Indian Journal of Experimental Biology Vol. 39. April 2001. pp. 339-343

Anti-inflammatory and analgesic activity of Indian Hypericum perforatum L.

Vikas Kumar l.2, P.N. Singh l & S.K. Bhattacharya2'

'Phannacology Laboratory, Department of Pharmaceutics, Institute of Technology. europharmacology Laboratory. Department of Pharmacology. Insti tute of Medical Sciences, Banaras Hindu University.

Varanasi 221 005. India

Receil'ed 8 Septelllber 2000; revised 27 Decelllber 2000

A standardi sed 50% aqueous ethanolic extract of the Indian variety of HypericlIlIl pe/fore/1II11l (IHp) was examined for its putative anti-inllammatory and analgesic activity at the do es of 100 and 200 mg/kg. po. The experimental paradigms used were carrageenan induced pedal edema and cotton pellet induced granuloma for anti-inllammatory activity. whereas the tail Ilick. hot plate and acetic acid induced writhing methods were used to asses analgesic acti vity. Indomethacin (20 mg/kg. ip) was used as the standard anti-inllammatory drug. Pentazocine ( 10 mg/kg. ip) and aspirin (25 mg/kg. ip). both clinically used ana lges ics. were used as standard analgesics for comparison. IHp extract showed significant anti ­inflammatory and analgesic activity at both dose levels. in all the paradigms used. Additionally. IHp potentiated the anti­inllammatory activity of indomethacin and analgesic activities of pentazoci ne and aspirin.

HypericulIl pe/foralum L. is a rhizomatous perennial herb growing upto a height of 3 feet and is native to Europe, West Asia and North Africa. In addition it is also found in parts of Asia, Africa, Australia and North America. The aerial portion of H. pe/forall./111 has been used extensively thoughout hi story as a medicinal plant. In folklore tradition the bright ye llow flowered herb has been believed to have supernatural medicinal properties. It is commonly known as St. John 's Wort and has been used in European. folk medicine for a variety of clinical uses. The most common uses of HypericulII have been for the treatment of depression, as an anthelmint ic, for bedwetting in children and a a diuretic ' -4. Although H. perfOrall/1II has been mentioned in Ayurveda and is known as Bassolll, its clinical use does not appear to include nervous disorders5

. Recently Indian H. pe/iorallllll (lHp) has been shown to exert several neuropsychopharmacological actions, including antidepressant, anxiolytic and nootropic activi t/'-'o. HypericulI/ has historically been one of the most relied upon botanicals for the treatment of wounds ". However, scientific data on its anti-inflammatory and analgesic activity is not available. Hence in the present study the anti-inflammatory and analgesic activity of rHp was investigated .

Materials and Methods Drug Ireatlll ents-The plant (JHp) was collected

* Correspondent author: E-mail : solid @benaras.ernet.in

during August from Company Garden, Saharanpur, India. A specimen of the plant is preserved with Indian Herbs, Saharanpur. 50% ethanolic extract (yield 26.75 % w/w, standardised for 4.5-5% hyperforin, HPLC) of the 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 mglkg, once daily for three con ecutive days. Indomethacin (lDPL, Ind ia) (20 mg/kg, i.p.) was used as the standard anti­inflammatory agent whereas pentazocine (Ranbaxy, India) (10 mg/kg, ip) and aspiri n (Astra- IDL Ltd., India) (25 mg/kg, ip) were used as standard analgesic agents . All standard drugs were administered to animals 30 min before experiments for comparison . Control animals were treated with the veh icle (0.3% CMC suspension). Experiments were conducted on day 3, one hour after the last drug or vehicle administration.

Animals-Adult inbred Charles Foster albino rats (l50±lOg) and Wistar mice (23±2g), of ei ther sex, were obtai ned from the Central Animal House, Institute of Medical Sciences, Banaras Hindu University and were randomly distributed into different experimental groups. The animals were housed in groups of six in polypropylene cages at an ambient temp of 25°± I °C and 45-55% RI-I , with a 12: 12 hr light/dark cycle. Animals were provided with commercial food pellets (Brooke Bond-Lipton, India) and water ad libitum. Experiments were conducted between 0900 and 1400 hrs. 'Principles of laboratory

3~0 INDIAN J EXP BIOL. APRIL 200 1

an imal care' (NIH publication number no. 85-23, rev ised 1985) guidelines were followed.

