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Inhibition of morphine tolerance and dependence by Withania somnifera in mice
Journal of Ethnopharmacology 57 (1997) 213 – 217
Short communication
Inhibition of morphine tolerance and dependence by Withania somnifera in mice Shrinivas K. Kulkarni *, Ipe Ninan Pharmacology Di6ision, Uni6ersity Institute of Pharmaceutical Sciences, Panjab Uni6ersity, Chandigarh 160 014, India Received 10 December 1996; received in revised form 1 April 1997; accepted 6 June 1997
Abstract Chronic treatment with Withania somnifera (Ws) (family: Solanaceae, 100 mg/kg) commercial root extract followed by saline on days 1–9 failed to produce any significant change in tailflick latency from the saline pretreated group in mice. However, repeated administration of Ws (100 mg/kg) for 9 days attenuated the development of tolerance to the analgesic effect of morphine (10 mg/kg). Ws (100 mg/kg) also suppressed morphine-withdrawal jumps, a sign of the development of dependence to opiate as assessed by naloxone (2 mg/kg) precipitation withdrawal on day 10 of testing. © 1997 Elsevier Science Ireland Ltd. Keywords: Withania somnifera; Morphine; Tolerance; Dependence; Naloxone
1. Introduction The development of physical dependence and tolerance with repeated use is a characteristic feature of all the opioid drugs and offers major limitations in their clinical use. Tolerance and dependence are thought to result from neural adaptations produced by repeated exposure. The daily administration of morphine or other opioids to rodents in one or more injections per day has * Corresponding author.
been used for many years to produce a tolerant state which develops at a rate dependent on the specific drug, the dosage schedule, the interval between doses and the sensitivity of pharmacological assay. The signs of tolerance is measured by the classical method of assessing the antinociceptive response whereas dependence is measured in tolerant animals by the evocation of abstinence signs by abrupt drug withdrawal or the administration of a narcotic antagonist or both. Blasig et al. (1973) suggested that jumping was the most suitable sign of measuring abstinence quantita-
0378-8741/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 3 7 8 - 8 7 4 1 ( 9 7 ) 0 0 0 6 4 - 0
214
S.K. Kulkarni, I. Ninan / Journal of Ethnopharmacology 57 (1997) 213–217
tively because jumps are easily counted and the jumping rate increased when dependence increased or dose of antagonist increased. Withania somnifera (Aswagandha, family: Solanaceae), a plant with profound central nervous system (CNS) depressant actions has been recently shown to possess anticonvulsant properties in acute models in rats and mice via a GABAergic mechanism (Kulkarni and Verma, 1993; Kulkarni et al., 1993; Kulkarni and George, 1996). Investigations on Ws revealed its beneficial effects in stress, adaptogenesis, convulsion, cognitive dysfunctions, immunomodulation and geriatric problems (Ziauddin et al., 1996). Ws is popularly known as Indian ginseng as it possesses biological activity similar to that of Panax ginseng (family: Araliaceae) (Shibita et al., 1985). Recently, it has been reported that ginseng blocks the tolerance and dependence produced by cocaine and morphine (Kim et al., 1987, 1990, 1994). Therefore, the present experiments were undertaken to study the protective effect of Ws root extract on the development of tolerance and dependence to morphine in mice.
(09:00 and 16:00 h) for 9 days. On days 1, 3 and 9, antinociceptive response was assessed by the tailflick test 30 min after morphine injection. Various treatment groups (pretreatment:treatment) included: (i) Saline:saline; (ii) saline:morphine (10 mg/kg); (iii) Ws (100 mg/kg):saline; and (iv) Ws (100 mg/kg):morphine (10 mg/kg) (Trujillo and Akil, 1991). On 10th day, treatments were reversed so that the animals that had received Ws followed by morphine on days 1–9 were challenged with saline followed by morphine and animals that had been treated with saline followed by morphine on days 1–9 were challenged with Ws followed by morphine. In addition, the animals that had received chronic treatment with Ws followed by saline for 9 days were challenged with Ws followed by morphine on day 10. Ws was studied on rotarod apparatus to see whether it causes motor incoordination or not. Ws was given twice daily (09:00 and 16:00 h for 9 days and once (09:00 h) on 10th day. Animals were tested after 30 min of Ws adminstration on days 1, 3, 9 and 10.
