NON-NARCOTIC ORALLY EFFECTIVE, CENTRALLY ACTING ANALGESIC FROM AN AYURVEDIC DRUG

Journal of Ethnopharmocology, ll(l984) 309-317 Elsevier Scientific Publishers Ireland Ltd. 309 NON-NARCOTIC ORALLY EFFECTIVE, CENTRALLY ACTING ANALGESIC FROM AN AYURVEDIC DRUG CX ATAL, M.A. SIDDIQUI, USHA ZUTSHI, V. AMLA, R.K. JOHRI, P.G. RAO and S. KQUR Regional Research Laboratory, Jammu Tawi 180001 (India) (Accepted May 9, 1984) Embelin, a p-quinone, is derived from Embelia ribes Burm. The analgesic effect of potassium embelate has been studied in rats and mice. The test drug was found to be effective by oral, i.m, and i.c.v. routes and the results compared well with morphine. Although potassium embelate acts centrally to produce analgesia, its effect is not antagonized by naloxone indicating a different central site of action. There is no precipitation of abstinence syndrome as observed with morphine. Peripheral site of action of the drug is ruled out as it lacks any demonstrable anti-inflammatory action. It can be concluded that high oral efficacy and non-narcotic properties of the test drug make it more acceptable than morphine. In addition, lack of any adverse effects, high therapeutic index and absence of abstinence syndrome confers a long term safety on potassium embelate for use as an analgesic. Introduction In Ayurveda, pain has been described as Vedna (mild) and shool (severe). Although analgesics were being used in this system of medicine, they have not been well defined in the Ayurvedic literature. It appears that in India morphine for control of pain was used much later than the early Ayurvedic era. Vidang (Embelia ribes Burm.) is a reputed Ayurvedic drug and has been employed in a variety of diseases. Embelia ribes is indigenous to the Indian subcontinent and grows up to 1525 m (Fig. 1). The fruits and roots of these plants are used by the tribals for various ailments. It is referred in Sush~ta as the most spoilt constituent of anti-aging, anthe~intics and a variety of skin disease fo~ulations. It has been further described for use in mental disorders, jaundice, heart diseases and bronchitis. However, its use as an analgesic was first mentioned by Kirtikar and Basu (1933). Certain other workers (Sastry, 1952; Nadkarni, 1954; Himes, 1963) have also described the fruits of this herbal as a very effective anthelmintic. 0378-8741/84/$03.00 o 1984 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

Fig. I. Branch of Embelia ribes Burm. Recent reports (Gupta et al., 1976) have further confirmed its anthelmintic activity which has been ascribed to p-quinone embelin - the major chemical constituent of this plant, Researchers in the Regional Research Laboratory, therefore became interested in examining the analgesic properties of Embelia ribes &n-m. and its active principle, embelin. During 1976-1977 work was carried out on this aspect (Gupta et al., 1977) and analgesic property was reported for embelin and its de~vatives with various amines, more p~icul~ly isobutyl amines. It was concluded that both embelin and its amine derivative

311 showed analgesia in experimental animals only when given intraperitoneally. The mechanism of action was not clear nor could it be explained why embelin failed to show analgesia by other routes. This imposed limitations on further study. However, there is now renewed interest in the same Institute for investigating the mechanism of action of embelin and also for exploring the possibility of making it active through other routes. Materials and methods Dry berries of E. ribes Burm. were powdered and extracted with n-hexane. The concentrated extract on cooling yielded embelin which was crystallised twice from ethanol. The pure embelin (m.p. 140-141 C) thus obtained was dissolved in acetone (1: 10) and a stoichiometric amount of aqueous KOH (2%) was added slowly. The reaction mixture was evaporated to dryness to obtain potassium embelate. Formulations with sustained release properties of both potassium embelate and morphine were prepared according to the method of Collier et al. (1972). Morphine was used in a single dose, i.e. 150 mg/lo ml per kg whereas two doses of potassium embelate, i.e. 0 mg/lo ml per kg and 400 mg/lo ml per kg were tested for production of rapidly induced physical dependence. Morphine and test drug were suspended in 0.75 ml of mannite mono-oleate and 4.25 ml of light liquid paraffin. This oily phase was emulsified with 5 ml of 0.9% w/v NaCl in water. The emulsion alone was used as vehicle for control animals. Animal experi nen ts The studies were conducted on albino rats and mice (both sexes) with average weight of 100-150 g and 25.-30 g, respectively. The animals were naintained at 27 + 2 C temperature, 12 h of light period and were fed with Hindustan Lever diet with water ad libitum. The animals were fasted overnight prior to the onset of the study. Analgesic activity (1) Tail flick response was recorded with the aid of a analgesiometer (Techno Instruments) at different time intervals (Gujral and Khanna, 1957). Test drugs were administered by oral, i.m. and s.c. routes. (2) Caudal immersion method was used as a criterion to test the drug in mice, at different time periods, administered by oral and S.C. routes. (3) The results on Eddy s hot plate were obtained at different time periods with the test-drugs given by i.c.v. route. Hypodermic needles (24 gauge, 10 mm length) were used as cannulae which were fixed in perspex cubes. Steel wires were inserted inside the needle to prevent blockade. The animals were anaesthetized with ether, a hole was pierced with a dental drill at the level

