadasivam (1996) was performed for estimation of vitamin C (L-ascorbic acid) content in the selected plant samples. The plant samples were initially extracted in oxalic acid (4%y w/v). The L-ascorbic acid reduces the 2, 6-dichlorophenol indophenol (a blue coloured dye) to a colourless leuco-base The ascorbic acid is oxidised to dehydroascorbic acid. The dye is pink coloured in acid medium. Oxalic acid was used as the titrating medium. The vitamin C content of selected plant extracts was expressed as mg/100 g of individual plant samples.

Trypsin inhibition assay

An anti-proteolytic activity of selected plant samples was determined by employing a method of Tandon (1982). The method is based on the measurement of inhibition of Irypsin induced hydrolysis of BSA. Trypsin (0.075 mg/mL) was initially incubated with individual concentrations of plant extract of 0.1 mL for 20 min. The substrate BSA (6 g/100 mL, in 0.1 M phosphate buffer, pH 7.6) was added after 20 min. The reaction mixture was incubated for 25 min at 37[degrees]C. The reaction was terminated by the addition 3 Ml of C[Cl.sub.3]COOH (5%, w/v). The acid soluble fractions were obtained by centrifuging the contents at 5000 RPM for 15 min. The amount of protein in the acid soluble fractions was estimated by a method described elsewhere (Lowry 1951). The trypsin activity in the absence of inhibitor was considered as 100%, and the concentration of each plant sample giving 50% inhibition ([IC.sub.50]) was then calculated. Salicylic acid ([IC.sub.50] = 0.061 mg/mL) was used as a reference drug.

[beta]-glucuronidase inhibition assay

The effect of the plant extracts on activity of [beta]-glucuronidase was studied using a method described by Demetrios (1998). Different concentrations of plant extract (0.1 mL) in 0.1 M acetate buffer pH 7.4 for 5 min at 37[degrees]C were preincubated with 0.8 mL of 2.5 mM p-nitrophenyl-[beta]-D-glucopyranosiduronic acid and 0.1 mL of [beta]-glucuronidase was added. The mixture was incubated for 30 min. Reaction was terminated by addition of 2 mL of 0.5 N NaOH. The reaction mixtures were observed spectrophotometrically at 410 nm. The activity of [beta]-glucuronidase in the absence of inhibitor was considered as 100%, and the amount of plant samples required to achieve 50% activity was then calculated as [IC.sub.50] values. Salicylic acid (1 MM, [IC.sub.50] = 0.053 mg/ml) was used as a reference compound.

Inhibition of hydroperoxides

RBC membrane solution was prepared as described by Dodge (1963) with slight modification in buffer (Quist 1980). Membrane solution (1.0 mL) was mixed with 5 mL of chloroform: methanol (2:1) followed by centrifugation at 1000 g for 15 min for separation of the two phases. The chloroform layer was taken in a test tube and dried at 45[degrees]C in a water bath. The lipid residue was dissolved in 1.5 mL of cyclohexane and the hydroperoxides generated were detected at 233 nm against a cyclohexane blank. Coumarin (1 mM, [IC.sub.50] = 0.031 mg/ml) was used as a standard drug.

Results and discussions

The results of the various antioxidant assays performed in the present investigations are presented in Table 1. Amongst the tested plants the extract of the Solanum virginianum ([IC.sub.50] = 1.3 mg/mL) was found to be an effective reducing agent compared with other plant samples which showed reducing activity in a [IC.sub.50] range of 1.6-2.3 mg/mL. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity, nevertheless the reducing agents may exhibit their antioxidant potential by acting as a hydrogen atom donor, free radical chain breaker, by binding with transition metal ion catalyst or by preventing continued hydrogen abstraction (Hudson 1990; Diplock 1991; Yildirim 2000). The DPPH radical scavenging effect was observed to be optimum in the extract of Vitex negundo ([IC.sub.50] = 0.7 mg/ml) followed by extract of Zingiber officinale ([IC.sub.50] = 0.93 mg/ml) and Argemone maxicana ([IC.sub.50] = 0.95 mg/mL), all other samples showed moderate activity.