2.3. Technique 2. Materials and methods
2.1. Animals Albino mice (Laka strain) of either sex bred in Central Animal House facility of the Panjab University, weighing 20 – 30 g were used. The animals were housed under standard light/dark cycle with food and water provided ad libitum. The experiments were performed between 09:00 and 17:00 h.
2.2. Treatment schedule In acute studies, the animals received saline or Ws root extract (100 mg/kg, p.o.) followed 30 min later by saline or morphine (10 mg/kg s.c.). The antinociceptive response to morphine was assessed by the tailflick test 30 min after the second injection. For induction of tolerance to morphine, animals received the opiate injection twice daily
Antinociceptive response was assessed by measuring tailflick latency to radiant heat as described by D’Armour and Smith (1941) and modified by Kulkarni (1980). A cut-off time of 10 s was observed to prevent any injury to the tail. A minimum of three trials was recorded for each animal. To assess the morphine withdrawal, mice were injected with naloxone (2 mg/kg i.p.) immediately after the tailflick test on day 10. The withdrawal syndrome was assessed by placing each mouse in a 30 cm high plexi glass box and recording the incidence of escape jumps for 15 min.
2.4. Drugs Commercial Ws root extract (Gufic, Bombay), morphine (Government Analytical Laboratory, Chandigarh) and naloxone (Endo, New York) were obtained from the respective sources.
S.K. Kulkarni, I. Ninan / Journal of Ethnopharmacology 57 (1997) 213–217
215
Fig. 1. Effect of morphine (10 mg/kg), Withania somnifera (Ws, 100 mg/kg) and combination of Ws and morphine on analgesic response on day 1 (A), day 3 (B), day 9 (C) and day 10 (D). ** PB 0.01 as compared with the saline:saline-treated control.
2.5. Statistical analysis The data expressed as mean 9S.E.M. were analysed by one way analysis of variance (ANOVA) followed by Student’s t-test. A value of P B0.05 was considered statistically significant.
3. Results
3.1. Effect of chronic administration of morphine on antinocicepti6e response Animals receiving chronic treatment with morphine (10 mg/kg) per se displayed maximal analgesia on day 1 and 3 (Fig. 1A,B). These animals exhibited tolerance to morphine on the 9th and
10th day of testing as reaction time was reduced (Fig. 1C,D).
3.2. Effect of chronic concomitant treatment with Ws and morphine on de6elopment of tolerance to morphine The animals treated repeatedly with Ws (100 mg/kg) showed significant antinociceptive response on days 1 and 3 of morphine (10 mg/kg) treatment (Fig. 1A,B). Pretreatment with Ws prevented the development of tolerance to the antinociceptive response of morphine on day 9 of testing (Fig. 1C). The animals that had been treated with Ws and morphine on days 1–9, then given morphine alone on day 10 also displayed considerable analgesia on the 10th day (Fig. 1D).
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S.K. Kulkarni, I. Ninan / Journal of Ethnopharmacology 57 (1997) 213–217
Table 1 Effect of Ws on naloxone-precipitated morphine withdrawal jumps in mice Group no.
Treatment (mg/kg)a
n
Number of jumps (Mean 9 S.E.)
1 2 3 4
Saline:saline Saline:morphine (10) Ws (100):saline Ws (100):morphine (10)
6 6 6 6
0 8.3 92.8 0 0
Naloxone (2 mg/kg i.p.) was given immediately after the tailflick test on day 10. See Section 2.
a
3.3. Effect of chronic treatment with Ws on antinocicepti6e response Chronic administration of Ws (100 mg/kg) followed by saline failed to elicit any analgesic response on days 1 and 3 in mice (Fig. 1A, B). Animals treated with Ws followed by saline displayed tailflick latencies not greater than salilne pretreated animals on days 1 – 9 (Fig. 1A,B,C). But on day 10 when these animals were challenged with morphine (10 mg/kg) following Ws, the antinociceptive response to morphine was blocked (Fig. 1D).
3.4. Effect of chronic treatment with Ws and morphine on nalaxone precipitated withdrawal jumps Animals that had received repeated administration of saline followed by morphine (10 mg/kg) displayed numerous escape jumps in response to an injection of naloxone (2 mg/kg) on day 10. In contrast, the animals treated with Ws (100 mg/kg) and morphine (10 mg/kg) on days 1 – 9, then with saline and morphine on day 10 failed to produce any jumps after naloxone (2 mg/kg) administration (Table 1). Animals that received Ws did not show any significant difference (P B0.05) in motor activity from control group when tested on days 1, 3, 9 and 10.
