Plant Foods Hum Nutr (2011) 66:9196 DOI 10.

1007/s11130-011-0217-5

ORIGINAL PAPER

Isolation, Characterization and Quantification of Tricin and Flavonolignans in the Medicinal Rice Njavara (Oryza sativa L.), as Compared to Staple Varieties
Smitha Mohanlal & Rathnam Parvathy & Vasantha Shalini & Antony Helen & Ananthasankaran Jayalekshmy

Published online: 5 March 2011 # Springer Science+Business Media, LLC 2011

Abstract Njavara is an important medicinal rice variety of Kerala, India, widely used in Ayurveda as a health food and in the treatment of rheumatoid arthritis, paralysis, neurodegenerative diseases and in rejuvenation therapy. Phytochemical investigations and spectroscopic studies of the diethyl ether fraction of methanolic extract of Njavara Black (NB) rice bran gave three important compounds namely, tricin and two rare flavonolignans- tricin 4-O(erythro--guaiacylglyceryl) ether and tricin 4-O-(threo-guaiacylglyceryl) ether. The EC50 values of these compounds in DPPH system were 90.39, 352.04 and 208.1 g/ ml, respectively. Quantification of the compounds by HPLC in NB and staple, non-medicinal rice varieties Sujatha (SJ) and Palakkadan Matta (PM) showed that tricin is present 39.64 and 16.12 fold higher in NB, compared to SJ and PM, respectively. This is the first report on the

occurrence of tricin at significantly higher levels in Njavara and occurrence of the two flavonolignans in Oryza sativa species. Of the three compounds, tricin and the threo- form of flavonolignan showed anti-inflammatory effect of >65% after 5 h, at 2 mg/kg, in carrageenan-induced, paw edema experiments in rats. The results of the study corroborate with the preferential use of Njavara in indigenous medicine, over staple varieties. Keywords Flavonolignans . Njavara . Rice bran . Tricin . Tricin 4-O-(erythro-- guaiacylglyceryl) ether . Tricin 4-O-(threo--guaiacylglyceryl) ether Abbreviations COX cyclooxygenase DPPH 2, 2- diphenyl-1-picrylhydrazyl EC50 amount of extract or compound needed to decrease the initial DPPH concentration by 50% HPLC-PDA high performance liquid chromatographphotodiode array IR infra red MS mass spectrometry NB Njavara Black ND not detected NMR nuclear magnetic resonance NO nitric oxide NSAIDs non-steroidal anti-inflammatory drugs PM Palakkadan Matta RAW 264.7 cell line of mouse macrophages ROS reactive oxygen species SD standard deviation SEM standard error mean

Electronic supplementary material The online version of this article (doi:10.1007/s11130-011-0217-5) contains supplementary material, which is available to authorized users. S. Mohanlal : R. Parvathy : A. Jayalekshmy (*) Chemical Sciences & Technology Division, National Institute for Interdisciplinary Science and Technology (NIIST), CSIR, Industrial Estate P.O., Thiruvananthapuram 695019 Kerala, India e-mail: jayalekshmy9@gmail.com A. Jayalekshmy e-mail: jaya@niist.res.in V. Shalini : A. Helen Department of Biochemistry, University of Kerala, Kariavattom Campus, Thiruvananthapuram 695 581, India

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SJ SPSS/PC+ UVVIS

Sujatha statistical programme software ultra violet-visible

study is an investigation on the anti-inflammatory compounds of black glumed Njavara grown in a certified Njavara farm, as compared with a popular white rice variety Sujatha (SJ) and a preferred pigmented variety Palakkadan Matta (PM) grown in the same region.