Experimental methods Anti-inflammatory activity

Carrageenan-induced pedal edel/la in rats-Male albino rats were injected with 0.1 ml of a 1 % carrageenam solution in saline into the sub-plantar region of the left hind paw l2

, The paw was marked with ink at the level of the lateral malleolus and immersed in mercury up to thi s mark. The paw volume was mcasured before and I , 2, 3, 4 and 6 hr after the injecti on of carrageenan by mercury di splacement method plethysmograph ical ly. The edema volume was determined and expressed as percentage swellings, compared with the initi al hind paw volume of each rat.

Cotton pellet induced granulol/la in rats­Subacute inflammation was produced by cotton pellet induced granul oma in rats l 3

• Steril e cotton pellets (50± 1 mg) soaked in 0.2 ml of disti lied water containing penicillin (0. 1 mg) and streptomycin (0. 13 mg) were implanted subcutaneously bilaterally in axi lla under the ether anaesthesia. The animals were sacrificed on the 7th day. The granu lati on tissue with cotton pellet was dri ed overnight at 600 e and then the dry weight was taken. The weight of the cotton pellet before implantation was subtracted fro m the weight of the dried, dissected pell ets.

Analgesic activity

Tail flick latent period-The technique described by Davies and co-workers I~ was adopted, usi ng a Techno analgesiometer. The rat was placed in a rat holder, with its tail coming out through a slot in the lid. The tail was kept on the bridge of the analgesiometer, called jacket wi th an electrically heated nichrome wire underneath . The tail received radiant heat fro m the wi re, heated by passing current of 6 mAo The time taken for the withdrawal of the tail after switching on the current, was taken as the latent period, in sec, of "tail flicking" response and was considered as the index of nociception. The cut off time for determination of latent period was taken as 30 sec to avoid injury to the skin IS. Three tail flick latencies were measured per rat at each time interval and the means of the tail-flick latencies were used for statistical analysis. Pentazocine (10 mg/kg, i .p.) was used as the reference standard.

Hot plate reaction til/le in l/lic~Mice were screened by placing them on a hot plate maintained at

55± I (Ie and recording the reacti on time in seconds for forepaw li cking or jumping ' 6. Only mice which reacted wi thin 15 sec and which did not show large variation when tested on four separate occasions, each 15 mi n apart, were taken for the tes, t. Pentazocine ( 10 mg/kg, i.p.) was used as the reference standard. The time for forepaw licking or jumping on the heated plate of the analgesiometer was taken as the reaction time.

Acetic acid induced writhing response in mice--Acetic acid solution (15 mg/ml) at the dose of 300 mg/kg body weight was injected i.p. and the number of writhes during the following 30 min period was observed ' 6. A significant reduction in number of writhes by drug treatments as compared to vehicle treated animals was considered as a pos itive analgesic response. The percent inhibition of writhing was calculated. Aspirin (25 mg/kg, i.p.) was used as the reference standard .

Statistical analysis-The values are expressed as mean ±SD. Statistical significance of the differences between control and treated groups was calculated using Kruskal Walli s one way analysis of vari ance (ANOYA) fo ll owed by Mann-Whitney U-test (two tail ed). P < 0.0 I was considered to be signi ficant.

Results

Carrageenall-illduced pedal edel/la in rats: The effect of IHp extract on carrageenan-induced edema is shown in Table I. IHp exerted significant anti­inflammatory effect against carrageenan-induced inflammation in doses of 100 and 200 mg/kg but was less than thar of indomethacin (20 mg/kg, i.p.). IHp at both doses potentiated the an ti-in flam matory activity of indomethacin.

Cotton pellet induced granulollla ill ratsi-The weight of the granulation tissue was signi ficantly reduced by both doses of IHp extract and indomethaci n. IHp at both doses potentiated the anti­inflammatory ac ti vi ty of indomethacin as shown by further reduction of granulation tissue (Table 2) .

Tailflick latellt period-IHp at both the dose levels had significant analgesic activi ty and poten ti ated the analgesic ac tivity of pentazocine (Table 3).

Hot plate reaction time in lIlice-IHp extract showed significant analge ic act ivity at both the dose levels . Additionally, lH p potentiated the analgesic activity of pentazocine ( 10 mg/kg, i.p.) (Table 4).