4. Discussion and conclusions In the present study, Ws inhibited the development of tolerance to analgesic response of mor-
phine and its physical dependence. Ws per se did not produce analgesia and acute treatment did not block morphine-induced analgesia as well. But after chronic treatment for 10 days Ws blocked morphine-induced analgesia. The mechanism of this observed inhibition of the abstinence syndrome in morphine tolerant and dependent animals by Ws remains unclear. Earlier, an antiaddictive profile of BR-16A (Mentat®), a safe non analgesic polyherbal preparation containing Ws as one of the components had been reported (Kulkarni and Verma, 1992). The present study shows that antiaddictive profile of BR-16A could be mainly due to the presence of Ws in it, as Ws per se has shown protection against development of tolerance and dependence to morphine. Ws has a wide spectrum of psychotropic and antistress activities, and being non analgesic per se, it may be used safely in deaddiction profile. Chronic treatment with Ws root extract inhibited development of tolerance to analgesic effect of morphine. It also inhibited development of withdrawal jumps. Ws is a safe non analgesic herbal preparation which can be used in the treatment of opiate addiction.
Acknowledgements The supply of Ws root extract (Aswal) by M/s Gufic, Bombay is acknowledged.
References Blasig, J., Herz, A., Reinhold, K., Zieglgansberger, S., 1973. Development of physical dependence on morphine in re-
S.K. Kulkarni, I. Ninan / Journal of Ethnopharmacology 57 (1997) 213–217 spect to time and dosage and quantifications of the precipitated withdrawal syndrome in rats. Psychopharmacologia 33, 19 – 38. D’Armour, F.E., Smith, D.L., 1941. A method for determining loss of pain sensation. Journal of Pharmacology and Experimental Therapeutics 72, 74–79. Kim, H.S., Oh, K.W., Park, W.K., Yamano, S., Toki, S., 1987. Effects of Panax ginseng on the development of morphine tolerance and dependence. Korean Journal of Ginseng Science 11, 182–190. Kim, H.S., Jang, C.G., Lee, M.K., 1990. Antinarcotic effects of the standardized ginseng extract G115 on morphine. Planta Medica 56, 158–163. Kim, H.S., Kang, J.G., Secong, Y.H., Nam, K.Y., Oh, K.W., 1994. Blockade of ginseng total saponin of the development of cocaine induced reversed tolerance and dopamine receptor supersensitivity in mice. Pharmacology Biochemistry and Behavior 50, 23–27. Kulkarni, S.K., 1980. Heat and other physiological stress induced analgesia: Catecholamine mediated and naloxone reversible response. Life Science 27, 185–188. Kulkarni, S.K., George, B., 1996. Anticonvulsant action of Withania somnifera (Aswagandha) root extract against
.
217
pentylenetetrazol-induced kindling in mice. Phytotherapy Research 10, 447 – 449. Kulkarni, S.K., Sharma, A., Verma, A., Ticku, M.K., 1993. GABA receptor mediated anticonvulsant action of Withania somnifera root extract. Indian Drugs 30, 305 – 312. Kulkarni, S.K., Verma, A., 1993. Aswagandha and Brahmi: Nootropic and deaddiction profile of psychotropic indigenous plants. Drugs of Today 29, 257 – 263. Kulkarni, S.K., Verma, A., 1992. Prevention of development of tolerance and dependence to opiate in mice by BR-16A (Mentat®). Indian Journal of Experimental Biology 30, 885 – 888. Shibita, S., Tanaka, O., Shoji, J., Saito, H., 1985. Chemistry and pharmacology of Panax ginseng. In: Wagner, H., Hikino, H., Fransworth, N.R., (Eds.), Economic and Medicinal Plant Research. Academic Press, London, pp. 325 – 380. Trujillo, K.A., Akil, H., 1991. Inhibition of morphine tolerance and dependence by the NMDA receptor antagonist MK-801. Science 251, 8587. Ziauddin, M., Phansalkar, N., Patki, P., Diwanay, S., Patwardhan, B., 1996. Studies on the immuno modulatory effects of Aswagandha. Journal of Ethnopharmacology 50, 69 – 76.
