PHYTOTHERAPY RESEARCH Phytother. Res. 28: 937–939 (2014) Published online 14 November 2013 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5081
SHORT COMMUNICATION
Inhibitory Effect of Bacopasides on Spontaneous Morphine Withdrawal Induced Depression in Mice Khalid Rauf,1 Fazal Subhan,2 Muzaffar Abbas,3 Syed Mobasher Ali,1 Gowhar Ali,2 Muhammad Ashfaq1 and Ghulam Abbas1* 1
Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan Department of Pharmacy, University of Peshawar, K.P.K., Pakistan 3 Department of Pharmaceutical Sciences, College of Pharmacy, South Dakota State University, Brookings, USA 2
Bacopa monnieri is a perennial herb with a world known image as a nootropic. We investigated the effect of Bacopa monnieri methanolic extract (Mt Ext BM) 10, 20, and 30 mg/kg body weight (b.w) on acquisition and expression of morphine withdrawal induced depression in mice. Locally available Bacopa monnieri (BM) was screened for contents of Bacoside A3, Bacopasaponin C, and Bacopaside II using HPLC with UV. Morphine dependence was induced in mice using twice daily escalating chronic morphine treatments (20–65 mg/kg b.w) for eight consecutive days. Morphine withdrawal induced depression was assayed in animals using forced swimming test (FST), three days after last morphine injection. The HPLC analysis revealed that Mt-ext BM contained Bacoside A3 as major component, i.e. 4 μg in each mg of extract. The chronic treatment with Met Ext BM 10, 20, and 30 mg/kg b.w. dosing significantly inhibited opioid withdrawal induced depression in mice. These findings imply a newer potential role of Bacopa monnieri in the clinical management of opioid withdrawal induced depression which can be attributed to Bacoside A3. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: bacopasides; morphine withdrawal; depression; forced swim test; Bacopa monnieri.
INTRODUCTION Opiates are currently the drug of choice for the management of both acute and chronic malignant and non malignant pains (Birnbaum et al., 2006). Due to the extensive clinical utility of opiate prescription, drug abuse is a major issue before scientists and clinicians using opiates as first line therapy in chronic pain management (Banta-Green et al., 2010). Discontinuation of chronic opiates usage induces depression due to neuronal plasticity induced by chronic exposure to opioids (Goeldner et al., 2011). Opioid abstinence induced depression is one of the limiting factors next to tolerance that narrow therapeutic window of opiates and complicate the management of chronic pain management via opiates (Angst and Clark, 2006). The opioid withdrawal depression and relapse is present in more than 80% of the patients. This depression is one major proponent for treatment failure and switches back to opiate usage (Nunes et al., 2004). Bacopa monnieri (BM) is a renowned ayurvedic herb found in shady marshy places across the globe including Pakistan. BM has been used in Indian subcontinent for the management of memory disorders, insomnia, epilepsy, and cognition problems treatment. Additionally, BM has been reported to inhibit morphine tolerance and has antinociceptive effects comparable to morphine. It has been reported to have strong antioxidant * Correspondence to: Dr. Ghulam Abbas, Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology, Abbottabad22060, Pakistan. E-mail: [email protected]
Copyright © 2013 John Wiley & Sons, Ltd.
properties and has been found to enhance morphine analgesia (Rauf et al., 2011). Due to its strong antioxidant effect, BM has been found to have protectant effect against morphine toxicity (Sumathi and Niranjali Devaraj, 2009). BM has recently been reported to inhibit morphine sensitization via its antidopaminergic. BM has been found to have adenosinergic effect also and it inhibits diabetic neuropathic pain via Adenosine 1A receptor activation (Sahoo et al., 2010). Keeping in view its known opioid modifying role, this study was designed to investigate, first, contents of Bacopasaponin C, Bacopaside II, and bacoside A3 contents in methanolic extract of BM and, second, to investigate effect of BM on opioid withdrawal induced depression in mice.