DPPH is a nitrogen-centred free radical. It reacts similarly to the peroxyl radical. Its reaction rates correlate directly with antioxidant activity, the higher the rate, the more effective the antioxidant (Wright 2003). The reducing ability and DPPH radical scavenging activity shown by the extracts of different plant samples may be due to interaction with diverse polyphenols as they are reported to possess free radical scavenging effects (Miller 1996).

The extracts of the Vitex negundo and Cuminum cyminum were found to be ineffective towards scavenging of OH radicals. Of the reactive samples the extract of Datura metel ([IC.sub.50] = 0.55 mg/ml) and Zingiber officinale ([IC.sub.50] = 0.6 mg/ml) were graded as most effective compared with other samples which showed considerable OH radical scavenging activity in an [IC.sub.50] range of 0.835-0.95 mg/mL. The OH radicals are generated in biological cells through the Fenton reaction. The reactivity of OH radicals is reported to be extremely high where they interact indiscriminately with important cell components which may disturb the normal physiological functions of the cells (Barry 1984). OH radicals are also attributed in the damage of membranes of the cells in inflamed areas (Darligton 2001).

Phenolic substances have been implicated in the scavenging of OH radicals; the possible mechanism may be related to the active hydrogen donor ability of hydroxyl substitution. High molecular weight and the proximity of many aromatic rings and hydroxyl groups is an essential structural configuration for the free radical scavenging by specific functional groups (Korycka-Dahl 1978).

The profile of inhibition of conjugated diens (hydroperoxides) by different plant samples shows that the extract of Zingiber officinale ([IC.sub.50] = 0.195 mg/mL) was the more effective agent towards inhibition of lipid peroxidation. All other samples showed inhibition activity in an [IC.sub.50] range of 0.385-0.685 mg/mL). Lipid oxidation leads to conjugated dime hydroperoxide formation as a result of the hydrogen capture from the unsaturated fatty acids (Senevirathne 2006). The process of lipid peroxidation is one of the contributory factors in inflammatory disorders, ultimately resulting in the loss of membrane integrity causing cell death (Zurier 1993). The inhibition of conjugated diene formation may be related to hydroperoxide interacting polyphenol compounds like flavones, coumarins, chalcones and cinnamic acids which may neutralise hydroperoxides by the reduction process (Tiwari 2004).

The amount of vitamin C and total polyphenols in different plant samples has been summarised in Table 2. The concentration of vitamin C and total polyphenols were estimated to be highest in Vitex negundo (34.75 mg/100g) and Solanum virginianum (81.75 mg/g) respectively. All other samples were found to contain an appreciable amount of vitamin C and phenolics. The importance of vitamin C as an antioxidant is indispensable. Besides its role as an antioxidant it has proved to be instrumental in the management of conditions such as cancer, cardiovascular disease, hypertension, diabetes mellitus, age related eye disease, AIDS and bone healing Bsoul 2004). Polyphenols are widely distributed in plants and phenolic antioxidants are described as free radical scavengers possessing several biological activities of therapeutic importance (Nijveldt 2001).

The results of the inhibition of trypsin, [beta]-glucuronidase and PPO by extracts of different plant samples are summarised in Table 3. The results show that the extracts of Vitex negundo [IC.sub.50] = 0.2 mg/ml), Solanum virginianum ([IC.sub.50] = 0.225 mg/ml) and Cuminum cyminum ([IC.sub.50] = 0.25 mg/ml) appear to be the most effective samples for inhibition of trypsin induced hydrolysis of BSA, where other extracts showed trypsin inhibition in an [IC.sub.50] range of 0.375-0.9 mg/mL. With the exception of the extract of Argemone maxicana, all samples were found to be reactive with [beta]-glucuronidase. The extract of Vitex negundo ([IC.sub.50] = 0.044 mg/ml) was greater than Zingiber officinale ([IC.sub.50] = 0.046 mg/ml) and greater than Solanum virginianum ([IC.sub.50] = 0.049 mg/ml) in the inhibition of [beta]-glucuronidase. The remaining plant samples were moderate to poor inhibitors of [beta]-glucuronidase activity. The activity of PPO was affected by the contents of all plant extracts. On average all plant samples were found to be equally potent inhibitors of PPO, which showed PPO inhibition in an [IC.sub.50] range of 1.2-1.9 mg/mL.