Short communication
Inhibition of morphine tolerance and dependence by Withania somnifera in mice Shrinivas K. Kulkarni *, Ipe Ninan Pharmacology Di6ision, Uni6ersity Institute of Pharmaceutical Sciences, Panjab Uni6ersity, Chandigarh 160 014, India Received 10 December 1996; received in revised form 1 April 1997; accepted 6 June 1997
Abstract Chronic treatment with Withania somnifera (Ws) (family: Solanaceae, 100 mg/kg) commercial root extract followed by saline on days 1–9 failed to produce any significant change in tailflick latency from the saline pretreated group in mice. However, repeated administration of Ws (100 mg/kg) for 9 days attenuated the development of tolerance to the analgesic effect of morphine (10 mg/kg). Ws (100 mg/kg) also suppressed morphine-withdrawal jumps, a sign of the development of dependence to opiate as assessed by naloxone (2 mg/kg) precipitation withdrawal on day 10 of testing. © 1997 Elsevier Science Ireland Ltd. Keywords: Withania somnifera; Morphine; Tolerance; Dependence; Naloxone
1. Introduction The development of physical dependence and tolerance with repeated use is a characteristic feature of all the opioid drugs and offers major limitations in their clinical use. Tolerance and dependence are thought to result from neural adaptations produced by repeated exposure. The daily administration of morphine or other opioids to rodents in one or more injections per day has * Corresponding author.
been used for many years to produce a tolerant state which develops at a rate dependent on the specific drug, the dosage schedule, the interval between doses and the sensitivity of pharmacological assay. The signs of tolerance is measured by the classical method of assessing the antinociceptive response whereas dependence is measured in tolerant animals by the evocation of abstinence signs by abrupt drug withdrawal or the administration of a narcotic antagonist or both. Blasig et al. (1973) suggested that jumping was the most suitable sign of measuring abstinence quantita-
0378-8741/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 3 7 8 - 8 7 4 1 ( 9 7 ) 0 0 0 6 4 - 0
214
S.K. Kulkarni, I. Ninan / Journal of Ethnopharmacology 57 (1997) 213–217
tively because jumps are easily counted and the jumping rate increased when dependence increased or dose of antagonist increased. Withania somnifera (Aswagandha, family: Solanaceae), a plant with profound central nervous system (CNS) depressant actions has been recently shown to possess anticonvulsant properties in acute models in rats and mice via a GABAergic mechanism (Kulkarni and Verma, 1993; Kulkarni et al., 1993; Kulkarni and George, 1996). Investigations on Ws revealed its beneficial effects in stress, adaptogenesis, convulsion, cognitive dysfunctions, immunomodulation and geriatric problems (Ziauddin et al., 1996). Ws is popularly known as Indian ginseng as it possesses biological activity similar to that of Panax ginseng (family: Araliaceae) (Shibita et al., 1985). Recently, it has been reported that ginseng blocks the tolerance and dependence produced by cocaine and morphine (Kim et al., 1987, 1990, 1994). Therefore, the present experiments were undertaken to study the protective effect of Ws root extract on the development of tolerance and dependence to morphine in mice.
(09:00 and 16:00 h) for 9 days. On days 1, 3 and 9, antinociceptive response was assessed by the tailflick test 30 min after morphine injection. Various treatment groups (pretreatment:treatment) included: (i) Saline:saline; (ii) saline:morphine (10 mg/kg); (iii) Ws (100 mg/kg):saline; and (iv) Ws (100 mg/kg):morphine (10 mg/kg) (Trujillo and Akil, 1991). On 10th day, treatments were reversed so that the animals that had received Ws followed by morphine on days 1–9 were challenged with saline followed by morphine and animals that had been treated with saline followed by morphine on days 1–9 were challenged with Ws followed by morphine. In addition, the animals that had received chronic treatment with Ws followed by saline for 9 days were challenged with Ws followed by morphine on day 10. Ws was studied on rotarod apparatus to see whether it causes motor incoordination or not. Ws was given twice daily (09:00 and 16:00 h for 9 days and once (09:00 h) on 10th day. Animals were tested after 30 min of Ws adminstration on days 1, 3, 9 and 10.