MATERIAL AND METHODS Animals. Mice (Balb C) weighing 23–28 g, of either sex, bred in the animal house facility, Department of Pharmacy, University of Peshawar were used in the experiment. All procedures were approved by the Ethical Committee, Department of Pharmacy, University of Peshawar, Pakistan. Animals were kept at approved standards, of temperature 22 ± 2 °C, with 12 h light/12 h dark cycle, with free access to food and water. Chemicals and drugs. Commercial grade methanol was purchased from Haq Chemicals Limited Peshawar. Bacoside A3, Bacopaside II, and Bacopasaponin C were gifted by Prof Dr Ikhlas.A.Khan, School of Pharmacy, Mississippi University, USA. Morphine sulphate Received 18 July 2013 Revised 19 August 2013 Accepted 16 October 2013
K. RAUF ET AL.
Plant material. The plant was collected from Rumalee stream near Quaide Azam University Islamabad Pakistan in April. The plant identity was authenticated (voucher no.7421) by Prof Dr Muhammad Ibrar, Department of Botany, University of Peshawar Pakistan. The plant aerial parts were shade dried coarsely ground. Then, 500 g of this coarsely ground powdered plant was extracted with n-hexane, and then with acetone to remove fats and chlorophyll type pigments. The powdered plant was then further extracted with commercial grade methanol in soxhelet apparatus. The methanolic fraction of the plant (yield 14.37 g) was used during the experiments. Chromatographic analysis of Bacopa monnieri Methanolic extract for Bacopaside. Bacopaside ll, Bacoside A3, and Bacopasaponin C were quantified in methanolic extract of the plant using high performance liquid chromatography with UV detection using Phrompitayyarat method with slight modifications as described by Rauf et al. (2011). The HPLC system consisted of LC-20AT double pump (Shimadzu, Japan) and SPD-20A UV Visible detector, and C18 column (250 × 4.6 mm, 5 μm particle size), a Rheodyne injector with 20 μL loop. Briefly, 50 mg of Mt-ext BM was dissolved in 10 mL methanol (HPLC Grade) and was then centrifuged for 10 min at 3000 rpm. Then, this solution was filtered through 0.45 u filter. The mobile phase consisted of phosphoric acid 0.2% and acetonitrile (60:40 v/v), pH adjusted to 3.0 with 3 M NaOH. The HPLC system was run at 0.6 mL/min flow rate using wavelength of 205 nm. All the peaks were secured in 22 min run time. The peaks were confirmed by addition of standards Bacosides to the analyzing samples. Induction of morphine dependence in mice. Albino mice were made physically dependent on morphine with some modification (Rauf et al., 2011). Animals (n = 8) were injected morphine twice daily through intraperitoneal (i.p.) injection for eight days at the following schedule. Day 1, 20 mg/kg, day 2, 30 mg/kg, day 3, 40 mg/kg, day 4, 50 mg/kg, day 5, 60 mg/kg, and 65 mg/kg for days 6, 7, and 8 day. Control group (n = 8) received equal volume of saline twice daily. Evaluation of morphine withdrawal induced depression in mice. Opioid dependence was induced in albino mice (n = 8) by the method as described in the induction of morphine dependence procedure above. The animals were administered fluoxetine 30 mg/kg or Mt Ext BM 10, 20, or 30 mg/kg b.w. orally 72 h post last morphine injection once (acute) and for eight days (sub-acute). Control animals received equal volume of saline. After 1 h, animals were subjected to forced swimming test as described by Porsolt et al. (1977). Briefly animals were forced to swim in a plexi glass jar having 42 × 19 × 19 as height, length, and width, filled with water up to 17 cm and maintained at 25 ± 2 °C. Immobility time was noted after 1 min of adjustment time for the next 5 min swim time. Immobility behavior was defined as animals maintain an upright position with movements only to keep its head above water. Copyright © 2013 John Wiley & Sons, Ltd.
