Indi an Jo urnal o f Experimental Bio logy Vol. 39. April 2001 , pp. 344-349

Anti-stress activity of Indian Hypericum perforatUin L.

Vikas Kumar" 2, P N Singh' & SK Bhattacharya2* 'Pharmacology Laborntory. Department of Pharmaceutics, Institute o f Techno logy, Banaras Hindu Uni versity,

europhannaco logy Laboratory, Department of Pharmacology, Ins titute o f Medical Sciences. Banaras Hindu University. Varnnasi 22 1 005, India.

Receilw l 8 Scp/ell/ber 2000; revised 27 Decell/ber 2000

Indian HypericulI/ pc/jora/IIIII (IHp) was in vestigated o n a 14-day mild, unpredictable and inescapable foo t shock stress (FSS) induced perturbati ons in be hav io ur (depress ion), suppressed male sex ual behavi our and cogniti ve dy~ func ti o n in a l­bino rats. Gastric ulcerati on, and adrena l g land and spleen we ights, were also used ns the stress indices. P a ll (LI" gillsellg (PG) was lI sed as the standard adaptogenic agent for co mpari son. FSS induced marked gastric ulcerat ion. s ignifi ca nt increase in adrenal g land weight with conco mitant decrease in spleen wcight. Chronic stress also suppressed male sex ual behaviour, in­duced behavioural depress ion (porsolt· s swim despair test and learned he lplessness test) and cogniti ve dys fun ctio n (a ((enu­ated retc ntion of learning in acti vc and pass ive avoidance tes ts). All these FSS induced perturbat ions were ancnuated dose dependently by IIlp (100 and 200 mg/kg, po) and PG (100 mglkg, po). The results ind icate that IHp has significant anti ­stress activity. qualitatively comparnble to PG, against a varie ty o f behavioural and physiological perturbations induced by chronic stress. which has been proposed to be a be tter indicator of clinical stress than acute stress, and may indicate adapto­genic ac ti vity .

HypericulII peljoratl./I/l L (HP) has been used since ancient times in European folklore medicine for its many medicinal properties. Modern usage is st ill quite di verse and includes wound healing, kidney and lung ailments, insomnia and depress ion'. This widely grown plant has been in use by tribals of this country for ailments such as fever, joint pain, general weak­ness, sleep disorders and delayed wound healing. Standardised ext racts of HP are widely used in the treatment of psychovegetative disorders and espe­cially for mild forms of depression in Europe2

. Sev­eral clinical studies have confirmed the antidepressant activity of the extracts derived from HP3A. The extract of Indian Hypericum peljoratulII (lHp) has also shown signi ficant neuropsychopharmacological ac­tions5

.9

, but has not been commercially in thi s coun­try. Since this plant is of great market va lue it be­comes important to investigate its putative neuro­pharmacological properties. In this study, the anti­stress adaptogen ic activity of IHp was investigated in view of its reported anxiolytic, antidepressant and

. .. 5-7 nootroplc actIvIty .

Materials and Methods

Drug treatlllents-The plant (IHp) was collected during August from Company Garden, Sahnranpur, India. A specimen of the plant is preserved with

* AUlhor for correspondence: E-mail: sa lil @banaras.erneLin

Indian Herbs, Sahnranpur. 50% ethanolic extract (yield 26.75% w/w, standardised for 4 .5-5% hyperforin , HPLC) of the dried leaves, flowers and stem of the plant was orally administered as 0.3% carboxymethy l cellulose (CMC) suspension , in the doses of 100 and 200 mg/kg, once daily for 14 consecutive days, 1 hr before the induction of stress. Experiments were conducted on day 14, 1 hI' after the last stress procedure and 2 hr after drug or vehicle administration. Panax ginseng (PG) (B .E. Ltd., India) was used as the standard adaprogenic agent for comparison. Control animals were treated with the vehicle (0.3 % CMC suspension).