2.3. Technique 2. Materials and methods
2.1. Animals Albino mice (Laka strain) of either sex bred in Central Animal House facility of the Panjab University, weighing 20 – 30 g were used. The animals were housed under standard light/dark cycle with food and water provided ad libitum. The experiments were performed between 09:00 and 17:00 h.
2.2. Treatment schedule In acute studies, the animals received saline or Ws root extract (100 mg/kg, p.o.) followed 30 min later by saline or morphine (10 mg/kg s.c.). The antinociceptive response to morphine was assessed by the tailflick test 30 min after the second injection. For induction of tolerance to morphine, animals received the opiate injection twice daily
Antinociceptive response was assessed by measuring tailflick latency to radiant heat as described by D’Armour and Smith (1941) and modified by Kulkarni (1980). A cut-off time of 10 s was observed to prevent any injury to the tail. A minimum of three trials was recorded for each animal. To assess the morphine withdrawal, mice were injected with naloxone (2 mg/kg i.p.) immediately after the tailflick test on day 10. The withdrawal syndrome was assessed by placing each mouse in a 30 cm high plexi glass box and recording the incidence of escape jumps for 15 min.
2.4. Drugs Commercial Ws root extract (Gufic, Bombay), morphine (Government Analytical Laboratory, Chandigarh) and naloxone (Endo, New York) were obtained from the respective sources.
S.K. Kulkarni, I. Ninan / Journal of Ethnopharmacology 57 (1997) 213–217
215
Fig. 1. Effect of morphine (10 mg/kg), Withania somnifera (Ws, 100 mg/kg) and combination of Ws and morphine on analgesic response on day 1 (A), day 3 (B), day 9 (C) and day 10 (D). ** PB 0.01 as compared with the saline:saline-treated control.
2.5. Statistical analysis The data expressed as mean 9S.E.M. were analysed by one way analysis of variance (ANOVA) followed by Student’s t-test. A value of P B0.05 was considered statistically significant.
3. Results
3.1. Effect of chronic administration of morphine on antinocicepti6e response Animals receiving chronic treatment with morphine (10 mg/kg) per se displayed maximal analgesia on day 1 and 3 (Fig. 1A,B). These animals exhibited tolerance to morphine on the 9th and
10th day of testing as reaction time was reduced (Fig. 1C,D).
3.2. Effect of chronic concomitant treatment with Ws and morphine on de6elopment of tolerance to morphine The animals treated repeatedly with Ws (100 mg/kg) showed significant antinociceptive response on days 1 and 3 of morphine (10 mg/kg) treatment (Fig. 1A,B). Pretreatment with Ws prevented the development of tolerance to the antinociceptive response of morphine on day 9 of testing (Fig. 1C). The animals that had been treated with Ws and morphine on days 1–9, then given morphine alone on day 10 also displayed considerable analgesia on the 10th day (Fig. 1D).
216
S.K. Kulkarni, I. Ninan / Journal of Ethnopharmacology 57 (1997) 213–217
Table 1 Effect of Ws on naloxone-precipitated morphine withdrawal jumps in mice Group no.
Treatment (mg/kg)a
n
Number of jumps (Mean 9 S.E.)
1 2 3 4
Saline:saline Saline:morphine (10) Ws (100):saline Ws (100):morphine (10)
6 6 6 6
0 8.3 92.8 0 0
Naloxone (2 mg/kg i.p.) was given immediately after the tailflick test on day 10. See Section 2.
a
3.3. Effect of chronic treatment with Ws on antinocicepti6e response Chronic administration of Ws (100 mg/kg) followed by saline failed to elicit any analgesic response on days 1 and 3 in mice (Fig. 1A, B). Animals treated with Ws followed by saline displayed tailflick latencies not greater than salilne pretreated animals on days 1 – 9 (Fig. 1A,B,C). But on day 10 when these animals were challenged with morphine (10 mg/kg) following Ws, the antinociceptive response to morphine was blocked (Fig. 1D).
3.4. Effect of chronic treatment with Ws and morphine on nalaxone precipitated withdrawal jumps Animals that had received repeated administration of saline followed by morphine (10 mg/kg) displayed numerous escape jumps in response to an injection of naloxone (2 mg/kg) on day 10. In contrast, the animals treated with Ws (100 mg/kg) and morphine (10 mg/kg) on days 1 – 9, then with saline and morphine on day 10 failed to produce any jumps after naloxone (2 mg/kg) administration (Table 1). Animals that received Ws did not show any significant difference (P B0.05) in motor activity from control group when tested on days 1, 3, 9 and 10.