Acetic acid induce writhing respollse ill lIIicc­Both doses of IHp and aspirin (25 mg/kg, i. p.) showed

KUMAR el af.: HYPERICVM PERFORA TVM & ANTII NFLAMMATORY & ANALG ES IC ACTIVIT Y 341

significant decrease in writhing response induce by acetic acid . In addition, IHp at both doses, potentiated the analgesic activity of aspirin as shown by further decrease in writhing response, when given in combination (Table 5) .

Discussion The presen t study demonstrated that IHp extract

was effective in animal models of acute inflammation. Among the many methods used for screen ing of anti­inflammatory drugs, one of the most commonly employed techniques is based upon the abi lity of such agents to inhibit the edema produced in the hind paw of the rat after injection of phlogi tic agents. The time course of edema development in canageenan-induced paw edema model in rats is generally represented by a

biphasic curve l2. The first phase occurs within an

hour of injection and is partly due to the trauma of injection and also to the serotonin component 17

.

Prostaglandins (PGs) pl ay a major ro le in the development of the second phase of reaction which is measured around 3 h 18

• The presence of PGE2 in the inflammatory exudates from the injected foot can be demonstrated at 3 h and periods thereafterl9

.

Canageenan induced paw edema model in rats is known to be sensitive to cyclooxygenase inhibitors and has been used to evaluate the effect of non­steroidal anti-inflammatory agents which primarily inhibi t the enzyme cyclooxygenase in prostaglandin synthe is2o. Based on these reports it can be inferred that the inhibitory effect of IHp extract on carrageenan- induced inflammation in rats cou ld be

Table I-Effeci of IHp on carrageenan-induced acute paw edema in rats

Treatment Dose(mg/kg) Mean % edema at 3 hr

Vehicle (10) 175.64 ± 6.70

IHp (6) 100 71.90 ± 2.80*

IHp (6) 200 62.48 ± 3. 10*

Indomethacin (6) 20 60.87 ± 2.45*

IHp+lndomethacin (6) 100+20 55.35 ± 1.90*

IHp+lndomethacin (6) 200+20 51.24 ± 1.84*

Values in parentheses indicate number of animals; IHp= Indian HypericlIlII perJorallllll ; * P <0.01 in compari son to vehicle (A OVA fo llowed by Mann-Whitney V-test).

Table 2-Effect of IHp on cotton pellet induced granuloma in rats

Treatment Dose( mg/kg) Wt of dry cotton pellet granu-

loma (mg)

Vehicle ( 10) 75 .00 ± 6.10

IHp (6) 100 34.00 ± 3.75*

IHp (6) 200 30.00 ± 2. 10*

Indomethacin (6) 20 28.00 ± 2.25*

IHp+lndomethac in (6) 100+20 26.00 ± 3. 15*

IH p+lndomethacin (6) 200+20 23.00 ± 2.05*

Values in parentheses indicate number of animal s; IHp=lndian HypericlIlII perforalwn; * P <0.0 I in compari son to vehicle (A OVA followed by Mann-Whitney V-test).

Table 3--Effect of IHp on tail nick latent period in rats in rats

% inhibition

59.06

64.43

65.34

68.49

70.83

% inhibition

56.40

61.75

64.26

67.48

72.15

Treatment Dose (mg/kg) Mean latent period of tai l flick response (sec)

Vehicle ( 10)

IHp (6)

IHp (6)

Pentazocine (6)

IHp+Pentazocine (6)

IHp+Pentazocine (6)

100

200

10

100+10

200+10

Initial

9.07 ± 1.42

10.45 ± 0.67

10.54 ± 1.10

10. 15 ± 0.62

9.75 ± 0.83

9.67 ± 0.87

Values in parentheses indi cate number of animal s; IHp= Indi an HypericlIlII perJorallllll ; * P <0.0 I in compari son to vehicle (A OVA fo llowed by Mann-Whitney V- test).

After 30 min

9.84± 1.15

14.92 ± 0.80*

15.85 ± 0.96*

16.08 ± 1.26*

23.40 ± 1.63*

25.64 ± 1.33*

342 INDIAN J EXP I310L. APR IL 200 1

due to inhibition of the enzyme cyc loxygenase leading to inhibition of prostaglandin synthesis.

In the cOllon pellet induced gran uloma model of sub-acute inflammation, IHp ex tract significantly reduced the weight of granulation tissue and potentiated the anti-inflammatory activity of indomethacin. This method was described first by Meier ef af.21 who showed that foreign body granulomas were provoked in rats by subcutaneous implantation of pellets of compressed cotton. This method has been useful for evaluation of steroidal and non-steroidal anti-inflammatory drugs22

• It has been considered that cotton pellet induced granuloma is closely related to the formation of ant ibodies .