Anilllals-Adult inbred Charles Foster albino rats (I50± 109), of either sex, were obtained from the Central Animal House, Institute of Medical Sciences. Banaras Hindu University and were randomly distributed into different experimental groups . Tht animals were housed in groups of six ir polypropylene cages at an ambient temp of 25°C :! 1°C and 45-55% RH, with a 12: 12 h light/dark cycle Animals were provided with commercial food pellet: (Booke Bond-Lipton, India) and water ad libitulII Experiments were conducted between 0900 and 140( hrs. 'Principles of laboratory animal care' (NIl publication number no. 85-23, revised 1985 guidelines werc followed.

Induction of chronic stress-The method ( Armando et al.'o was used. The rats were random I

KUMAR et al.,: HYPERICUM PERFORATUM & ANTI-STRESS ACTIVITY 345

assigned to the unstressed control , stress and drug treated stress groups. Those assigned to the vehicle or drug treated groups were subj ected daily (including Sundays) to I hr of footshock through a grid floor in a standard conditioning chamber with the escape route closed. The duration of each shock (2 mA) and the intervals between the shocks were randomly programmed between 3 and 5 sec and 10 and I 10 sec, respectively in order to make them unpredictable. Animals were sacrificed on day 14, I hr after the last shock procedure on completion of the test procedure involved.

Techniques used for assessment of stress intensity-The following parameters were used to assess the intensity of stress-induced effects:

(a) Gastric ulceration: The stomach was removed and split open along the greater curvature. The numbers of discrete ulcers were noted by the help of a magnifying g lass. The severity of the ulcers was scored after histological confirmation as, O=no ulcers, I=changes limited to superficial layers of the mucosa with no congest ion, 2=half the mucosal thickness showing necrotic changes and congestion, 3=more than two-third of mucosal thickness showi ng necrotic changes and congestion , and 4=complete destructi on of the mucosa with marked haemorrhage. Thereafter, the period ulcer severity score was calcu lated after add ing up individual scores II.

(b) Adrenal cortex alld spleen weights: The adrenal g land and spleen were removed and weighed 12.

Methods used to assess stress-induced perturbations

(a) Stress-induced 'behavioural depression: The following methods were used to a sess behavioural depression

(i) Stress-induced 'behavioural despair' test: Rats were forced to swim individually in a polypropylene ves. el (45x40x30 cm) with a water level of 20 cm, which ensured that the rat's feet did not touch the floor of the vessel and that it could not climb out of it. The rat was allowed to swim for 10 min. Thereafter, during the next 5 min, the total period of immobility, characterized by complete cessation of swimming with the head float ing above water level, was noted . This immobility peri od, after initial frenz ied attempts to escape, is postulated to represent 'behavi oural despair ' as an experimental model of endogenous d

. \3 epress lon . . (ii) Learned helplessness test: On day 12 of the

inves ti gation, rats were subj ec ted to footshock (60 scrambled shocks, 15 sec duration, 0.8 mA, every

min) in a two compartment jumping box (Techno) with the escape door to the adjoining unelectrifi ed compartment closed. The exercise conti nued for I hr. On day 14, 48 hr later, the rats were subj ec ted to avoidance training, using the same apparatus but keeping the escape route to the unelectrified chamber open. During thi s avoidance training the rats were placed in the electrified chamber and allowed to acclimatize for 5 min before being subjected to 30 avoidance trials, with an inter-trial interval of 30 sec. During the first 3 sec of the trial , a buzzer stimul us (conditioned stimulus, eS) was presen t followed by electroshock (u nconditioned stimulus, UeS) (0.8 mA) delivered via the grid floor for the next 3 sec. The avoidance response was characterized by escape to the adjoining 'safe' chamber during es . Failure to escape during ues within 15 sec assessed as 'escape' failure which is postulated to indicate despair or d

. 14 epress lon . (i ii ) Stress-induced inhibition of male sexual

behaviour: A male rat was placed in a cage in a dimly room for 10 min with 2 oest rini sed (sequenti ally treated with oestradiol valerate 5 Mgirat, followed 48 hr later by hydroxyprogesterone 1.5 mg/rat, sc) female rats. The total numbers of mounts were counted l5.