Statistical analysis. The data is expressed as mean ± S.E. M. of immobility time. Differences between various means were computed using one-way ANOVA. *p < 0.05, **p < 0.01, and ***p < 0.001 as compared to control.
RESULTS Quantification of Bacoside A major components in Mt-ext BM The HPLC analysis revealed that Mt-ext BM contained Bacoside A, major component. Our results further indicated that the quantity of these Bacopasides was 1.3 μg (Bacopasaponin C), 4 μg (Bacoside A3), and 1.3 μg (Bacopaside ll), in each mg of extract.
Effect of acute and sub-acute administration of Bacopasides on spontaneous morphine withdrawal induced depression Acute administration of Ext BM failed to arrest expression of opioid withdrawal induced depression in all three doses as shown in Fig. 1. However, the sub-acute (eight days) treatment dose dependently decreased in immobility time as compared to control (Fig. 2). Immobility time in Seconds
(Germany) was generously gifted through proper channel by M/S Punjab Drug House laboratories Lahore.
150 SAL MP
100
Meth-ext BM 10mg/kg(Acute) Meth-ext BM 20mg/kg(Acute) Meth-ext BM 20mg/kg(Acute)
50
FLX 30 mg/kg (Acute)
0
Figure 1. Effect of acute administration of saline, Met Ext BM, and fluoxetine on opioid withdrawal induced depression in mice. The figure showed the immobility time (mean ± S.E.M.) of morphine withdrawal mice in forced swim test following acute administration of methanol extract of Bacopa monnieri (Met Ext BM, 10, 20, or 30 mg/kg) and fluoxetine (30 mg/kg).
Immobility time in Seconds
938
150
SAL MP
100
50
Meth-ext BM 10mg/kg(Chronic)
***
Meth-ext BM 20mg/kg(Chronic)
***
Meth-ext BM 30mg/kg(Chronic)
***
***
FLX 30 (Chronic)
0
Figure 2. Effect of sub-acute administration of saline or Met Ext BM or fluoxetine on opioid withdrawal induced depression in mice. The figure showed the immobility time (mean ± S.E.M.) of morphine withdrawal mice in forced swim test following subacute (eight days) administration of methanol extract of Bacopa monnieri (Met Ext BM, 10, 20, or 30 mg/kg) and fluoxetine (30 mg/kg). *p < 0.05, **p < 0.01, and ***p < 0.001 as compared to control. Phytother. Res. 28: 937–939 (2014)
BACOPASIDES HEALS MORPHINE WITHDRAWAL INDUCED DEPRESSION
939
opioid withdrawal induced depression which can be attributed to Bacoside A3.
DISCUSSION Opioid withdrawal depression is one major proponent to relapse as chronic morphine usage enhances vulnerability to stress and this acute stress then translates into depression (Goeldner et al., 2011). Bacopa monnieri has been reported to have a strong antidepressant effect evidenced in various preclinical models and this effect has been reported to be mediated via multiple pathways, like nitric oxide synthase inhibition, serotonergic and dopaminergic and adenosinergic pathways (Sairam et al., 2002). Keeping this in mind, the standardized methanolic BM extract was evaluated for their antidepressant potential in morphine withdrawal depression. Our data showed that the chronic (Table 2), not acute (Table 1), administration of BM extract significantly reduced the immobility time in forced swim test. The current investigation suggests the potential role of Bacopa monnieri in the clinical management of
Acknowledgements We sincerely thank the Higher Education Commission of Pakistan for sponsoring the PhD scholar. We are also thankful to Prof. Dr. Ikhlas. A.Khan, the National Center for Natural Products Research, Mississippi, USA for the gift of HPLC standards of Bacosides. We sincerely thank and acknowledge the Ministry of Narcotics Control and the Ministry of Health for their help and support in acquisition of morphine sulphate. We acknowledge and appreciate the Punjab Drug House for the gift of morphine sulphate through proper channel.
Conflict of Interest The authors have declared that there is no conflict of interest.
