Indian Journal of Experimental Biology Vol. 41, February 2003, pp. 135- 140

Enzymatic and non-enzymatic antioxidants in selected Piper species

J Karthikeyan

Department of Biochemistry, PSG College of Arts and Science, Coimbatore 641 004, Ind ia

and

P R a ni *

Department of Biotechnology, PSG College of Technology, Coimbatore 641014, Indi a

Received 30 April 2002; revised 9 Decelnber 2002

Piper species, commonly used in diet and traditional medicine were asse~sed for their antioxidant potential. Cata lase activity was predominated in Piper langum, followed by Piper cllbeba, green pepper, Piper brachystachywll and Piper ni· grum. P. nigrum was richest in g lutathione peroxidase and glucosc-6-phosphate dehydrogenase, green pepper was ri chest in peroxidase and vitamin C while vi tami n E was more in P. lal/gum and P. nigrum. P. brachystachyum and P. longUlII were rich sources of vita min A. All the Piper species had GSH content of around I to 2 nM/g ti ssue. The antioxidant components of Piper species constitute a very efficient system in scavenging a wide variety of reactive oxygen species. Antiox idant po­tential of Piper species was further confirmed by their ability to curtail ill vitro lipid peroxidation by around 30-50% with concomitant increase in GSH content.

Reactive Oxygen Species (ROS) are generated from leakage of electrons onto oxygen from mitochondrial electron transport chain, microsomal cytochrome P450

and their electron donating enzymes and other sys-I ? • -

tems '-. For useful purposes, ROS (e.g., O2 , HOCl , and H20 2) are produced from activated phagocytes3

.

Inactivation and removal of ROS depend on reactions involving the antioxidative defense system. The en­dogenous antioxidant defense includes enzymatic (e.g. superoxide dismutase, catalase, peroxidase etc.) and non-enzymatic (e.g., ascorbic acid, a-tocopherol, glutathione etc.) systems4

.

Oxidative stress is a state of imbalance between generation of reactive oxygen species (ROS) like hy­droxyl and superoxide radicals, and the level of anti­oxidant defense system. Oxidative stress results in the damage of biopolymers including nucleic acids, pro­teins, polyunsaturated fatty acids and carbohydrates. Lipid peroxidation (LP) is ox ida~ive deterioration of polyunsaturated lipids and it involves ROS and transi­tion metal ions5

. It is a molecular mechanism of cell injury leading to generation of peroxides and lipid hydroperoxides which can decompose to yield a wide range of cytotoxic products, most of which are alde-

*Correspondent author: Email: ranirangaraja@yahoo.co.uk

psgtechbio@yahoo.com Phone: 91-0422-2572177 Fax: 91-0422-2573833

hydes, like malondialdehyde (MDA), 4-hydroxynonenal (HNE) etc.

ROS and free radical mediated processes have also been implicated in the pathogenesis of a variety of diseases like Alzheimer' s disease, Parkinson 's di s­ease, atherosclerosis , cancer, liver damage, rheuma­toid arthritis, immunological incompetence, neurode­generative disorders6

.7 etc. Nutritional antioxidant de­

ficiency also leads to oxidative stress8, which signifies

the identification of natural antioxidative agents pre­sent in diets consumed by the human population . Pep­per is widely incorporated in the diet of Asian and Western countries and it is also an important constitu­ent of more than 150 Ayurvedic formulation s. Piper­ine, a well identified alkaloid of Piper species, is re­ported to have a antimicrobial and hypoglycemic ef­fects9

. While Piper species are reported to have wide spectrum of biological acti vity, its antioxidant poten­tial has not been established so far, making it impor­tant and interesting to study the antioxidant potenti al of Piper species.

The main objectives of the present study is to: (l ) establish the antioxidant potential of the Piper species by evaluating both enzymatic and non-enzymatic an­tioxidants in selected Piper species; and (2) study the efficacy of Piper species in preventing in vitro lipid peroxidation using liver mitochondria as a model system.