Stress-induced coglllti ve dysfunction: The following parameters were used to assess the effect of stress on retention of a learned task as memory :

(i) Active avoidance test: Rats were trained for an active avoidance task before subjecting them to stress. During training, the rat was placed in the ri ght electri fied compartment of a shuttle box (Techno) and allowed to acclimatize for 5 min . Thereafter, the animal was subjected to 15 sec of a buzzer stimulus(eS) which was followed by elec tri c shock ( I mA, 50 Hz) g iven through the grid floor (UeS). The rats were given at least 10 trials, with an inter-trial interval of 60 min , until they reached the criterion of 100% avoidance response of jumping to the unelectrified left chamber of the shuttle box during es. The test was repeated on day 14 in order to assess the retention of the active avoidance learning l6 .

(ii) Passive avoidance test: The test appara tus was a rectangular box (45x30x40) with an e lec trified grid floor. An 8 cm high platform (17x 12 cm) was fix ed to the centre of thc floor. A rat was placed on the platform and allowed to step down. 24 hI' later, on day I of the experimcnt, the rat was aga in placed on the platform and on stepping down , received footshock (0.75 mA, 2 sec) through the grid floor. The rat was

346 INDIAN] EXP BIOL, APRIL 200 1

g iven 3 more tlials until the latency of step down had stabili sed. The test was repeated on day 14 and retention o f learning as memory, for each rat was recorded 17.

Statistical analysis: The values are expressed as mean ±SD. Statistical s ignificance of the differences between control and treated groups was calculated using Kruskal Wallis one way analys is of variance (A OVA) followed by Mann-Whitney U-test (two tailed). P <0.0 I was considered to be significant.

Results

( I ) Gastric ulceration: Chronic stress markedly increased the incidence, number and severity of gastric ulcers. IHp (l00 and 200 mg/kg, po) and PG ( 100 mg/kg, po) significant ly reduced these strcss­induced gastric indices (Table I).

(2) Adrenal cortex alld spleen weights: Chronic stress signi ficantly increased adrenal g land weight and reduced that of spleen. These stress- induced changes were attenuated by IHp ( 100 and 200 mg/kg, po) and PG (100 mg/kg, po) (Table 2).

(3) Stress-indllced 'behaviollral despair ' and 'Ieamed helplessness' tesT : Chronic stress increa ed the duration of immobility tes t, while increasing escape failures with concomitant decrease in avoidance response in the learned helplessness test, features indicative of depress ion. IHp ( laO and 200 mg/kg, po) and PG (100 mg/kg, po) tended to reverse the stress-i nduced behavioural changes

(Tables 3 and 4). (4) Stress- induced inhibition of male sexllal

behaviour: Chronic stress significan tly decreased the sexual behaviour of male rats, as indicated by decrease in the number o f mountings . This stress effect was reversed by IHp ( 100 and 200 mg/kg, po) and PG (laO mg/kg, po) (Table 3).

(5) Active and passive ([\ oidallce tests: Chron ic stress produced significant decrease in the retention of acquired active and passive learning. These stress induced memory deficits were reduced by IHp (100 and 200 mg/kg, po) and PG (lOa mg/kg, po) (Tables 3 and 5).

Discussion

Stress research in laboratory animals has assumed an importan t role in understanding the biological and behavioural consequences of external or int ernal stressors, which threaten to perturb homeostasis, and may induce a number of clinical diseases when the body fail s to counter the stre s s ituation 18. A va ri ety of stre sful situat ions have been employed and the lack of consistency of the sI re S protocols is astounding l8

. Likew ise, there is wide variation in the physiological consequences of the stressors utilized in ani mal research I'>. However, it is now widely accepted that chron ic inescapable intermittent stress, particularly of an unpredictab le pattern, is more likely to induce neural , endocrine and biochemical perturbations than either acute or chronic stress of a