Introduction Contemporary importance of free radicals and reactive oxygen species (ROS) as causative agents in degenerative diseases and conditions like atherosclerosis, cancer, inflammation, rheumatism, aging, etc., can not be overlooked [1]. Foods are a safe, natural source of potent antioxidants [2]. India, especially the southern state of Kerala, is well known for its rich biodiversity, medicinal plants, spices and practice of Ayurveda. Recently, there is greater acceptance of holistic treatment regimes to fight degenerative disease conditions using herbal extracts that are rich in antioxidant phytochemicals [3, 4]. The ancient texts of Ayurveda report special rice described as Shaashtikam-a variety of red rice reaped in 60 days timethat is medicinal (http://www.njavara.com/aboutnjavara). Njavara is a unique pigmented rice cultivar of extra short duration (6090 days), endemic to Kerala. It is widely used in ayurvedic system of medicine and experts in the field consider Njavara to be the medicinal variety cited in ancient literature [http://www.njavara.com/aboutnjavara, 5]. This is the only cultivar traditionally used in specific treatments like Panchakarma that includes protocols like Njavara kizhi and Njavara theppu for rheumatoid arthritis, neurological problems, paralysis and rejuvenation therapy. Njavara is also recommended as health food for people of all ages and its brown rice is the main ingredient in the nutraceutical kit (Karkitaka Kanji) for making Medicinal Porridge, recommended by physicians to boost immunity against diseases, during monsoon season, in this region (http://www. njavara.com/aboutnjavara). There are two major types of Njavara grown in Kerala for medicinal use viz. the black glumed and golden yellow glumed types, depending on the colour of the outer husk of paddy, both having red-pigmented rice grains (http://www.njavara.com/aboutnjavara). There are no previous scientific reports on the bioactive compounds of Njavara and available reports are on agronomic aspects, genetic characteristics, proximate composition, lipid profile and starch characteristics [57]. As our group has been working on natural antioxidants and bioactives of indigenous sources, we undertook a study of the methanolic extract of Njavara rice bran and rice that showed higher antioxidant activity for Njavara compared to staple varieties. These interesting results made us to investigate the nutraceutical relevance of Njavara as a health food. Here, we present the first report on this important aspect. Our Materials and Methods Chemicals 2, 2- diphenyl-1-picrylhydrazyl (DPPH) and quercetin were procured from Sigma-Aldrich Co., USA. Deuterated solvents for nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC) grade acetonitrile and water were purchased from Merck, Mumbai, India. Sephadex LH20 (particle size 25100 m) was purchased from Pharmacia Fine chemicals AB, Uppsala, Sweden. All chemicals and solvents were of analytical grade. The inflammatory agent used for the study was Type IV Lamda carrageenan from Spectrochem Ltd., India. Diclofenac (Voveran) was procured from Novartis India Ltd., Mumbai, India. Plant Material and Extraction Authentic Njavara black (NB) samples were collected directly from a certified farm namely, the ECO FARM Karukamanikalam at Chittoor, Palakkad, Kerala. Samples of staple varieties SJ and PM were also collected from the same farmer, for comparison. Plant specimens were verified by Dr. Maya C. Nair, Department of Botany, Government Victoria College, Palakkad- 678 001, Kerala, India and voucher specimens are available at the herbarium of above mentioned Department. Freshly milled bran samples were stabilized by heating 100 g lots of bran, spread on a petri dish, at 100 C for 30 min in air oven (Sri Rudran Instruments Co., Chennai, India). 100 g lots of stabilized rice bran (NB, SJ, PM) were extracted using 800 ml of petroleum ether solvent for about 16 h in a Soxhlet extractor. The residual rice bran was further extracted with 800 ml of methanol as described above. These extractions were repeated for 250 g of bran of NB, SJ and PM. Methanolic extract residue was suspended in 200 ml water and partitioned with (5100 ml) of diethyl ether. The diethyl ether extracts were evaporated to dryness on a rotary evaporator (Laborota 4000-Heidolph, Germany) and the dried residue was made up to definite volume in methanol and stored in the refrigerator until further work up. DPPH Radical Scavenging Activity DPPH solution of 0.1 mM was prepared in methanol. Different concentrations (1001,000 g/ml) of the dif-