136 INDIAN J EXP BIOl, FEBRUARY 2003

Materials and Methods Preparation of the pepper eXlract--The Piper spe·

cies- Piper nigrum, Piper cubeha, Piper longum and Pipa brachyslachyum were obtained in the dried ,"orm from the ayurvedic drug shops. Green pepper, a Ileshy form of P. nigrum was obtained freshly from the local vegetable market, and stored under refriger­ated condition. The pepper species were weighed separately antI homogenised with 50% ethanol at a concelltration of I g in 2mL The extracts were centri ­fuged at 10,000 rpm for 10 min and the supernatants were kept under refrigerated condition and used for biochemical estimations within 4 hL

Biochemical eSlimations - The activities of the an­tiox idant enzymes namely superoxide dismutase lO

,

1 II ·d 12 I h· .. 13 I cala ase ,peroxl ase ,g utat lOne peroxluase ,g u-cose-6-phosphate dehydrogenase 14, ascorbate ox i­dase ls

, and the non enzymatic antioxidants viz., glu­ta th ione l6

, ascorb ic acid 17, vitamin 1\1 7 and EI 7 were

determined in the pepper extracts by suitable colori ­metric procedures. The values are expressed as mean ± SO. The data were statistically analysed by the method of Snedecor and Cochran 18.

Isolalion of goat liver mitochondria-The goat li ve r mitochondria was prepared according to the method of Li et al l 9

. Lipid peroxidation level in the mitochondria was measured as thiobarbituric acid re­ac ti ve substances (TBARS) and diene conjugates, using the method of Buege and Aust2o. In vitro lipid peroxidation was induced by the addit ion of 1.5mM FeS04.

Results and Discussion The energetic benefit of aerobic metabolism is as­

sociated with the generation of reactive oxygen spe­cies which are implicated in variety of diseased cOl1di-

. 21 D· . I b h lions. Jet contains severa su stances t at are capa-ble of scavenging ROS directly or indirectly by pro­moting mechanism which enhance detoxification22

.

Strong evidence sugges ts that consumption of fruits and vegetables results in decreased incidence of all types of cancer23. They are known to contain varie ty of non-enzymatic antioxidants, namely carotenoids, tocopherols, ascorbic acid and plant polyphenols, which exert their antimutagenic activity , even after subjected to the cook ing process. Pepper whi ch is widely included in the diet is evaluated for its anti ox i­dant property in the present study.

Enzymatic antioxidants in Piper species - The level of antioxidant enzymes assessed in different Piper species are collectively represented in Table I . The highest SOD activity was found in P.longum (23.79 units/mg protein) compared to the other spe­cies included in the present study implicating that this source could be exploited for commercia l purification of SOD. SOD's are reported widely in plant sources and superoxide scavenging effect of fresh juice and methanolic extract of Emilia sonchiJofia leaves was reported24. Alcoholic extract of Hypericum petfora­tum also showed reasonable superoxide anion scav­engi ng activitl5

. Currelltly, purified SOD is therapeu­tically used in the treatment of antioxidative and anti ­inflammatory diseases.

Table I - Enzymatic anti uxidants in Piper species

[Values are means of 3 replic3tes in 2 repetitive experiments]

Sample SOD Catalase Peroxidase Glutathione Glucose-6-P A eorbate oxidase peroxidase dehydrogenase

Unils/Illg Unils/g Units/Illg Uni ts/g Un its/mg Unils/g Units/mg Units/g Units/mg Units/g Units/mg Units/g protein ti ss l!e protein lissue prolein tissue protein tissue protein tissue protein lissue

PI 9.14c 9.96 3.72" 4.05 3 1.57" 34.40 1772.2c 1931.6 54.45c 59.30 0.01 4" 0.015

P2 3.49" 19.90 22.30b 128.00 0.99" 5.66 281.9" 161~ .. 5 17.6 1c 100.70 0.056c 0.320