Table I-Effecls of IHp and Pun(LI" gil/seng compared to vehicle on chronic stress- induced gastric ulcerations in rats

Treatment Dose (mg/kg) Ulcer inc idence Number o f ulcers ~)everity of ulcers

Vehicle+Stress(VS)( 10)

IHp+VS (6)

IHp+VS (6)

Pal/ax ginseng+ VS(6)

100

200

100

(%)

100

67"

33"

67"

8.7± 1.7 16.8±3.3

4 .S±2.3" 8.7±4.5"

2.3± 1.7" 4 .8±3.S'

2.3±1.0' 4.8±2.0"

Values in parentheses indicates number of animal s: • indi cates difference with VS (ANOVA followed by Mann-Whitney V­test).

Table 2--Effeet s o f IHp and Panax g inseng compared to vehicle on chronic stress- induced changes in adrenal gland and spleen weights in rats

Treatment Dose (mg/kg) Adrenal gland wt Spleen wt (mg/100g) (mgIIOOg)

Vehicle( 10) 24. IS±2. 10 19S.70± 11.00

Vehicle+Stress(VS)( 10) 41.10±3.7S" 128.6S±8.S4"

IHp+VS (6) 100 30.74±2.90b IS7 .7S±7.0S h

IHp+VS(6) 200 27.0S±2.S4b 178.48±6. IOb

Pal/ar gil/sel/g+ VS(6) 100 28.4S±2.70b 170. 10±9. IS b

Values in parentheses indicates number of animals; " indicates difference with vehicle treated group; b indicates difference with VS (ANOV A followed by Mann-Whitney V-test).

KUMAR el 01 .. : HYPERICUM PERFORATUM & A TI -STR ESS ACTIVIT Y 347

predictable nature 's. The validity of the method used in the present study is demonstrated by the biological effects induced by it , which include gastric ulceration , increase in adrenal gland weight and decrease in the weight of the spleen. All these parameters have been conclusively shown to be stress-induced effects2o.

The preventi on and management of stress di sorders remains a major clinical problem. Benzodi azepines (BDZs) appear to be effecti ve against acute stress but fail to prevent the consequences of chronic stress' s .. In additi on the problems of tolerance and phys ical dependence ex hibited by BDZs, on prolonged use, limit thei r uti lity's. An answer to this vex ing problem was fir. t prov idcd when Brekhman and Dardymov2

'

reported that some plant-deri ved agcnts could induce a state of non-spec ifi c increase of resistance to affect internal homeostas is. These agents, named adaptogens, appeared to be effecti ve only when the physiological perturbations were discernible fo llowing prolonged illness, old age and exposure to chronic stress2

' . Adaptogens like Pal/ax ginseng (PG) were shown to be effecti ve in attenuating stress induced adverse effects in astronauts, soldiers and athletes in the USSR2' . PG, the first clinically used adaptogen, has been extensively investi gated experimentally and clinically for its stress-attenuating

. . 2? actI vIty -.

Both IHp and PG prevented chronic stress-induced

gastric ulcerations, in the term of the incidence and severity of the ulcers. Involution of the spleen and increase in adrenal gland weight, are also consequences of chronic stress' S, both responses being reversed by IHp and PG .

There is considerable ex perimental and clin ical evidence to suggest that chroni c stress induces endogenous depression23

. A number of animal models of depression are based on the use of uncontroll able stress and the biochemi cal correlates of such tests are consonant with those seen in chronic stress, including monoamine deficiency and increased activity of the cort icotrophin-releasing factor23

. Both IHp and PG were able to reverse chronic stress-induced indices validated as animal models of depress ion. Chronic stress is known to affect other endocrine responses as well , which can induce sexual debi lity in male 24 and perturb glucose metabolism25

. Maturity-onset diabetes melli tus may represent a state of stress-induced disturbance in glucose homeosta is25

. IHp and PG reversed chronic stress-induced inhibition of male sexual behav iour.