Trypsin is a member of the serine proteases family. These proteases are involved in initiation of inflammation; serine protease inhibition has been considered one of the targets for design of anti-inflammatory drugs (Bilfinger 2002). The lysosomes of the polymorphonuclear neutrophils are rich in [beta]-glucuronidase. This enzyme is attributed as one of the mediators for initiating the process of inflammation (Ito 1982, Savill 1995). Flavonoids, a member of plant polyphenols have been reported to inhibit proteolytic enzymes involved in recruiting the process of inflammation (Middleton 1992). The plant samples showing inhibition of [beta]-glucuronidase and trypsin may contain bioactive flavonoids as they are known to possess anti-inflammatory activity (Nijveldt 2001).

In present investigations PPO has been studied as a model metal containing oxidizing enzyme. PPO (EC 1.14.18.1) is a copper containing metalloenzyme reported to occur in all plant species. The enzyme utilises molecular oxygen for conversion of a series of polyphenols to highly reactive quinones by an oxidation process (Gacche 2004). Metal catalysed formation of free radicals is reported to play a critical role in the development of diseases such as rheumatoid arthritis and cancer (Barry 1984). A system involving transition metal ions and molecular oxygen may generate free radicals thereby may be altering the normal function of cells. Phytochemicals, especially phenolic substances, have been reported to act as metal chelators (Sanchez-Mareno 1999).

The interaction between metal (copper) chelating polyphenols and the copper moiety present in the active site of PPO could be a possible mechanism for the inhibition of PPO activity.

Conclusion

Recent trends in the herbal market show that about one third of all prescriptions issued in developed countries, such as the United States, Canada and in Europe, contained a herb or a purified extract of an active component derived from medicinal plants. In the less developed countries where synthetic chugs are expensive, such as China, India and the countries of the Far East, approximately 70% to 90% of prescriptions include herbal drugs. Plant extracts used for centuries without harmful side effects became a source of hope for pharmaceutical companies and clinicians. But clinical based evidence, standardisation and authentication of botanicals has still a long way to go. The results of the present studies may be of use in the current research of herbal medicines.

Acknowledgements

Authors are thankful to UGC India for financial assistance and Director, School of Life Sciences, SRTM University Nanded (MS) India, for providing the necessary facilities during this work.

References

Ambasta SP. 1992. The useful plants of India. New Delhi: Publication and Information Directorate, CSIR.

Barry H, John MCG. 1984. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J 219:1-14.

Bartolome B, Nunez V, Monagas M, Gomez-Cordoves C. 2004. In vitro antioxidant activity of red grape skins. Eur Food Res Technol 218:173-7.

Bhattacharjee SK, De LC. 2005. Medicinal Herbs and Flowers. Jaipur: Aavishkar Publishers and Distributors.

Bilfinger TV, George BS. 2002. The role of protease inhibition with emphasis on the effects of inflammation and vascular immune phenomena. Curr Pharm Design 8:125-33.

Bray HG, Thorpe WV. 1954. Analysis of phenolic compounds of interest in metabolism. Meth Biochem Anal 1:27-30.

Bsoul SA, Terezhalmy GT. 2004. Vitamin C in health and disease. J Contemp Dent Pract 5:2;1-14.

Carmia Borek. 2004. Antioxidant and radiation therapy. J Nutr 134:3207S-3209S.

Chatterjee A, Pakrashi SC. 1995. The treatise on Indian medicinal plants Vol 4. New Delhi: Publication and Information Directorate CSIR.

Christos K, Dimitra HL. 2003. Biological evaluation of several coumarin derivatives designed as possible anti-inflammatory/antioxidant agents. J Enzyme Inhib Med Chem 18:1;63-9.

Darligton LG, Stone TW. 2001. Antioxidants and fatty acids in the amelioration of rheumatoid arthritis and related disorders. Bri J Nutra 85:251-69.

Demetrios NN, Konstantina CF, Konstantinos EL, Dimitra HL. 1998. Synthesis and biological evaluation of several coumarin-4-carboxamidoxime and 3-(coumarin-4yl)-1,2,4-oxadiazole derivatives. Eur J Med Chem 33:715-24.

Diplock AT. 1997. Will the 'good fairies' please prove to us that vitamin E lessens human degenerative disease? Free Radic Res 27:511-32.