P3 23.79c 29.73 83.19c 103.90 1.80b 2.25 94Ll c 1176.4 6.07· 7.58 0.123" 0. 154

P4 4.67d 19.90 48.39d 82.70 7.49c 12.80 828.7b 1417.1 12.2 1b 20.80 0.032b 0.054

P5 5.65b 10.00 39.72c 70.30 75.61c 133.80 1285.7" 2275.7 24.40c 43.10 0.057c 0.100

SED = 0.05 SED = 0. 19 SED = 1.94 SED = 0.00 19 SED = 0.24 SED = 0.28

LSD(5%) = 0.10 LSD(5%) '" 0.39 LSD(5 %) = 4 LSD(5%) = 0.004 LSD(5 %) = 0.50 LSD(5%) = 0.57

LSD( I%) = 0. 13 LSD( I%) =0.53 LSD( I%) =5.4 1 LSD(I %)=O.OO6 LSD( I %) ~ 0.68 LSD(I %) = 0.77

I Un it=inhibitiun I Unit'" l).lmoleof 1 Unit = l).lmoleof I Unit= l).lgofGSH I Unit=increasein I Unil=O.OI O.D. of 50% nitrite 1-1 20 2 consumed/min pyrogallol oxi- utilized/min O.D. of 0.0 I/min change/min

formation dized/min

In a column , means fo llowed by a common leIter are not sign ificantly different at 5% level by DMRT. PI: Plj~er Iligrum; P2 : Piper bruchyslachYIIn!: P3: Piper Icngum; P4: Piper cubeba; P5: Green pepper

KARTHIKEYAN & RANI: EVALUATION OF ANTIOXIDANTS IN SEL ECTED PIPER SPECIES 137

The highest actIvity of catalase observed in P. longum (83units/mg protein) coincides very well with the highest activity of SOD noted in the same species, indicating that the H20 2 formed by SOD is effecti vely removed by the catalase. Plant catalases are reported to be very sensiti ve to environmental conditions and has a rapid turnover rate26.

Among the five species studied P. nigrum showed highest GSH-Px activity (1772 ullits!mg protein) and it is interesting to note that the highest GSH-Px activ­ity observed in P. nigrum was associated with very low catalase acti vity observed emphasizing that GSH­Px activity pl ays the major role in peroxide removal in thi s pepper species, whereas, the highest catalase activity in P. longum was associated with low activity of GSH-Px indicating the role fo r catalase in peroxide removal in P. longum. In green pepper and P. cllbeba, both catalase and GSH-Px acti vity were reasonable, ind icating a role for both the enzymes in peroxide removal. The presence of GS H-Px was a lso reported in cultured plant cells27

. GSH-Px iso la ted from Nico­tiana SylvesTrii8 has shown homology to anim;]1 GSJI-Px .

The ascorbate ox idase(AO) activity in P. [ongum was 0.123 uni ls/g ti ssue \vhich could be related with the highest SOD acti vity noted in thi s species. Di ffer­ent for ms of AO has bee n reporled in chloroplast, mi­tochondrial , cy tosol , perox isomcs and glyoxysomes~9 . In the present study, the highest AO activi ty in P. IO'1gUln emphasizes the role of ascorbate syste m in cont ro lling H20 2 concentration in thi s species. Green pepper was fo und to contain highest peroxidase acti v­ity (75 units/mg protein). Th is observation po ints OUl.

the importance of perox idase system in fresh tissue rather than in dried fru its.

T he observed high activity of gl ucose-6-phosphatc dehydrogenase in P. nigrum and green pepper coin­ciJes very well with high GSH-Px activities. Wh ile GSH-Px is invol ved in the reduction of H20 l. and or­gan ic peroxide, a continuous flow of reducing equ iva­lents through glutathione system necessarily has to be balanced by continuous format ion of NADPH main­t;lining the steady state. It is established by increased g iucose· 6-phosphale-dehydrogenase activity noted in the above species.