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ferent extracts and compounds were prepared keeping the sample volume to be 0.5 ml and 5 ml of DPPH solution was added to each test tube and shaken well. The test tubes were kept at ambient temperature (302 C) in the dark for 30 min. A control was prepared with 0.5 ml methanol and DPPH solution (5 ml). Percentage of radical scavenging activity and EC50 (amount of extract or compound needed to decrease the initial DPPH concentration by 50%) were determined spectrophotometrically at 517 nm, using Shimadzu UV-1601, according to Sanchez-Moreno et al. [8]. Isolation of Compounds 1, 2 and 3 and their Quantification by HPLC-PDA Analysis Three compounds were isolated from 2 g of diethyl ether residue chromatographed over (a) silica gel column with peteroleum ether:ethyl acetate gradient, (b) Sephadex LH-20, and (c) analytical and preparative HPLC, followed by crystallization to afford pure compounds 1 (24 mg), 2 (3.1 mg) and 3 (3 mg), respectively. Isolated compounds 1, 2 and 3 were quantitated in NB, SJ and PM by analytical, reverse phase high performance liquid chromatography-photodiode array (HPLC-PDA) (details given in supplementary). Anti-inflammatory Activity For the experiment, the rats (details given in supplementary) were divided into six groups (IVI) with six rats in each group (n=6). Group I received saline. Acute inflammation was produced by the sub-plantar administration of 0.1 ml of carrageenan (1% in normal saline) from group IIVI into the right hind paw of the rats. The animals were pre-treated intra-peritoneally in groups IIV, with the compound 1, 2, 3
Table 1 EC50 (amount of compound needed to decrease the initial DPPH concentration by 50%) and content (mg/100 g dry weight) of tricin (Compound 1), tricin 4-O-(erythro--guaiacylglyceryl) ether Compound EC50 (g/ml)

(2 mg/kg each) and diclofenac (20 mg/kg) in saline respectively, 30 min before the administration of carrageenan. The volume of each paw was measured by means of a plethysmograph at 0, 3rd, 5th h after carrageenan injection [9]. The percentage of paw edema was calculated according to Winter et al. [10]. Statistical Analysis Values were represented as meanstandard deviation (SD) and standard error mean (SEM) of two analyses from three replications (n=6). Analysis of variance was performed for quantification by HPLC and in vivo anti-inflammatory experiments using the statistical program software (SPSS/ PC+), version 11.0 (SPSS, Chicago, IL, USA). Duncans multiple range test was conducted for comparison of means at P<0.05.

Results and Discussion Antioxidant Activity Rice bran of NB, SJ and PM were extracted first with petroleum ether (6080 C) and subsequently with methanol to yield residue of NB (16.77 g), SJ (9.78 g) and PM (13.98 g). Rice was also extracted in a similar manner. The petroleum ether and methanolic extracts of rice bran and rice were evaluated by DPPH scavenging assay which showed that methanolic extracts of rice bran were more active (unpublished). For example, at 200 g/ml, the methanolic extract of NB rice bran showed 93.0% activity whereas SJ and PM showed 65.56 and 41.50%, respectively. Corresponding activity of rice extracts were: 34.64% (NB), 9.07% (SJ) and 7.03% (PM). The petroleum ether
(Compound 2) and tricin 4 O-(threo--guaiacylglyceryl) ether (Compound 3) in Njavara Black (NB), Sujatha (SJ) and Palakkadan Matta (PM) rice bran

Content (mg/100 g) NB SJ 4.860.01c 1.880.08b 2.710.01b PM 11.980.01b NDi NDi

Compound 1 Compound 2 Compound 3 Quercetin

ac

90.390.03g 352.040.16e 208.010.09f 42.980.02h

193.050.03a 57.150.09a 64.620.03a

Meanstandard deviation of two analyses from three replicate (n=6) determinations followed by different letters in a row are significantly different in the Duncans test at P<0.05

Meanstandard deviation of two analyses from three replicate (n=6) determinations followed by different letters in a column are significantly different in the Duncans test at P<0.05
i