Hyperic in has been reported to inhibit the growth of glioma cell lines ill vitro and to be a potent inducer of gli oma cell death due to inhibition of protein kinase C (PKC) as measured by e H] thymidine uptake. In this regard the glioma inhibitory acti vity is reported to be equal to or greater than tamoxi fen and additi onally was reported to be increased by approxi mately 13% upon exposure to visible light23

. Other researchers report PKC-inhibitory activity with both hypericin and pseudohypericin24

. Receptor tyrosine kinase activi ty of epidermal growth factor has been reported to inhibited by hypericin25

. The PKC inhibition may contribute to the anti-inflammatory effects associated

with HypericlIm as hyperici n has been found to inhibit the release of arachidonic acid and leukotri ene 8 / 6

.

IHp extract also ex hibited analgesic activity in rodents and synergised with the analgesic acti vity or pentazocine. The extract was found to sign ificantly increase the tail flick reaction time in rats. Th is test is very useful for discriminating between centrally acting morphine- like analgesics and non-opiate analgesics, giving posi tive response with the former only.

The hot plate method was ori gi nally described by Woolfe and Mac Donald27

. This test has been found to be suitable for evaluation of centrally but not of peripherally acting analgesics. The validity of thi s test has been shown even in the presence of substantial .. f f "'8 TI Impamnent 0 motor per ormance" . le present findings indicate that IHp may be centrally acting.

In order to distinguish between the central and peripheral analgesic action of IHp, acetic acid induced writhing response in mice was used to examine the effect. This method is not only simple and reliable but also affords rapid evaluation of peripheral type of analgesic action . In this test, the animals react with characteristics stretching behaviour which is called writhing. It was found that IHp significantly inhibited the acetic acid induced writhing response and potentiated the anti-inflammatory activity of aspirin

Table 4-Effcct of IHp on hot plate reaclion time in mice

Treatment Dose (mg/kg) Mean lalenllime (sec)

Initi al

Vehicle ( 10) 11 .45 ± 0.95

IHp (6) 100 12.04 ± 1.05

IHp (6) 200 11.80 ± 1.38

Pentazocine (6) 10 11 .40 ± 1.90

IHp+Penlazocine (6) 100+10 10.85 ± 0.91

IHp+Penlazoc inc '(6) 200+10 11.28 ± 1.17

Values in parentheses indicate number of animals; IHp= Indian HypericlIlII perforall/lIl; * P <0.0 1 in compari son 10 vehicle (ANOVA followed by Mann-Whitncy U-Iest).

Table 5-Effect of IHp on acelic acid induced writhi ng in mice

Treatmcnt Dose( mg/kg) Mean number of wrilhes in

30 min

Vehicle ( 10) 72.4 1 ± 3.40

IHp (6) 100 46.50 ± 3.27*

IHp (6) 200 31.00 ± 1.4 1*

Aspirin (6) 25 26.83 ± 2.40*

IHp+Aspirin (6) 100+25 22.16 ± 1.94*

IH p+Aspirin (6) 200+25 19.83± 1.72*

Values in parentheses indicate number of an imals; IHp= Indi an HypericulIl perfora/lIl11; * P <0.0 1 in compari son 10 vehicle (A OVA followed by Mann-Whitney U-Iest).

After 30 min

10.78 ± 1.10

45.28 ± 2.70*

48.90 ± 2.45*

51.65 ± 3.15 *

53.20 ± 3.68*

55.84 ± 4. 15*

% inhibition

35.78

57. 19

62.95

69.40

72.6 1

KUMAR e/ al. : HYPERICUM PERFORATUM & ANTIINFLAMMATORY & ANALGESIC ACTIVITY 343

as well. The abdominal constriction IS related to the sensltlsation of nociceptive receptors to prostaglandins. It is therefore possible that lHp exerts an analgesic effect probably by inhibiting synthesis or action of prostaglandins.

Based on the results of the present study it can be concluded that IHp has potenti al an ti-inflammatory activity against both exudative-proliferative and sub­chronic phases of inflammation. The extract also has analgesic activity which is both centrally and peripherally mediated .

Acknowledgement Yikas Kumar is grateful to lCMR, New Delhi for

award of Senior Research Fellowship.

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