Non-enzymaTic anti-ox idants in Piper species­Apart from enzymatic antioxidants, spectrum of non­enzymatic antioxidants namely vitamin A, C, E and g lutathione are important in cellular system in curtail­ing ROS . The levels of these antioxidants were as­se~sed and the results are represented in Table 2.

The vitamin C level was lowest in P. brachystachyu!1I

(0.88 ~!g/g tissue) while highest le vel of 2.7 /lg/g ti s­sue was observed in P. longwl1 . Ascorb ic ac id is re­ported to be associated with better sca venging aC li \'i­ties in vivo than the antioxidant enzymes, because they are present both intracellul arly as well as ill the extracellular f1uid 30

. As an antioxidant, it is reported that ascorbate reacts with sllperoxide, hydrogen per­oxide or the tocopheroxyl rad ica l to form monodehy­droascorbic acid andlor didehydroascorbi c acid . The oxidi sed fo rms are recycled back to ascorbic ac id.

The estimated vitamin E Icvd in different Piper

species ranges wide ly from 18 to 66 JJ~g/g tissue (Table 2). The antioxidant properties of tocopherol are result of its ability to quench both sing let ox ygen and peroxi des31

• Within the membrane, tocophero l is the only protective agent ihat can act against the tox ic effects o f oxygen radical s32

The vitamin A content in d ifferent Piper species ranges form 16 to 70 /lg/g ti ssue. Piper bracllystacllYl// 1/ had the highest level. whereas lowest level was ob­served in P. lungum . Caroteno id s exhibit a central role against cancers, card iovascular d isease, HI V 111 -

fection and other age-related disorders33. Recent re­

ports suggests that a ll the caroteno ids except r3-carotene are very effici ent antiox idants.

T he glu tath ione val ues in Piper spec ies varied fro Ill 0.92 to 1.8 nM/g ti ssue . G iutathione reduced the for­mation of toxic lipid peroxide and hydrogen perox ide in biological system by acting as substrate for g lu­tathione peroxidase34

• GSH can fu nction as an anl i­ox idant in the fo llowing ways:

o It can react chemically with sing let oxygen, supcr­oxide and hydroxy l rad ica ls and therefore functi on directly as a free radical cave nger.

• GSH may stabil ize membrane structure by reIllOV­ing acyl peroxides fo rmeJ by lipid peroxidation 1'1':­

actionsJ) .

~ GSI-I is the reduc ing agent that recycles ascorb ic acid from its oxidized to its reduced form by the enzyme dehydroascorbate reductase29

.

Upon comparison of antioxidant status of green pepper (fleshy form) and P.nigrum (dried for m) the following results were observed :

• No significant change in Iota; activity was ob­served for SOD and glucose-6-phosphate dehydro­genase , whereas specific activity increased by 1.6 fold in dried form .

• Both specific acti vity and total activity of catalase. peroxidase and ascorbate oxidase decreased in dried

138 INDIAN J EXP BIOL, FEB RUARY 2003

form. Only 10-25% of ac tIVIty was observed in dried form when compared to fleshy form.

• The flesh form of pepper is more predominant in vitami n C and GSH while dried form is enriched with vitami n C and E. The above observed variation in antioxidant status

could be attributed to different maturation stages of pepper fruit and also could be due to sun ~drying proc­ess.

Evaillation oj efficacy oj antioxidant potential oj Piper species - Peroxidation of membrane system are the foremost consequences of free radical damage and the efficiency of plant extracts in inhibiting lipid perox idation in vitro is a very good measure of as­sessment of antioxidant potential. The presence of iron in the reduced form was found to be necessary

for lipid peroxidation and this was generall y achieved by the addition of ferrous salts or by adding Fe3

+ and reducing agents like ascorbate26

.36

.

To evaluate the antioxidant potential of different Piper extracts, liver mitochondria was selected as model system based on the following considerations: I . It has been well established that isolated mitochon­

dria produces H20 2 and O2 • - in presence of com­pounds like NADH , anti mycin A, etc37

.