eh

not detected

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extract of NB bran showed <1.0% scavenging activity at 1,000 g/ml whereas similar extracts of other samples including NB rice, did not show any measurable activity at the same concentration. Hence, the methanolic extract of rice bran was chosen for further phytochemical investigations. The methanolic extracts of NB, SJ and PM were fractionated using diethyl ether to investigate flavonoid type of compounds. The diethyl ether extract of NB (934.237.47 mg/100 g), SJ (390.323.12 mg/100 g) and PM (844.076.75 mg/100 g) were evaluated for DPPH scavenging activity. The EC50 of the three extracts were in the order NB (259.83 g/ml)<PM (340.26 g/ml)<SJ (457.36 g/ml) which showed that NB had better radical scavenging activity. Isolation of Compounds 1, 2 and 3 and their Quantification by HPLC-PDA Analysis Isolation procedures gave three compounds 1, 2 and 3 and were identical with the reported compounds [11, 12] based on mass spectrometry (MS), ultra violet (UV) absorption and NMR spectroscopic data including 1H and 13C. Thus, compounds 1, 2 and 3 are identified and reported as tricin, tricin 4-O-(erythro--guaiacylglyceryl) ether and tricin 4 O-(threo--guaiacylglyceryl) ether. The DPPH scavenging assay of the isolated compounds was also carried out and Table 1 shows the EC50 values. For comparison, a known antioxidant flavonoid quercetin was used as reference. Of the three compounds, compound 1 was more efficient, followed by compound 3> compound 2. The diethyl ether extract of NB, SJ and PM were analyzed by HPLC. Figure 1 shows the HPLC-PDA chromatograms of NB, SJ and PM with corresponding ultra violet-visible (UVVIS) spectra. Quantification of each compound in the diethyl ether extract was carried out using pure tricin and tricin 4-O-(erythro-guaiacylglyceryl) ether. The peaks corresponding to the compounds 1, 2 and 3 were quantified using tricin and tricin 4-O-(erythro--guaiacylglyceryl) ether as standards and their solutions were prepared in acetonitrile in the concentration range of 2, 3, 4, 5, 6, 7 and 8 g/20 l and run by HPLC. The standard solutions exhibited linear relationship between area response and concentration of sample injected, with the correlation coefficient (R2) of 0.9976 and 0.9997, respectively. The detection limit for tricin was 0.47 g/20 l and % recovery was between 99.05 and 100.37, whereas for tricin 4-O-(erythro--guaiacylglyceryl) ether, detection limit was 0.23 g/20 l and % recovery was between 99.12 and 100.02. The identification of compound peaks in the experimental samples was based on the congruence of retention times and UVVIS spectra with those of pure compounds in literature [13]. Compounds 2 and 3 were checked for their absorption characteristics and found comparable. Hence, compound 2 which was available

Fig. 1 HPLC-PDA chromatographic profiles of compounds: tricin (1), tricin-4-O-(erythro--guaiacylglyceryl) ether (2) and tricin-4-O(threo--guaiacylglyceryl) ether (3) with corresponding UVVIS spectra of diethyl ether extracts of rice bran, at 330 nm in a) Njavara Black (NB) b) Sujatha (SJ) and c) Palakkadan Matta (PM)

in more quantity was chosen for quantification purpose. Table 1 shows the quantification of the compounds 1, 2 and 3 in NB, PM and SJ rice bran. Tricin is present 39.64 and 16.12 fold higher in NB, compared to SJ and PM,

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respectively. HPLC profiles of SJ and PM are magnified in Fig. 1 to facilitate locating the peaks of 2 and 3. Anti-inflammatory Activity The methanolic extract and diethyl ether fraction of NB showed anti-inflammatory effect of about 80% at 5 mg/kg dose. Hence, the compounds isolated were evaluated for this biological effect. The anti-inflammatory activity of compounds 1, 2 and 3 against acute edema (induced by carrageenan) is shown in Table 2 and the results are comparable to that of the standard drug diclofenac. At 3rd h, compounds 1 and 3 showed 50% edema inhibition whereas compound 2 showed only 20%. Of the three compounds, compound 1 inhibited edema formation maximum, to an extent of 70%, followed by compound 3 with 66.6% and 2 with 44.4% at 5th h. Compared to standard drug diclofenac, the three compounds showed better inhibition at lower concentration. Carrageenan-induced local inflammation (paw edema or pleurisy) is a commonly used method to evaluate and compare the efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) and also in determining the role of mediators involved in vascular changes associated with acute inflammation [14]. The initial phase of inflammation (edema, 01 h) has been attributed to the release of histamine, 5-hydroxytryptamine and bradykinin, followed by a late phase (16 h) mainly sustained by prostaglandinrelease, attributed to the induction of cyclooxygenase-2 in the tissue [15]. In the present study, all the compounds showed edema inhibition as compared to diclofenac. The edema inhibition by compound 1 and 3 was maximum and it was more pronounced at the second phase, suggesting its inhibitory effect on prostaglandin production as a major mechanism by which compounds exert anti-inflammatory effect. Moscatelli et al. [16] have studied the in vitro antiinflammatory activity of tricin, based on prostaglandin E2 levels, cyclooxygenase (COX) and phospholipase activity. Chang et al. [17] mention about the anti-inflammatory effect of salcolin B (identical with tricin 4-O-(erythro-guaiacylglyceryl) ether) only, based on inhibition of nitric oxide (NO) in a cell line of mouse macrophages (RAW