312 of right lateral ventricle. A small volume of CSF was allowed to flow and then the cannula was cemented in the place with acrylin-r dental cement. Correct implantation of the cannula was checked by injecting 10 ~1 of Evan s blue and looking for dye in the proper area after autopsy. Micro-injections were given in awake, hand-held rats. (4) Assessment of analgesia was also done by observing the effect of drugs on writhing syndrome. Mice were induced to writhe by intraperitoneal administration of mg/kg of 3% (Koster et al., 1959). Test drugs were administered by p.o., i.m. and S.C. routes in different doses and the total number of stretching episodes for a period of 30 min were scored and recorded. (5) Development of Tolerance: ADloo dose of morphine as well as of potassium embelate was administered (twice daily) for 12 days. Analgesic activity was checked by Eddy s hot plate method after morning injection every day and decrease of percentage analgesia was noted. (6) Physical dependence: Rapidly induced physical dependence was produced by single S.C. injection of sustained release formulations of morphine and potassium embelate. The experimental details are mentioned below. Group I, vehicle only; Group II, morphine 150 mg/lo ml per kg; Group III, potassium embelate 0 mg/lo ml per kg and Group IV, potassium embelate 400 mg/lo ml per kg. After 24 h of administration, withdrawal syndrome was precipitated by challenging with naloxone-iicl (10 mg/kg s.c.) and observations were made 30-60 min after naloxone administration. The following observations regarding abstinence were made: writhing, squeal, diarrhoea, teeth chattering, ptosis, wet dog shaking, goose flesh, rhinnorhea and head shake. (7) E.Ds0 values and their standard errors were calculated according to graphic method of Karber (1931). Results The test drng showed dose dependent production of analgesia as assessed by analgesiometer. The response was displayed between 10 min and 4 h. A comparison with morphine-induced analgesia showed that the test drug produced a highly significant (P < 0.01) delayed effect. At l-5 min, when morphine displayed maximum analgesia, insignificant analgesia was monitored with the test drug. After 10 min when morphine-induced analgesia had already worn out, highly significant (P < 0.01) effect was observed with the test drug. Table 1 shows that the test drug is effective using all routes of administration (Lm., S.C. and i.c.v. ). Potassium embelate displays significant analgesia (P < 0.05) with oral admin~t~tion at two dose levels, i.e. mg/kg and 40 mg/kg (Eddy s hot plate). On the other hand, it is effective by S.C. route at 10 mgfkg dose in comparison to 5 mg/kg of morphine. Using caudal immersion technique for testing analgesia, it was observed that potassium embelate