2. Various Fe-S proteins and NADH dehydrogenase have also been implicated as possible sites of su­peroxide and H20 2 formation.

3 . Mitochondrial membrane was found to contain large amounts of PUFA in their phospholipid which include fatty acids with 2,4 ,5, and 6 double bonds. The rate of peroxidation both in vivo and in

Table 2 - Non-enzymatic antiox idants in Piper species

rValues are means of 3 replicates in 2 repetitive experiments]

Sample Vitamin C Vitamin E Vitamin A GSH (!1g/g ti ssue) (!1g/g ti ssue) (!1g!g ti ssue) (nMIgti ssue)

PI 1.68c 66.98c 43.24c 0.92" P2 0.88" 46.30c 69.46c 1.03b

P3 1.72e 63 .62d 63.64c l.l8c

P4 1.37b 18.1 2" 30.93b 1.27d

P5 2.70d 38.95b 16.81 " 1.84c

SED = 0.05 SED = 0.39 SED = 0.27 SED =0.Or)4 LSD(5%) =0.1 I LSD(5 %) = 0.81 LSD(5 %) = 0.56 LSD(5 %) = 0.01 LSD(I %) = 0.14 LSD( I%) = 1.09 LSD( I%) = 0.76 LSD( I%) = 0.02

In a column, means followed by a common letter arc not significantly different at 5% level by DMRT. PI : Piper nigrulIl; P2 : Piper brachystachyum; P3: Piper longum; P4: Piper cubeba; P5: Green pepper

Table 3 - Effect of pepper extracts on Lipid peroxidation and reduced GSH content in the goat li ver mitoc hondria

I Values are means of 3 replicates in 2 repetitive experi ments. Figures in parentheses are % inhibition of lipid perox idati on and thiol oxi­dation by the different pepper extracts]

Experimental group

Group I Liver mitochondria

Group II Liver mitochondria + 1.5 mM FeS04

Group III Li ver mitochondria + 1.5 mM FeS04 + pepper extract Piper Iligrum Piper brachystacliyulIl Piper /ongwlI Piper cllbt:ba

Green pepper

TBARS" (!1Mlmg protein)

0.25 ± 0.001

2.09± 0.0 13

1.480 ± 0.002 (29%) 1.235 ± 0.002 (4 1%) 1.700 ± 0.060 (50%) 0.860 ± 0.002 (59%) 1.0 10 ± 0.040 (50%)

Conjugated dienes* (!1mole/mg protein)

57.725 ± 0.476

3 18. 16±0.354

12.4.18± 0.274 (6 1%) 125.23 ±0.2 16 (60%) 149.42±0.346 (50%) 188.17 ± 0.460 (40%) 157.36± 0.302 (50%)

* The levels of TBARS and conjugated dienes were compared between Group II and Group III ** GSH level was compared between Group I and Group III

GSH"" (!1M1mg prote in)

6.42± 0.32

3. 17± 0.10

5.27±0.26 (66%) 5.24± 0.29 (65 %) 4.R7± 0.27 (54%) 5.01 ± 0. 19 (58%) 4.93± 0. 15 (55 %)

KARTHIK EYAN & RANI: EVALUATION OF ANT IOXIDANTS IN SELECTED PIPER SPECIES 139

vitro increase with increasing number of double bonds in fatty acids 37.

4. Damage to mitochondri a due to lipid peroxidation can have profound effect on the cell. Lipid perox i­dation correlated weil with swelling and finally with lysis and di sintegrati on of mitochondria'7.

Lipid peroxidation was induced in the model sys-tem by incubating the liver mitochondria in the pres­ence of 1.5mM FeS0 4 for 30 min with and without different Piper extracts. In earlier studies for an­tilipidperoxidative property , the EDso value of 58 )..lg was reported for vitamin E2S

• Hence in the present study, the ethanolic extracts of Piper species were concen.trated so as to give around 60)..lg of vitamin E in 0.5ml of the concentrated extract.