264.7). Our study gives a comparison of tricin and the two flavonolignans in carrageenan-induced rat paw edema model (in vivo). Tricin is previously reported in rice bran but the rare flavonolignans (2 and 3) are first time identified and quantified in Njavara and in the Oryza sativa species [11]. Compounds 2 and 3 are found to be stereoisomers due to the presence of two adjacent chiral centers. These compounds were originally reported in the aerial parts of Salsola collina (Chenopodiaceae) and also in Avena sativa L. of the Poaceae (Graminae) family [13, 18]. Tricin is widely distributed in Gramineae plants. A recent review by Zhou & Ibrahim [19] on tricin highlights the potential of the compound as a multifunctional nutraceutical. In this review, the beneficial health effects of tricin such as antioxidant effect, inhibition of lipid peroxidation, sparing effect on vitamin E in erythrocyte membrane, antiviral, immunomodulatory, antitubercular, antiulcerogenic, antimutagenic, mildly estrogenic, anti-inflammtory and potent anticancer effects are cited. Hudson et al. [11] have proven tricin as a potential chemopreventive constituent of rice bran and their preliminary (in vitro) study showed it to interfere potently with the survival of human derived breast and colon cancer cells. Later, Verschoyle et al. [20] suggested tricin may be considered safe enough for clinical development as a cancer chemopreventive agent. Our studies show that Njavara bran contains tricin, in significantly higher concentration compared to staple rice varieties, which give scientific support to its medicinal use. Njavara is generally used as brown rice, retaining about 80% of bran on it. We can see that Compounds 2 and 3 are present only in minute quantities in SJ compared to NB (Table 1). In PM, the peaks 2 and 3 (Fig. 1c) showed max at 328 nm which is different from the max of 338 nm of the peaks 2 and 3 (Fig. 1a) representing compounds 2 and 3 of NB. Hence, the compounds 2 and 3 are absent in PM. The peaks with max at 328 nm in the HPLC of PM were not further investigated owing to their very low concentration. The diethyl ether fraction of the methanolic extract of NB is having greater radical scavenging activity and the quantification of the compounds 1, 2 and 3 in the extracts showed that they are at higher concentration in NB. The higher

Table 2 Anti-inflammatory effect of tricin (compound 1), tricin 4-O-(erythro--guaiacylglyceryl) ether (compound 2), tricin 4 O-(threo-guaiacylglyceryl) ether (compound 3) and diclofenac on carrageenan induced paw edema in rats Time Inhibition (%) of different treatment groups (dose 2 mg/kg) Compound 1 3rd h 5th h
ac

Drug diclofenac (dose 20 mg/kg) Compound 3 50.01.44b 66.61.91

b

Compound 2 20.00.57c 44.41.27

c

50.01.44b 70.02.02
b

72.02.08a 86.02.48a

Meanstandard error mean of six determinations followed by different letters in a row are significantly different in the Duncans test at P<0.05