Stress is known to interfere with cognit ive functi ons, tending to retard the memory engram rather than the acqui sition of learning26

. The mechanisms involved in the memory-attenuating effect of stres remains conj ectural but a similar neurochemi cal basis operating in the induction of stress-induced depression, may be responsible26

. IHp and PG

Table 3--Effccls o f II-!p and Pal/ax g il/sel/g compared to vehicle on chronic stress- induced inc rea. e in swim stre s immobi lity. suppression of sexual behaviour and memory deficil in acti ve avoidance response in rats

T reatment Dose Duration of Immobility Number o f mountings (N) Acti ve avoidance response (mg/kg) (sec) on day 14 (%)

Vehicle (10) I 17 .OS± I 0 . 15 6. IS±0.SO 75

Vehicle+Stress(VS)( I 0) 260.92±7.3S" 1.70±0.74" 20"

Il-!p+VS (6) 100 IS l .47±S.6Sb 3 .2S±0.7Sb 50

IHp+VS(6) 200 139.2S±6. lOb 4 .00±0.9Sb 70b

Pal/ox g il/sel/g+VS(6) 100 142.4S±6.SSb 4 . IS±0.86h 70b

Values in parentheses indicates number o f an imals; " indicates diffe rence with vehicle treated group; b indi cates difference wilh VS (ANOVA followed by Mann-Whilney U-test).

Table 4--Effects o f II-!p and Pal/ax g il/sel/g compared to vehic le chronit: stress-induced changes in learned helplessness test in rats

Treatment Dose (mg/kg) Escape failures (N) Avoidance response (N)

Vehicle (10) 14.7S±1.80 3.70±0.71

Vehicle+Stress(VS)( 10)

Il-!p+VS (6) 100

Il-!p+VS(6) 200

Pal/ax g insel/g+VS(6) 100

26.0S± 1.95"

IS.3S± I.SSb

IS .70±1.24b

IS.9S± 1.20h

0.S3±O. IS"

I.4S±O.SSh

2.70±O.64h

2.6S±0.S8h

Values in parentheses indicates number of animals: " indicates difference with vehicle treated group: b indicates difference wi th VS (A NOV A followed by Mann-Wh itney U-tesl).

348 INDIAN J EXP BIOL, APRIL 2001

Table 5 -- Effects of IIlp and Pallax gillsellg compared to vehic le on chronic stress-induced mcmory defic it in passive avoidance response in rats

Treatment

Vehic le (10)

Vehiclc+Stress(VS)( I 0)

IHp+VS (6)

IH p+VS(6)

PWIlI.\" gillsellg+ VS(6)

Dosc (mg/kg)

100

200

100

Latcncy (Step-through) in sec

Day I Day l4

12.35±2.10 IS.70±2.45

11.74± 1.85 6.4 7±O.SS"

11.58±1 .90 16.24±2.05h

12.4S±2.00 17.50± 1.94"

11.70±1.91 I U ;7±1.96h

Values in parenthcses indicates number of animal s; :I indicates diffcrence with vehiclc treated group; h ind icates differcnce with VS (ANOVA fo llowed by Mann-Whitney U-tcst).

attenuated the stress-induced deficit of retention of learned tasks, both in the acti ve and passive avoidance parameters , thus facilitating memory and its recall.

The findings indicate that, li ke the standard adaptogen PG, IHp can attenuate chronic stress­induced biochemical , behavioural and physiological perturbations in rats. PG has earlier been reported to reverse chronic stress induced effects in humans21

.

Japanese traditional medicinal plant formula tions, like Gosya-jillki-gan, KYIlSizill and Reiollsall, essenti ally based on Ginkgo biloba, have been reported to reduce the adverse effects of chronic hanging stress on sexual and learning behaviours in mice27

.