Effect of Piper extracts on lipid peroxidation of liver mitochondria- The effect of ethanolic extracts of different Piper species on in vitro lipid peroxida­tion of liver mitochondria was assessed by estimating both TSARS and diene conjugates. The results were represented in Table 3.

Piper cubeba was found to be more effective in curtailing lipid peroxidation by 59%, while the extent of inhibition was around 50% for P. longum and green pepper and around 41 % for P. brachystachyum. P. nigrum was found to be less effective with only 29% inhibition. The observed high levels of TSARS in the model system in the presence of P. nigrum ex­tract would be contributed by the inherent piperine content of the pepper extract. Khajuria et al. 38 also reported increased TSARS of rat intestinal mucosa and epithel ial cells upon piperine treatment although conjugated diene levels were not altered.

The conjugated diene (initi al peroxidative product and accurate indicator of lipid peroxidation) levels were also assessed in the present study. The extent of inhibition of lipid peroxidation exerted by different pepper extracts were ranged from 40-60%, the lowest being contributed by P. cubeba and highest by P. ni­grum and P. brachystachyul1l. The lowest anioxidant efficacy of P. cubeba is correlated with the lowest level of vitamin E (18.12)..lg/g tissue) in this species, compared to that of vitamin E in P. nigrum (66.98 )..lg/g tissue).

The inhibition of in vitro lipid peroxidation by the pepper extracts observed in the present study can be attributed to the presence of known antioxidants like vitamin C, E, A and glutathione, and also the various enzymic antioxidants in the extracts. The observed effects could also be due to the presence of other

secondary metabolites in Piper spec ies. The piperine content has been reported to vary from 1.9 to 3. 1 % (moisture free base), depending on fruit maturation stages. It is ill teresting to note that piperine, a princi­ple alkaloid of Piper species increased the serum re­sponse i.e., the serum ~-carotene level when it is sup­plemented with oral ~-carotene39 .

Effect of Pepper extracts on GSH COli tent du ring lipid peroxidation- In the li ver mitochondri a, upon the induction of lipid perox idation with Fe2

+, GSH level decreased signi ficantly by 50%, whereas when mitochondri a was preincubated with pepper extracts, only around 18-25% depl etion was seen. It is interest­ing to note that, even in the presence of promoters of lipid peroxidation, different pepper extracts protected the GSH depletion by 20%, when compared to that in their absence.

It has been reported that reduced GSH is a better index of assessing the lipid peroxidati on than the quantificaiton of TBARS4o. An inverse relationship was establi shed between TSARS and reduced GSH content. The protective effect of pepper extracts on lipid peroxidation with concomitant increase in GSH noted in the present study could be attributed to the inbuilt antioxidant system present in them. The results clearly pinpoints the antioxidant potential of diffe rent pepper species. In vivo studies should be carried out in the experimental animals in order to ascertain their antioxidant potential in biological system.

References I Beal M F, Oxidative damage in ncurodegenerative di scases,

Neurosciel1list,3 (1997) 2 1. 2 Hansford R G, Hoguc B A & Mildaziene V. Dcpendence of

H20 2 formation by rat heart milochondria on substrate ava il ­ability and donor age, J Bioenerg Biomemb, 29 ( 1997) 89.

3 Prakash p. Kumar G P, La!oraya M. Hemnani T & Pari harm M S, Superoxide aniol] radical generation as a temperalU re stress response in the gill s of frcsh water catfi sh Hel­eroplleusles!ossilis: Role in mucus exudation under elevated tcmpcralUre, Comp Biochem Physiol, I 19 ( 1998) 2 1 I.

4 Chatterjce I B, Proc Illdiall Natll Sci Acad. 1364 ( 1998) 2 13. 5 Khajuri a A, Lipid perox idation , Everyman 's Sci, 32 ( 1997),

109. 6 Wiseman H & Halliwell B, Damagc to DNA by reacti ve

oxygen and nit rogen species: Role in inO ammatory disease and progression to cancer, Biochem J, 3 13 ( 1996) 17.