REFERENCES Angst MS, Clark JD. 2006. Opioid-induced Hyperalgesia: A Qualitative Systematic Review. Anesthesiology 104: 570–587. Birnbaum HG, White AG, Reynolds JL, et al. (2006). Estimated costs of prescription opioid analgesic abuse in the United States in 2001: a societal perspective. Clin J Pain 22: 667–676. Banta-Green CJ, Von Korff M, Sullivan MD, Merrill JO, Doyle SR, Saunders K. 2010. The Prescribed Opioids Difficulties Scale: A Patient-centered Assessment of Problems and Concerns. Clin J Pain 26: 489–497. Goeldner C, Lutz P-E, Darcq E, et al. 2011. Impaired Emotional-Like Behavior and Serotonergic Function During Protracted Abstinence from Chronic Morphine. Biol Psychiatry 69: 236–244. Nunes EV, Sullivan MA, Levin FR. 2004. Treatment of depression in patients with opiate dependence. Biol Psychiatry 56: 793–802.
Copyright © 2013 John Wiley & Sons, Ltd.
Porsolt RD, Pichon ML, Jalfre M. 1977. Depression: a new animal model sensitive to antidepressant treatments. Nature 266: 730–732. Rauf K, Subhan F, Abbas M, Badshah A, Ullah I, Ullah S. 2011. Effect of Bacopasides on acquisition and expression of morphine tolerance. Phytomed 18: 836–842. Sumathi T, Niranjali Devaraj S 2009. Effect of Bacopa monniera on liver and kidney toxicity in chronic use of opioids. Phytomed 16: 897–903. Sahoo PK, Pradhan D, Behera P 2010. Neuroprotective Effect of Bacopa monnieri leaf extract targeted at Adenosine Receptor In Diabetic Neuropathic Pain. J Pharm Res 3: 1806–1809. Sairam K, Dorababu M, Goel R, Bhattacharya S. 2002. Antidepressant activity of standardized extract of Bacopa monniera in experimental models of depression in rats. Phytomed 9: 207–211.
Phytother. Res. 28: 937–939 (2014)
SHORT COMMUNICATION
Inhibitory Effect of Bacopasides on Spontaneous Morphine Withdrawal Induced Depression in Mice Khalid Rauf,1 Fazal Subhan,2 Muzaffar Abbas,3 Syed Mobasher Ali,1 Gowhar Ali,2 Muhammad Ashfaq1 and Ghulam Abbas1* 1
Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan Department of Pharmacy, University of Peshawar, K.P.K., Pakistan 3 Department of Pharmaceutical Sciences, College of Pharmacy, South Dakota State University, Brookings, USA 2
Bacopa monnieri is a perennial herb with a world known image as a nootropic. We investigated the effect of Bacopa monnieri methanolic extract (Mt Ext BM) 10, 20, and 30 mg/kg body weight (b.w) on acquisition and expression of morphine withdrawal induced depression in mice. Locally available Bacopa monnieri (BM) was screened for contents of Bacoside A3, Bacopasaponin C, and Bacopaside II using HPLC with UV. Morphine dependence was induced in mice using twice daily escalating chronic morphine treatments (20–65 mg/kg b.w) for eight consecutive days. Morphine withdrawal induced depression was assayed in animals using forced swimming test (FST), three days after last morphine injection. The HPLC analysis revealed that Mt-ext BM contained Bacoside A3 as major component, i.e. 4 μg in each mg of extract. The chronic treatment with Met Ext BM 10, 20, and 30 mg/kg b.w. dosing significantly inhibited opioid withdrawal induced depression in mice. These findings imply a newer potential role of Bacopa monnieri in the clinical management of opioid withdrawal induced depression which can be attributed to Bacoside A3. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: bacopasides; morphine withdrawal; depression; forced swim test; Bacopa monnieri.