Increased generati on of oxidative free radicals (OFR), or impaired antioxidant defence mechanisms , have been implicated in chronic stress induced perturbed homeostasis including immunosuppression, inflammation, diabetes mellitus, peptic ulceration and other stress-related diseases28

. IHp have been shown to exert signi ficant antioxidant activ ity induced by augmented activ ity of OFR scavenging enzymes, superoxide dismutase, catalase and glutathione peroxide29

.Jo. Thus, the observed adaptogenic

antistress effect of IHp may be atleast partly due to its antioxidant activi ty .

The present investigation indicates that IHp has significant adaptogenic activity as shown by its mitigating effects on several chronic stress ind uced physiological and behaviour perturbations, comparable to that induced by the we ll accepted adaptogenic agent, POI/ax gil/selig.

Acknowledgement Vikas Kumar is g rate ful to ICMR. New Delhi for

award of Senior Research Fellow hip.

References I COli J M. III I' il /"() r<:c.:ptor binding and cn/.ym<: inhibitioll by

HypericulII peljfll"(f/UIII cxtract. Pi/(/rlll(/copsychialrv 30 ( 1997) 108.

2 De-Smet P A & Nolen W A. SI. John' s wort as an <lntidepres­sant (editorial commcnt). Br J Pharlllacol . 313 (1996) 241.

3 Harrer G & Sommcr H, Therapie leichtcr/millelschwcrer de­pressioncn mit HypericulII, Mllllcheller Medizillische Wo­chellschriji, 135 (1993) 305.

4 Ernst E, St. John ' s wort, an antidepressant? A systematic. critcri<l based study, Phylollledicille. 2 ( 1995) 67.

S Kumar V, Singh P N. Jai swal A K & Bhattacharya S K. Anti­depressant acti vity of Indian HypericlI/I/ IX ljoralll/l/ Linn in rodents. Illdiall J Erp Bioi. 37 ( 1999) 1171 .

6 Kumar V, Ja iswnt A K. Singh P N & Bhattacharya S K. Anxiolytic activity of Indian HypericIIJIl peljoralll lli Linn: An ex perimenta l study, Illdiall J Exp Bioi, 38 (2000a) 36.

7 Kumar V, Singh P N, Muruganandam A V & Bhattacharya S K, Effect of Indinn J-lyperiCII/l/ peljoral 1111 I Linn on animal models of cogn itive dysfunction, J ElllllOphar/l/acol. 72 (2000b) 11 9.

8 Kumar V, Singh P N & BhattacharYJ S K. Neu rochcmical studies on Indian HypericlIlII pc/jorallllll Linn, Illdiall J Erp Bioi. (2000) (in prcss).

9 Kumar V, Khanna V K, Seth P K. Singh P N & I3hnttacharya S K, Brain neurotransmitter receptor bind ing and nootropic studies on Indian HypericIIIII peljorallllll Linn. PhylOlher Res. (2000) (communicated).

10 Armando I , Lcmoine A P, Segura. E T & I3nrontini M B, Thc stress-induced reduction in monoamine oxidase (MAO) A activity is revcrsed by benzodiazepines: ro le of peripheral benzodiazepine receptors, Cell Mol Nellro/Jiol, 13 (1993) 593.

II Bhargava K P & Singh N, Anti-stress activ ity or Ocillll//I/ sm/CIIIIII Linn, Illdiall J E\"p Bioi, 10 ( 198 1) 443.

12 I3hallacharya, S. K., Bhattacharya, 1\ . & Chakrabarti , A, Adaptogenic activity of Siotone, a polyherbal formul ati on of Ayurvedic rasayanas, Illdiall J Exp Bioi. 38 (2000) 11 9.

13 Porso lt R D. Bert in A & Jalfre M, Behav ioral despair in mice: A primary sc reen ing test for antidepress:lIl ts. Arch 1111 Pi/(/r· /l/ucodYII Th er 229 (1977) 327.

14 Thiebot M II. Martin r & Puech A J. An imal behavioural studies in thc evalunt ion of antidepressant drugs. Hr J P.\'· ('hial. 160 (1992) 44.