7 Adams J D (Jr), in Burger'S medicinal chemistry and drug discol'ery, 51h ed ition, vo l. 3, eJited by M E Wolff (John Wiley and Sons, New York) 1996, 26 1.

3 Gutleridge J M & Halliwell B, Alllioxidallts ill lIulriliol1. heallh alld disease (Oxford University press, Oxford), 1994.

9 Dhar M L. Dhawan B N. Prasad C R, Ras togi R P, Singh K K& Tandon J S, Screening of Indian plants fo r biological ac­ti vity, Pan V, Illdiall J Ex!, Bioi, 12 ( 1974) 512.

140 INDIAN J EXP BIOL. FEBRUARY 2003

!O Das K, Samnnta L & Chai ny G B N, A modifi~d spectropho­tOmetric assay of superoxide di~mutase using nit rite fOrlm.­tion by superoxide radi ca ls, Illdiall J Bior:hem Biophys, 37 (2000) 201.

II Si nha A K. Colorimetric assay of cata lase. 111101 Biochelll. 47 (J9"/2) 389.

!2 Addy S K & Gooclll1al~ R N, Pol yphcnoi oxidase and perox i­dase in 'ipplc leaves inocu latcd with a virule nt or an avirulent str<lin for F:nt'inirl alll\'lI lo\'Ora, Illdiall PhrlOpmhol. 25 (1972) 575 .

13 Rotrud. J T, Pope A L Gallther I{ [, Swanson A B. Hafe­man D G & I ioekstra W G. Selcnium: l3im:ht: mical role as a component 01' glutathione p~!'Ox idast" Sciellce, 179 ( 1973) 588.

14 Ballnsky D & Be,nstein R E. The purification and propcrties of Glueose-()-Phosphatc dchydrogenase rrom human eryt hro­cytes, Hinchell! f3iuphys Ac/(/ G7( 196:<) 311 .

15 Obcrbaehel M F & Vine:; H M . Respon ~c f)f oxidation & phosph')ryla ti on in citrus !nitochondl'ia to arsenate. ,""({I II re. 206 (1%5) W).

16 Tietze I:. Enzymatic mcthod for quantitativ(, dcterm;. lati on of nal10gr:lm alllOUIllS of total and oxidizcd glutath ione: Appli ­cation !o r.lammalian blood and other tissues. AII({I Biochelll . 27 ( 190') 502 ,

17 Varley H, Goweniock A 1\ &. Bell M 1;1 Pmc!ical ciilliwl biochelll i.lt ry, 5'" edition, Volul11e 2 (Willi ,!lll Heinemann Medical Books Ltd., London) 1984, 215.

I R Snedecor G W & Cochran W G, S/(I l islicolll,clhods (Oxf0rd & IBH Publi shing Co Pvt Ltd, New Delhi ) 196R. 301.

19 Li H L, Moreno-S: anehez R, & Rott('nberg H. Alcohu! inhib­its the activation of NAD- li nked dehydrogenasr.s by ca lciu 111

in brain & heart mitochondria, Bio('hilll Biophys ;\('/(1. 1236 (1995) 306.

20 Buege J A & Aust S D. Microsomal lipid perox ida tion , Me!llOds EII ;'VIIIOI , 52 ( 1978) 302.

21 Hall iwei l B. in Oxygell radicals alld diseasc pmcess, ed ited by C E T!lomas and 13 Kalyanaraman. (Hard wood Academic Publishers, The Netherland,) 1997, I.

22 Goud V K, Polasa K & Krishn aswa my K, Effect or lLIrmeric on x~n()bi o!ic mc taboliz ing e rr l.y mes, PlolI/ Food HIlII/OII Null', 44 (1993) 214.

D Mathur P, Naturai an tiox idants in our diet , Nutr i lioll, 31 (1997) 10.