INTRODUCTION Opiates are currently the drug of choice for the management of both acute and chronic malignant and non malignant pains (Birnbaum et al., 2006). Due to the extensive clinical utility of opiate prescription, drug abuse is a major issue before scientists and clinicians using opiates as first line therapy in chronic pain management (Banta-Green et al., 2010). Discontinuation of chronic opiates usage induces depression due to neuronal plasticity induced by chronic exposure to opioids (Goeldner et al., 2011). Opioid abstinence induced depression is one of the limiting factors next to tolerance that narrow therapeutic window of opiates and complicate the management of chronic pain management via opiates (Angst and Clark, 2006). The opioid withdrawal depression and relapse is present in more than 80% of the patients. This depression is one major proponent for treatment failure and switches back to opiate usage (Nunes et al., 2004). Bacopa monnieri (BM) is a renowned ayurvedic herb found in shady marshy places across the globe including Pakistan. BM has been used in Indian subcontinent for the management of memory disorders, insomnia, epilepsy, and cognition problems treatment. Additionally, BM has been reported to inhibit morphine tolerance and has antinociceptive effects comparable to morphine. It has been reported to have strong antioxidant * Correspondence to: Dr. Ghulam Abbas, Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology, Abbottabad22060, Pakistan. E-mail: [email protected]
Copyright © 2013 John Wiley & Sons, Ltd.
properties and has been found to enhance morphine analgesia (Rauf et al., 2011). Due to its strong antioxidant effect, BM has been found to have protectant effect against morphine toxicity (Sumathi and Niranjali Devaraj, 2009). BM has recently been reported to inhibit morphine sensitization via its antidopaminergic. BM has been found to have adenosinergic effect also and it inhibits diabetic neuropathic pain via Adenosine 1A receptor activation (Sahoo et al., 2010). Keeping in view its known opioid modifying role, this study was designed to investigate, first, contents of Bacopasaponin C, Bacopaside II, and bacoside A3 contents in methanolic extract of BM and, second, to investigate effect of BM on opioid withdrawal induced depression in mice.
MATERIAL AND METHODS Animals. Mice (Balb C) weighing 23–28 g, of either sex, bred in the animal house facility, Department of Pharmacy, University of Peshawar were used in the experiment. All procedures were approved by the Ethical Committee, Department of Pharmacy, University of Peshawar, Pakistan. Animals were kept at approved standards, of temperature 22 ± 2 °C, with 12 h light/12 h dark cycle, with free access to food and water. Chemicals and drugs. Commercial grade methanol was purchased from Haq Chemicals Limited Peshawar. Bacoside A3, Bacopaside II, and Bacopasaponin C were gifted by Prof Dr Ikhlas.A.Khan, School of Pharmacy, Mississippi University, USA. Morphine sulphate Received 18 July 2013 Revised 19 August 2013 Accepted 16 October 2013
K. RAUF ET AL.
Plant material. The plant was collected from Rumalee stream near Quaide Azam University Islamabad Pakistan in April. The plant identity was authenticated (voucher no.7421) by Prof Dr Muhammad Ibrar, Department of Botany, University of Peshawar Pakistan. The plant aerial parts were shade dried coarsely ground. Then, 500 g of this coarsely ground powdered plant was extracted with n-hexane, and then with acetone to remove fats and chlorophyll type pigments. The powdered plant was then further extracted with commercial grade methanol in soxhelet apparatus. The methanolic fraction of the plant (yield 14.37 g) was used during the experiments. Chromatographic analysis of Bacopa monnieri Methanolic extract for Bacopaside. Bacopaside ll, Bacoside