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Plant Foods Hum Nutr (2011) 66:9196 5. Deepa G, Venkatachalam L, Bhagyalakshmi N, Shashidhar HE, Singh V, Naidu KA (2009) Physicochemical and genetic analysis of an endemic rice variety, Njavara (Oryza sativa L.), in comparison to two popular south Indian cultivars, Jyothi (PTB 39) and IR 64. J Agric Food Chem 57:1147611483 6. Deepa G, Singh V, Naidu KA (2008) Nutrient composition and physicochemical properties of Indian medicinal rice-Njavara. Food Chem 106:165171 7. Simi CK, Abraham TE (2008) Physicochemical rheological and thermal properties of Njavara rice (Oryza sativa) starch. J Agric Food Chem 56:1210512113 8. Sanchez-Moreno C, Larrauri JA, Sauro-Calixto F (1998) A procedure to measure antiradical efficiency of polyphenols. J Sci Food Agric 76:270276 9. Ghosh MN, Singh H (1974) Inhibitory effect of a pyralizidine alkaloid, crotalaburnine, on rat paw oedema and cotton pellet granuloma. Br J Pharmacol 51:503508 10. Winter CA, Risley EA, Nuss GV (1962) Carrageenin-induced edema in hind paw of the rat as an assay for anti-inflammatory drugs. Proc Soc Exp Biol Med 111:544547 11. Hudson EA, Dinh PA, Kokubun T, Simmonds MSJ, Gescher A (2000) Characterization of potentially chemopreventive phenols in extracts of brown rice that inhibit the growth of human breast and colon cancer cells. Cancer Epidemiol Biomark Prev 9:11631170 12. Bouaziz M, Veitch NC, Grayer RJ, Simmonds MSJ, Damak M (2002) Flavonolignans from Hyparrhenia hirta. Phytochemistry 60:515520 13. Wenzig E, Kunert O, Ferreira D, Schimid M, Schuhly W, Bauer R, Hiermann A (2005) Flavonolignans from Avena sativa. J Nat Prod 68:289292 14. Cuzzocrea S, Nocentini G, Di Paola R, Agostini M, Mazzon E, Ronchetti S, Crisafulli C, Esposito E, Caputi AP, Riccardi C (2006) Proinflammatory role of glucocorticoid-induced TNF receptor-related gene in acute lung inflammation. J Immunol 177:631641 15. Mazzon E, Esposito E, Di Paola R, Muia C, Crisafulli C, Genovese T, Caminiti R, Meli R, Bramanti P, Cuzzocrea S (2008) Effect of tumour necrosis factor-alpha receptor 1 genetic deletion on carrageenan-induced acute inflammation: A comparison with etanercept. Clin Exp Immunol 153:136149 16. Moscatelli V, Hnatyszyn O, Acevedo C, Megias J, Alcaraz MJ, Ferraro G (2006) Flavanoids from Artemisia copa with antiinflamatory activity. Planta Med 72:7274 17. Chang C, Zhang L, Chen RY, Kuo LY, Huang J, Huang H, Lee K, Wu Y, Kuo Y (2010) Antioxidant and anti-inflammatory phenylpropanoid derivatives from Calamus quiquesetinervius. J Nat Prod 73:14821488 18. Syrchina AI, Gorshkov AG, Shcherbakov VV, Zinchenko SV, Vereshchagin AL, Zaikov KL, Semenov AA (1992) Flavonolignans of Salsola collina. Khim Prir Soed 2:182186, Engl. Trans., Chem Nat Compd 28:155158 19. Zhou J, Ibrahim RK (2010) Tricin-a potential multifunctional nutraceutical. Phytochem Rev 9:413424 20. Verschoyle RD, Greaves P, Cai H, Borkhardt BM, DIncalci M, Riccio E, Doppalapudi R, Kapetanovic IM, Steward WP, Gescher AJ (2006) Preliminary safety evaluation of the putative cancer chemopreventive agent tricin, a naturally occurring flavone. Cancer Chemother Pharmacol 57:16

activity of diethyl ether extract of NB can be attributed to the higher concentration of these compounds compared to SJ and PM. Hence, the anti-inflammatory property of Njavara can be correlated with the presence of the three compounds in higher quantity, especially tricin, in Njavara compared to staple varieties.

Conclusions In summary, the study establishes the isolation of three compounds tricin (compound 1), tricin 4-O-(erythro-guaiacylglyceryl) ether (compound 2) and tricin 4-O(threo--guaiacylglyceryl) ether (compound 3) in Njavara bran and the presence of rare flavonolignans (compounds 2 and 3) in rice bran for the first time. The in vivo study confirmed and compared the anti-inflammatory action of compounds 1, 2 and 3. The bioactive compound tricin, a promising multifunctional nutraceutical, is present in higher concentration in Njavara compared to the staple varieties. The rare flavonolignans, which are antioxidant and antiinflammatory, also occur in higher quantity in Njavara compared to staple varieties. These findings corroborate with the preferential use of Njavara in Ayurveda, over staple varieties.
Acknowledgements The authors are grateful for the funding assistance provided by Kerala State Council for Science, Technology and Environment (KSCSTE), Government of Kerala. The author Smitha Mohanlal wishes to thank Council of Scientific and Industrial Research (CSIR), India for the financial support as Senior Research Fellowship (SRF). Thanks are also due to Director, NIIST, CSIR and Head, Department of Biochemistry for constant encouragement and support.

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