24 Shylcsh B A & Pad ikkala H, Anti ox idant and <1ilt i­inilammatory act iv it y of Elllili!: SOllcil i(o/ia, Filolerapia, 7 1 ( 1999) 275.

25 Tripathi Y 13, Pandey E & Dubey G P, An ti ox idant properly of HypericulII pellor{/{ulII (L) of Indian origin and its COI1l -

parison with establi shed Medhya raS(/),alla.l· of ay urvedic medicine, CUlT Sci , 76 ( 1999,27.

26 Hartwig 8 , Sten P & Feierabend J. Lighl dependence of Gita­lase sy nthesis and degrndatic n in leaves and the influence of interfering stress conditions, Plall! Physio l , 100 (1992) 1547.

27 Drotar A, Phelps P & Fall R, Ev idence for g!utathione per­cx idase acti vit ies in cultured plant ce ll s, Plwlf Sci, 42 (J 985) 35.

28 Criq ui M C, Namet E, Parmen tier Y, Marbach .I , Darr A & Flec k J, Iso lation and characteri zat ion of' a plant eDNA show ing homology to ,mind glu tat hi one peroxidases . Phlilf Mol Bioi, I R ( 1902) 623.

29 Loewus F A, A:corh ie ac id and it s l11etJbnlic procIucts. in Biocilf! lII isfly (!fplall!s. Vol 14. ed ited hy J. Preiss (Academic Press, New York ) 1988, 85.

30 Chatteljee I B & Nandi A . Ascorbic aeilt: A scavenger of oxyradi ea ls, Ilidiall J fiiochelll lJiophys, 28 ( 1991) 233 .

31 Frye;' M J, The antioxidant effects of tllylak id Vi tamin E (<x-tocophe rol), Plan! Cell EII\lI/'IIII. 15 ( j 992) 38 i.

32 Suntress Z A & Shek P N, Prevention of lung injury by a.­ttlcopht:rol li posome, J Drug tarflei, 3 ( 1995) 2(): .

33 Gerster H, Potent ia: role of ~-carolr.ne in the prevcntion or cardiovascular di s('ase, ilil . J \fi l Nu!r Res, 6! (1997) 277.

34 Thurnham I D, Functionally important ant iox ida lI t'i and free radical scave nge rs in foods, Food Sci Techllol Today. 6 ( 1994)42.

3S Price A, Lucas P W & Lea P J. Age dependent damage and glutathione me!abolism in owne fumigated barley : A leaf sect ion approaclI, J EX/I BOI, 41 ( 1900) 1309.

36 Selv3m R, Lalitha SlIbramanian , Gayathri R & Angayarkanni N. The antioxid:lnt ac tivity of tLIrIlleric (CII:'C/III !a IOllga). J El /II wplwnliocol . 47 ( 199.'i) 59.

37 Ri eh P R & Bonner W D (1r). The ~ ite~ of supcruxide anion generation in higher plant mi toc hondria, Arch Biochelll Bio­pilys, 188 ( ! 978) 206.

3X Khajuria A, Johri R K & Zut , ili U, Piperine med iated al!e!'a­tions in lipid peroxidation and cePu lar thi ol status or rat in­testinal mucosa and epithe \: ,d ce ll s, Phr!(JIllcdicine , 6:5 ( 1999) 35 I.

39 Badmaev V, Majecd M & Norkus E P, Piperi ne, an alkaloid derived from blaek pepper increases serum response: or beta­carotene during 14-days of oral beta-earotenL supp lementa­tion, Nurrilioll-Res, 19:3 ( 1999) 38 1.

40 Suja S, Latha S, Sh:1fI11il a & Shyarna la Devi C S. Protective elTect of L1V 52 and L1V 100 ay urvedi fo rmulation 011 lipid peroxidat lon in rat liver bQl11ogcnate. An ill vir ro study, ' lidiall J EX[I Bio: , 35 (1997) 50.