A3, and Bacopasaponin C were quantified in methanolic extract of the plant using high performance liquid chromatography with UV detection using Phrompitayyarat method with slight modifications as described by Rauf et al. (2011). The HPLC system consisted of LC-20AT double pump (Shimadzu, Japan) and SPD-20A UV Visible detector, and C18 column (250 × 4.6 mm, 5 μm particle size), a Rheodyne injector with 20 μL loop. Briefly, 50 mg of Mt-ext BM was dissolved in 10 mL methanol (HPLC Grade) and was then centrifuged for 10 min at 3000 rpm. Then, this solution was filtered through 0.45 u filter. The mobile phase consisted of phosphoric acid 0.2% and acetonitrile (60:40 v/v), pH adjusted to 3.0 with 3 M NaOH. The HPLC system was run at 0.6 mL/min flow rate using wavelength of 205 nm. All the peaks were secured in 22 min run time. The peaks were confirmed by addition of standards Bacosides to the analyzing samples. Induction of morphine dependence in mice. Albino mice were made physically dependent on morphine with some modification (Rauf et al., 2011). Animals (n = 8) were injected morphine twice daily through intraperitoneal (i.p.) injection for eight days at the following schedule. Day 1, 20 mg/kg, day 2, 30 mg/kg, day 3, 40 mg/kg, day 4, 50 mg/kg, day 5, 60 mg/kg, and 65 mg/kg for days 6, 7, and 8 day. Control group (n = 8) received equal volume of saline twice daily. Evaluation of morphine withdrawal induced depression in mice. Opioid dependence was induced in albino mice (n = 8) by the method as described in the induction of morphine dependence procedure above. The animals were administered fluoxetine 30 mg/kg or Mt Ext BM 10, 20, or 30 mg/kg b.w. orally 72 h post last morphine injection once (acute) and for eight days (sub-acute). Control animals received equal volume of saline. After 1 h, animals were subjected to forced swimming test as described by Porsolt et al. (1977). Briefly animals were forced to swim in a plexi glass jar having 42 × 19 × 19 as height, length, and width, filled with water up to 17 cm and maintained at 25 ± 2 °C. Immobility time was noted after 1 min of adjustment time for the next 5 min swim time. Immobility behavior was defined as animals maintain an upright position with movements only to keep its head above water. Copyright © 2013 John Wiley & Sons, Ltd.
Statistical analysis. The data is expressed as mean ± S.E. M. of immobility time. Differences between various means were computed using one-way ANOVA. *p < 0.05, **p < 0.01, and ***p < 0.001 as compared to control.
RESULTS Quantification of Bacoside A major components in Mt-ext BM The HPLC analysis revealed that Mt-ext BM contained Bacoside A, major component. Our results further indicated that the quantity of these Bacopasides was 1.3 μg (Bacopasaponin C), 4 μg (Bacoside A3), and 1.3 μg (Bacopaside ll), in each mg of extract.
Effect of acute and sub-acute administration of Bacopasides on spontaneous morphine withdrawal induced depression Acute administration of Ext BM failed to arrest expression of opioid withdrawal induced depression in all three doses as shown in Fig. 1. However, the sub-acute (eight days) treatment dose dependently decreased in immobility time as compared to control (Fig. 2). Immobility time in Seconds
(Germany) was generously gifted through proper channel by M/S Punjab Drug House laboratories Lahore.
150 SAL MP
100
Meth-ext BM 10mg/kg(Acute) Meth-ext BM 20mg/kg(Acute) Meth-ext BM 20mg/kg(Acute)
50
FLX 30 mg/kg (Acute)
0
Figure 1. Effect of acute administration of saline, Met Ext BM, and fluoxetine on opioid withdrawal induced depression in mice. The figure showed the immobility time (mean ± S.E.M.) of morphine withdrawal mice in forced swim test following acute administration of methanol extract of Bacopa monnieri (Met Ext BM, 10, 20, or 30 mg/kg) and fluoxetine (30 mg/kg).