mug Route Dose Initial 1 min 5 min 10 min 30 min 60 min 1 min 240 min (mg/w 7.29 7.23 5.42 * 0.23 2 0.16 + 0.31 13.43 8.42 4.37 * 0.13 + 0.25 f 0.27 15.37 12.31 5.47 * 0.16 * 2.3 t 0.34 8.31 5.47 2.19 t 0.16 f 0.17 5 0.12 15.47 10.21 7.35 f: 0.21 f. 0.23 * 0.26 17.35 11.29 5.34 t 0.29 f 0.37 ir 0.26 15.29 10.37 9.21 +_ 0.16 f 3.1 t 0.15 25.35 18.31 15.47 t 0.52 f. 0.117 + 0.19 W TABLE 1 MEAN (+_ S.E.) REACTION TIME IN SECONDS AFTER DIFFERENT TIME INTERVALS OF ADMINISTRATION (EDDY S HOT PLATE) Six animals per point were used. Group A Embelate Embelate Embelate Group B Morphine Group C Embelatfz Embelate Group D Embelate Embelate i.m. i.m. km. S.C. S.C. S.C. p.0. p.0. 5 10 5 10 40 2.31 3.23 5.29 15.31 11.32 f 1.15 f 0.19 f 0.2 x 0.24 f. 0.23 2.31 3.26 3.55 16.0.21 t 0.31 t 0.29 fr 0.25 c 0.35 t 0.16 3.15 4.19 10.71 25.34 19.81 + 0.25 t 0.19? 0.13 t: 0.16 t 0.25 2.35 11.31 30.21 15.29 9.81 * 0.113 e 0.21 * 0.27 4. 0.25 c 0.19 2.45 5.29 6.32 7.59 15.29 f 0.13 t 0.17 * 0.15 + 0.13 t 0.15 2.34 5.41 10.11 10.34 18.47 + 0.23 ). 0.17 f 0.21 t 0.24 _c 0.15 2.92 7.51 10.35 19.21 28.31 + 0.23 ck 0.15 t 2.7 c 0.15 t 0.19 3.17 5.31 17.53 23.29 29.25 * 0.19 t 0.27 + 2.3 f 2.5 f: 0.41

0 5 IS 30 6~~N TES / 240 400 TIME IN Fig. 2. Analgesic effect of potassium embelate and morphine after oral and subcutaneous admin~tration in mice by caudal immersion method. at doses of 5, 10 and mg/kg S.C. and mg/kg oral produced a dose dependent increase in reaction time similar to morphine (Fig. 2). It is interesting to note that potassium embelate by oral route ( mg/kg) produced an equally significant analgesia as morphine (s.c.). The analgesic effect with the test drug persists up to 4 h by all the routes studied, while in the case of morphine the effect lasted up to 2 h only. There is no effect of naloxone on the analgesia produced by potassium embelate. On i.c.v. administration, dose-dependent analgesia is observed. Single injection of 75 pg of the test drug is more effective than 50 hg of morphine (Fig. 3). Potassium embelate is unable to produce tolerance which is a common phenomenon with morphine (F ig. 4). The percentage analgesia was 60% in comparison to 80% of morphine on the first day of the experiment, dropping 0 5 10 15 30 60 I 240 360 TiME NV MtNlJTES Fig. 3. Analgesic effect of potassium embelate and morphine (hot plate method) after intracerebro ventricular administ~tio~ in rats.

315 - Morphine -Potassium Emb&te 0 3 6 9 12 TIME IN DAYS Fig. 4. Study of development of tolerance (hot plate method) with morphine and potassium embelate in rats. only by 10% within 3 days and then stabilising at this level up to the end of the experiment (12 days). In the case of animals where morphine was administered the fall was very rapid (50% within 3 days) and continued further to the end of experiment where analgesia was not observed at all. This shows that the test drug cannot induce tolerance, a feature inherent with narcotics. TABLE 2 EFFECT OF MORPHINE AND POTASSIUM EMBELATE ON PROTECTION FROM WRITHING PRODUCED BY ACETIC ACID ( mg/kg). Six animals (mice) per point were used. -68 Treatment Route Dose (mg/kg) Wriths/30 min per rat % protection Acetic acid Potassium embelate + i.m. Potassium embelate + i.m. Potassium embelate + i.m. Potassium embelate + p.0. Potassium embelate + p.0. Naloxone-HCl + S.C. potassium embelate + i.m. Morphine + S.C. Naloxone-HC I + S.C. morphine S.C. amean f SE. *Significant P < 0.05 **Highly significant P < 0.01. 5 10 40 10 5 10 5 75.00 r 4.30a 0 35.49 + 4.6* 53.55.31 + 6.5** 73.35 15.34 * 2.3** 80.9 35.31 f 2.9* 53.14 23.45 * 3.1** 69.14 33.35 f 4.5* 55.71 30.47 f 3.7* 60.4 68.39 + 3.9* 9.3