IS Mori shita S, Shoji M. Oguni Y. Hirai Y, Sugimoto C & Ito C. Effects of crude drugs derived from animal sourccs on sexual and learning in chemically ~tressed mice, I'hwolher RC.I. 7 ( 1993) 57.

16 Jai slI'al A K. Upaclhyay S t 1 & I3hatla 'har)':t S K. Ff 'eel fir piracet:ll1l, a nOOlropic ageili. 011 discriminat ion learning cicli­ci ts inciuc.:d by par<:ntal undernul rition and environmental impuverish mcnl in young rat s. Illdiall .I Erp LJiol. :'71.) ( 19,'9) 269.

KUM AR et 01 .. : HYPERICUM PERFORATUM & ANTI -STRESS ACTIVITY 349

17 Sen A P & Bhattacharya S K, Effect o f selecti ve muscarinic receptor agoni sts and antago nists o n active-avo idance learn­ing acqui sition in rats, Illdiall J Exp Bioi. 29 (1991 ) 136.

18 McCarty R, in : Stress lI eurochemical alld humoral mecha· lIisms, edited by G .R., Van Loon & R .. R. Kvetnansky, (Gor­dan & Breach Sc ie nce Publishers, ew Yo rk) 1989.3.

19 Vogel W H. Coping, stress. tressors and health co nse­quences. Neuropsychabiology. 13 ( 1985) 129.

20 ate l. o n B H. Tapp W N, Adamus J E, Mittle r J C & Levin B E, Humoral indic ies of stress in rats, Physial Belwv. 26 ( 198 1) 1049.

2 1 Brekhman II & Dardymo v I V, ew substances o f plant o ri­g in which increase nonspec ific resistance. Amlll Rev Pharma­cal Toxicol. 9 ( 1969) 419.

22 Shi bata S, T anaka O. Shoji J & Saito H, EcO//Omic alld me­dicillal plallt research. edited by B .. Wagner. H. Hikino & N.R. Fransworth . (Academic Press, Londo n), 1985, 217 .

23 Caroll B J. fc inberg M & G rede n J F. A rch Cel/ Psychiat . 33 ( 1976) 1039.

24 Saito H. ishi yama N, Fuj imori H. Hinata K, Kamegaya T. Kato Y & Bao T. in Stress: The role oj catecholamille.~ alld lIeurotral/Smillers. edited by F Usd in. R Kvetnansky & I J Kopin . (Gordan and Breach Scie nce Publishers. New York)

1984 p. 467. 25 Shoji M, Sato H. Hirai Y. Og ulli Y, Sugimoto C, Mori shita S.

& Ito C , Phannaco logical e ffec ts o f Gosha-jinki-gan-ryo ex­trac t: Effect s on experimental di abetes. Nippall Yakllrigaku Zasshi, 99 (1992) 143.

26 Bhattacharya S K, in Evaluation o f adaptogenic ac ti vity of Trasina, an ayurvedic herbal fo rmulat io n, edited by Mukher­jee B, Traditiol/olmedicille. (O xford & IBH Publi shers, New Delhi ). 1993, 320.

27 Mori shita S, Shoji M. Oguni Y. Hi rai Y. Sug imoto C & Ito C. Effects o f traditional medic ines, C osya-jillki-goll . KVIIShill a nd Reiollsall on sexual and learning behav io ur in chemically stressed mice, Phytather Res. 7 ( 1993) 179.

28 Maxwe ll S R J, Prospects fo r the use o f anti oxidants thera­pies, Drugs 49 (1995) 345.

29 Tripathi Y B, Pandey E & Dubey G P, Antiox idant property o f Hypericum peljomtum (L.) o f Indian o rig in and its com· pari son with establi shed Medhya rasayanas o f Ayurved ic med ic ine, Cllrr Sci. 76 ( 1999) 27.

30 Tripathi Y B & Pandey E. Ro le o f alcoho lic extract o f shoot o f Hypericum peljoratl/II/ Linn on lipid pe riox ida ti on and vario us species o f free radicals in rats. Illdiall J Exp Bioi. 37 (2000) 567.