4. Discussion and conclusions In the present study, Ws inhibited the development of tolerance to analgesic response of mor-
phine and its physical dependence. Ws per se did not produce analgesia and acute treatment did not block morphine-induced analgesia as well. But after chronic treatment for 10 days Ws blocked morphine-induced analgesia. The mechanism of this observed inhibition of the abstinence syndrome in morphine tolerant and dependent animals by Ws remains unclear. Earlier, an antiaddictive profile of BR-16A (Mentat®), a safe non analgesic polyherbal preparation containing Ws as one of the components had been reported (Kulkarni and Verma, 1992). The present study shows that antiaddictive profile of BR-16A could be mainly due to the presence of Ws in it, as Ws per se has shown protection against development of tolerance and dependence to morphine. Ws has a wide spectrum of psychotropic and antistress activities, and being non analgesic per se, it may be used safely in deaddiction profile. Chronic treatment with Ws root extract inhibited development of tolerance to analgesic effect of morphine. It also inhibited development of withdrawal jumps. Ws is a safe non analgesic herbal preparation which can be used in the treatment of opiate addiction.
Acknowledgements The supply of Ws root extract (Aswal) by M/s Gufic, Bombay is acknowledged.
References Blasig, J., Herz, A., Reinhold, K., Zieglgansberger, S., 1973. Development of physical dependence on morphine in re-
S.K. Kulkarni, I. Ninan / Journal of Ethnopharmacology 57 (1997) 213–217 spect to time and dosage and quantifications of the precipitated withdrawal syndrome in rats. Psychopharmacologia 33, 19 – 38. D’Armour, F.E., Smith, D.L., 1941. A method for determining loss of pain sensation. Journal of Pharmacology and Experimental Therapeutics 72, 74–79. Kim, H.S., Oh, K.W., Park, W.K., Yamano, S., Toki, S., 1987. Effects of Panax ginseng on the development of morphine tolerance and dependence. Korean Journal of Ginseng Science 11, 182–190. Kim, H.S., Jang, C.G., Lee, M.K., 1990. Antinarcotic effects of the standardized ginseng extract G115 on morphine. Planta Medica 56, 158–163. Kim, H.S., Kang, J.G., Secong, Y.H., Nam, K.Y., Oh, K.W., 1994. Blockade of ginseng total saponin of the development of cocaine induced reversed tolerance and dopamine receptor supersensitivity in mice. Pharmacology Biochemistry and Behavior 50, 23–27. Kulkarni, S.K., 1980. Heat and other physiological stress induced analgesia: Catecholamine mediated and naloxone reversible response. Life Science 27, 185–188. Kulkarni, S.K., George, B., 1996. Anticonvulsant action of Withania somnifera (Aswagandha) root extract against
.
217
pentylenetetrazol-induced kindling in mice. Phytotherapy Research 10, 447 – 449. Kulkarni, S.K., Sharma, A., Verma, A., Ticku, M.K., 1993. GABA receptor mediated anticonvulsant action of Withania somnifera root extract. Indian Drugs 30, 305 – 312. Kulkarni, S.K., Verma, A., 1993. Aswagandha and Brahmi: Nootropic and deaddiction profile of psychotropic indigenous plants. Drugs of Today 29, 257 – 263. Kulkarni, S.K., Verma, A., 1992. Prevention of development of tolerance and dependence to opiate in mice by BR-16A (Mentat®). Indian Journal of Experimental Biology 30, 885 – 888. Shibita, S., Tanaka, O., Shoji, J., Saito, H., 1985. Chemistry and pharmacology of Panax ginseng. In: Wagner, H., Hikino, H., Fransworth, N.R., (Eds.), Economic and Medicinal Plant Research. Academic Press, London, pp. 325 – 380. Trujillo, K.A., Akil, H., 1991. Inhibition of morphine tolerance and dependence by the NMDA receptor antagonist MK-801. Science 251, 8587. Ziauddin, M., Phansalkar, N., Patki, P., Diwanay, S., Patwardhan, B., 1996. Studies on the immuno modulatory effects of Aswagandha. Journal of Ethnopharmacology 50, 69 – 76.