Dodge JT, Mitchell C, Hanaban DJ. 1963. The preparation and chemical characteristics of Hb-free ghosts of human erythrocytes. Arch Biochem Biophys 100:119-30.

Esenberg DM, Kesseler RC, Foster C, Norlock FE, Calkins DR, Delbanco TL. 1993. Unconventional medicine, costs and patterns of use. New Engl J Med 328:246-52.

Gacche RN, Warangkar SC, Ghole VS. 2004. Glutathion and cinnamic acid: Natural dietary components used in preventing the process of browning by inhibition of polyphenol oxidase in apple juice. J Enz Inhi & Med Chem 19:2;175-9.

Gupta V, Sharma S. 2006. Plants as natural antioxidants. Nat Prod Radiance 5:4;326-34.

Gulcin I, Oktay MO, Kufrevioglu OI, Aslan A. 2002. Determination of antioxidant activity of lichen Cetaria islandica (L.). Ach J Ethanopharmacol79:325-9.

Gutteridgde JMC. 1995. Free radicals in disease processes: A complication of cause and consequences. Free Radic Res Comm 19:141-58.

Hudson BJF. 1990. Food Antioxidants. London: Elsevier.

Ito K, Kagaya H, Tsukamoto G, Nose T. 1982. The studies of the mechanism of anti-inflammatory action of 2-(ethylpyridin-2-yl)-benzimidazole (KB-1043). Drug Res 32:1;117-22.

Jose N, Janardhan KK. 2000. Antioxidant and anti-tumor activity of Pleurotus florida Curr Sci 79:7;941-3.

Korycka-Dahl M, Richardson M.1978. Photogeneration of superoxide anion in serum of bovine milk and in model systems containing riboflavin and aminoacids. J Dairy Sci 61:400-7.

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measurement with folin phenol reagent. J Biol Chem 193;265-75.

MacLennan AH, Wilson DH, Taylor AW. 1996. Prevalence and cost of alternative medicine in Australia. Lancet 347;569-73.

Maxwell S. 1997. Antioxidant therapy: does it have a role in the treatment of human diseases? Exp Opin Invest Drugs 6:211-36.

Middleton EJ, Kandaswami C. 1992. Effect of flavonoids on immune and inflammatory cell functions. Biochem Pharmacol 43:1167-79.

Miller AL. 1996. Antioxidant flavonoids: structure, function and clinical usage. At Med Rev 1;103-11.

Naguchi N, Watanabe A, Shi H. 2000. Diverse functions of antioxidants. Free Radic Res 33:809-17.

Nijveldt RJ, Nood Els van, Hoorn DEC, Boelens PG, Norren KV, Leeuwen PAM. 2001. Flavonoides: a review of probable mechanisms of action and potential applications. Am J Clin Nutr 74:418-25.

Pathak SU, Karyekar SK, Ghole VS, Hegde MV. 1992. Effect of activation methods on affinity chromatography of potato polyphenol oxidase. Photochem 31:5;1481-3.

Quist KH. 1980. Regulation of erythrocyte membrane shape by calcium ion. Biochem Biophys Res Commun 92;631-7.

Sadasivam S, Manickam A.1996. Biochemical Methods. 2nd edn. New Delhi: New Age International.

Sanchez-Moreno C, Larrauri JA, Saura-Calixto F. 1999. Free radical scavenging capacity and inhibition of lipid oxidation of wines, grapes juices and related polyphenolic constituents. Food Res Int 32:407-12.

Sasaki K, Matsumato I, Beppu M. 1991. In Affinity Chromatography. Tokyo: Tokyo Kagaku Dojin.

Savill J, Haslett C. 1995. Granulocyte clearance by apoptosis in the resolution of inflammation. Semin Cell Biology 6;385-93.

Scandalios JG. 1997. Oxidative stress and the molecular biology of antioxidant defenses. New York: Cold Spring Harber.

Scartezzini P, Speroni E. 2000. Review on some plants of Indian traditional medicine with antioxidant activity. J Ethnopharmacol 71;23-43.

Senevirathne M, Kim S-H, Siriwardhana JHH, Lee K-W, Jeon Y-J. 2006. Antioxidant potential of Ecklonia cava on reactive oxygen species scavenging, metal chelating, reducing power and lipid peroxidation inhibition. Food Sci Tech Int 12:1;27-38.