316 TABLE 3 PRECIPITATION OF ABSTINENCE SYNDROME BY NALOXONE IN MORPHINE AND POTASSIUM EMBELATE TREATED RATS Morphine Potassium embelate Writhing ++ + Squealing ++ - Diarrhoea +i-+ - Teeth chattering + - Ptosis + + Wet dog shaking + - Goose flesh + -I- Rhinnorhea + - Head shake + f It is quite clear that the test drug offers a significant degree of protection (P < 0.05) to animals which were induced to writhe by administering mg/kg of (Table 2). This protection is not antagonised by naloxone-hcl (10 mg/kg s.c.) which again confirms that this compound does not possess characteristics of narcotic analgesics. The study on precipitation of withdrawal symptoms by naloxone administration to the dependent animals shows that unlike morphine potassium embelate does not exhibit any of the withdrawal symptoms (Table 3). The test drug displays an EDSO of 8.2 mg/kg on oral administration and only 5 mg/kg by i.m. administration (Fig. 5). To ascertain the LDso of the compound, a dose as high as 00 mg/kg was tried in rats and mice and no mortality or adverse effect were noticed except for local i~~tion at the DOSE REPRESENTATION IN mz/s Fig. 5. Graphic representation of ED,, of potassium embelate administration. after oral and

317 site of injection. Reversible respiratory depression and fall in blood pressure were not observed in the case of dogs. Potassium embelate, however, does not show any anti-inflammatory activity in carrageniq-induced oedema, and does not potentiate the hypnotic effect of pentobarbitone sodium. Discussion The present results suggest that potassium embelate possesses a high degree of analgesic potency. Several lines of evidence suggest that this is a non-narcotic analgesic, although it acts centrally. A high degree of analgesia comparable to that of morphine is produced in rats by a single i.c.v. injection (50 pg). Moreover, this effect is not antagonised by naloxone-hcl indicating that potassium embelate behaves like a centrally acting, non-narcotic analgesic. Other pharmacological effects, i.e. lowering of blood pressure, depression of respiration and constriction of pupils characteristic of narcotic analgesics are absent in the case of potassium embelate. There is no significant antiinflammatory effect displayed by this compound confirming the finding that it has no peripheral action. This compound is also free from the dependence syndrome much associated with narcotics. A high degree of analgesia observed on oral administration of potassium embelate shows that orally effective formulations of embelin can be made. It can therefore be concluded that this compound acts centrally at certain unidentified distinct sites other than opiate receptors. These sites are in close proximity of opiate binding sites due to the fact that during i.c.v. administration the same site is used for this compound as well as morphine. A high therapeutic index confers a wide margin of safety on potassium embelate. References Ben-Bassat, J. Finney, D.J. and Goodwin, LG. (1956) Archives Internationales de Pharmacodynamie et de Therapie 122,434-447. Collier, H.O.J., Francis, D.L. and Schneider, C. (1972) Nature 237,221-223. Eddy, N.B. and Leimback, D.J. (1953) Pharmacology and Experimental Therapeutics 107,385-393. Gujral, M.L. and Khanna, B.K.J. (1957) Scientific and Industrial Research 16C, 11-13. Gupta, O.P., Anand, K.K., Mohd Ali, Ghatak, Ray, B.J. and Atal, C.K. Indian Journal of Experimental Biology 14,356-357. Gupta, O.P., Mohd Ali, M., Ghatak Ray, B.J. and Atal, C.K. (1977) Indian Journal of Physiology and Pharmacology 21,33. Himes, N.E. (1963) Medical History of Contraceptives, Gamut Press Inc., New York. Karber, G. (1931) Archiu fiir Experimentelle Pathologie und Pharmakologie 162, 480-484. Kaviraj, K.B. (1963) Sushruta Somhita, 2nd edn., Chowkhamba Sanskrit series, Varanasi. Kirtikar and Basu (1933) Indian Medicinal Plants 1933,1-4. Koster, R.M., Anderson and De Beer, E.J. (1959) Federation Proceedings 18,412-414. Nadkarni, A.K. (1954) Indian Materia Medica, p. 478. Sastry, B.N. (1952) The Wealth ofindia, Vol. 3, New Delhi, p. 167.

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