Immobility time in Seconds
938
150
SAL MP
100
50
Meth-ext BM 10mg/kg(Chronic)
***
Meth-ext BM 20mg/kg(Chronic)
***
Meth-ext BM 30mg/kg(Chronic)
***
***
FLX 30 (Chronic)
0
Figure 2. Effect of sub-acute administration of saline or Met Ext BM or fluoxetine on opioid withdrawal induced depression in mice. The figure showed the immobility time (mean ± S.E.M.) of morphine withdrawal mice in forced swim test following subacute (eight days) administration of methanol extract of Bacopa monnieri (Met Ext BM, 10, 20, or 30 mg/kg) and fluoxetine (30 mg/kg). *p < 0.05, **p < 0.01, and ***p < 0.001 as compared to control. Phytother. Res. 28: 937–939 (2014)
BACOPASIDES HEALS MORPHINE WITHDRAWAL INDUCED DEPRESSION
939
opioid withdrawal induced depression which can be attributed to Bacoside A3.
DISCUSSION Opioid withdrawal depression is one major proponent to relapse as chronic morphine usage enhances vulnerability to stress and this acute stress then translates into depression (Goeldner et al., 2011). Bacopa monnieri has been reported to have a strong antidepressant effect evidenced in various preclinical models and this effect has been reported to be mediated via multiple pathways, like nitric oxide synthase inhibition, serotonergic and dopaminergic and adenosinergic pathways (Sairam et al., 2002). Keeping this in mind, the standardized methanolic BM extract was evaluated for their antidepressant potential in morphine withdrawal depression. Our data showed that the chronic (Table 2), not acute (Table 1), administration of BM extract significantly reduced the immobility time in forced swim test. The current investigation suggests the potential role of Bacopa monnieri in the clinical management of
Acknowledgements We sincerely thank the Higher Education Commission of Pakistan for sponsoring the PhD scholar. We are also thankful to Prof. Dr. Ikhlas. A.Khan, the National Center for Natural Products Research, Mississippi, USA for the gift of HPLC standards of Bacosides. We sincerely thank and acknowledge the Ministry of Narcotics Control and the Ministry of Health for their help and support in acquisition of morphine sulphate. We acknowledge and appreciate the Punjab Drug House for the gift of morphine sulphate through proper channel.
Conflict of Interest The authors have declared that there is no conflict of interest.
REFERENCES Angst MS, Clark JD. 2006. Opioid-induced Hyperalgesia: A Qualitative Systematic Review. Anesthesiology 104: 570–587. Birnbaum HG, White AG, Reynolds JL, et al. (2006). Estimated costs of prescription opioid analgesic abuse in the United States in 2001: a societal perspective. Clin J Pain 22: 667–676. Banta-Green CJ, Von Korff M, Sullivan MD, Merrill JO, Doyle SR, Saunders K. 2010. The Prescribed Opioids Difficulties Scale: A Patient-centered Assessment of Problems and Concerns. Clin J Pain 26: 489–497. Goeldner C, Lutz P-E, Darcq E, et al. 2011. Impaired Emotional-Like Behavior and Serotonergic Function During Protracted Abstinence from Chronic Morphine. Biol Psychiatry 69: 236–244. Nunes EV, Sullivan MA, Levin FR. 2004. Treatment of depression in patients with opiate dependence. Biol Psychiatry 56: 793–802.
Copyright © 2013 John Wiley & Sons, Ltd.
Porsolt RD, Pichon ML, Jalfre M. 1977. Depression: a new animal model sensitive to antidepressant treatments. Nature 266: 730–732. Rauf K, Subhan F, Abbas M, Badshah A, Ullah I, Ullah S. 2011. Effect of Bacopasides on acquisition and expression of morphine tolerance. Phytomed 18: 836–842. Sumathi T, Niranjali Devaraj S 2009. Effect of Bacopa monniera on liver and kidney toxicity in chronic use of opioids. Phytomed 16: 897–903. Sahoo PK, Pradhan D, Behera P 2010. Neuroprotective Effect of Bacopa monnieri leaf extract targeted at Adenosine Receptor In Diabetic Neuropathic Pain. J Pharm Res 3: 1806–1809. Sairam K, Dorababu M, Goel R, Bhattacharya S. 2002. Antidepressant activity of standardized extract of Bacopa monniera in experimental models of depression in rats. Phytomed 9: 207–211.
Phytother. Res. 28: 937–939 (2014)