Tandon M, Tandon P, Barthwal JP, Bhalla TN, Bhargava KP. 1982. Anti-inflammatory and anti-proteolytic activities of newer indolyl-isoquinilines. Drug Res 32(11):10;1233-5.

Tiwari AK. 2001. Imbalance in antioxidant defense and human disease: multiple approach of natural antioxidant therapy. Curr Sci 81:1179-87.

Tiwari AK. 2004. Antioxidants: New-generation therapeutic base for treatment of polygenic disorders. Curr Sci 86;1092-1102.

Thomas CE, Kalyanaraman B. 1997. Oxygen radicals and the disease process. Netherlands: Hardwood Academic Publishers.

William D, Warden CJH, Hopper D. 2005. Pharmacographia Indica Vol 1. New Delhi: Srishti Book Distributors Ltd.

William D, Warden CJH, Hopper D. 2005a. Pharmacographia Indica Vol 2. New Delhi: Srishti Book Distributors Ltd.

Wright JS. 2003. Searching for fountain of youth. Chem Br 39;25-7.

Yagi K. 1987. Lipid peroxides and human diseases. Chem Phy Lipids 45;337-51.

Yildirim A, Mavi M, Oktay AA, Kara OF, Algur, Bilaloglu V. 2000. Comparison of antioxidant and antimicrobial activities of Tilia (Tilia argentea Desf Ex DC), Sage (Salvia triloba L.), and Black Tea (Camellia sinensis) extracts. J Agric Food Chem 48;5030-4.

Zurier RB. 1993. Fatty acids, inflammation and immune responses. Prostaglandins, Leukotrienes & Essential Fatty Acids 48;57-62.

RN Gacche *, NA Dhole, AD Jadhav

School of Life Sciences, Swami Ramanand Teerth Marathwada University 

Nanded-431 606 (MS), India

* Corresponding author email: [e-mail]

Table 1
Results of the effect of ethanol extracts of selected medicinal
plants on reducing activity, scavenging of DPPH and OH radicals
and inhibition of hydoperoxides (OOH)

Sr No       Name of plant        RA      DPPH      OH       OOH

                                      [IC.sub.50] (mg/mL)

1        Vitex negundo          1.60      0.7      NR      0.490
2        Argemone maxicana      1.70     0.95     0.835    0.570
3        Morin a oleifera       1.80      1.9     0.850    0.575
4        Solanum virginianum    1.30      1.1     0.950    0.385
5        Datura metel           2.30     1.025    0.550    0.685
6        Cuminum cyminum        2.20      1.1      NR      0.434
7        Zingiber officinale    1.90     0.93     0.600    0.195
8        Glutathione             ND      0.52      ND       ND
9        Ascorbic acid          0.049     ND       ND       ND
10       Coumarin               1 ND      ND      0.95     0.031

NR--no reaction, ND--not determined.

Table 2
Vitamin C and total phenolic content of the selected medicinal plants

Sr No   Name of plant         Vitamin C (mg/100g)   Phenolics (mg/g)

1       Vitex negundo                34.75               69.00
2       Argemone maxicana            30.40               11.20
3       Moringa oleifera             32.37               39.30
4       Solanum virginianum          26.64               81.75
5       Datura metel                 21.72               27.56
6       Cuminum cyminum              17.37               48.80
7       Zingiber officinale          13.32               22.75

Table 3
Effect of ethanol extract of plant samples on the activity of
trypsin induced hydrolysis of BSA, [beta]-glucuronidase and PPO

                                              [beta]-
Sr No    Name of plant          Trypsin    glucuronidase     PPO

                                       [IC.sub.50] (mg/ml)

1        Vitex negundo           0.200         0.044         1.9
2        Argemone maxicana       0.375          NR           1.2
3        Moringa oleifera        0.396         0.650         1.4
4        Solanam virginianum     0.225         0.049         1.3
5        Datura metel            0.800         0.120         1.4
6        Cuminum cyminum         0.250         0.054         1.2
7        Zingiber officinale     0.900         0.046         1.6
8        Salicylic acid          0.061         0.053         ND
9        L-cysteine               ND            ND          10.91

NR--no results under experimental conditions, ND--not determined.

All results are the mean values of duplicate measurements