Nature cures nature: Hypericum perforatum attenuates physical withdrawal signs in opium dependent rats

Abstract

Context: Hypericum perforatum Linn. (Hypericaceae) (St. John’s wort) attenuates opium withdrawal signs.

Aim: To explore the therapeutic potential of Hypericum perforatum in the management of opium-induced withdrawal syndrome.

Materials and methods: The effect of the Hypericum perforatum hydro-ethanol extract was investigated for potential to reverse naloxone (0.25 mg/kg)-induced opium withdrawal physical signs. Rats received opium extract (80–650 mg/kg) twice daily for 8 days along with Hypericum perforatum (20 mg/kg, orally) twice daily in chronic treatment and the same single dose 1 h before induction of withdrawal syndrome in the acute treated group.

Results: Hypericum perforatum reduced stereotype jumps and wet dog shake number in the chronic treatment compared to the saline control group (F(2, 24) = 3.968, p < 0. 05) and (F(2, 24) = 3.689, p < 0.05), respectively. The plant extract in the acutely treated group reduced diarrhea (F(2, 24) = 4.850, p < 0. 05 vs. saline). It decreased rectal temperature by chronic treatment at 30 min (F(2, 24) = 4.88, p < 0.05), 60 min (F(2, 240 = 5.364, p < 0.01) and 120 min (F(2, 24) = 4.907, p < 0.05).

Discussion and conclusion: This study reveals that the extract of Hypericum perforatum attenuates some physical signs of opium withdrawal syndrome possibly through direct or indirect interaction with opioid receptors. Further study is needed to clarify its mechanism.

• Diarrhea
• hypothermia
• jumps
• opium withdrawal signs reversal
• St. John’s wort
• wet-dog shakes

Introduction

Drug addiction is a major problem in the modern world. Opiates (opium, morphine and heroin), stimulants (cocaine, amphetamine), alcohol, marijuana and tranquilizers (benzodiazepines) are some of the major classes of abused drugs. An intense search for agents or combinations of agents that can prevent or modify dependence/addiction is of immense importance. Thus, numerous drugs or combinations of drugs have been proposed to modify or relieve the symptoms of drug dependence. In recent years, medical scientists have explored methods for the treatment of opioid dependence, but except for the substitution therapeutic agents like methadone and similar agents to detoxify opioid addiction, no reliable remedy is available to date. There is undoubtedly a need for safer and more effective treatments or measures that can provide some alleviation of the symptoms of a patient undergoing withdrawal from drugs of abuse. Numerous herbs have a range of sedative actions, encompassing analgesic, hypnotic, anti-depressant, anxiolytic activities, often combining two or more actions. They are designated generically as “nervenes”. Unlike most centrally acting pharmaceutical agents, nervene herbs are mild and gentle in activity, with complex and poorly understood multiple pharmacological effects. Available sedative herbs are usually used to treat moderate depression, insomnia and sleep disturbances. Hypericum perforatum is one such remedy.

Crude opium is still used as a pain killer and cough suppressant in the rural communities of countries like India, Pakistan and Afghanistan. Users of opium thus suffer from opioid tolerance, dependence and abstinence syndrome on nonavailability of dose. We are working on herbal plants in the management of opioid withdrawal syndrome and observed that H. perforatum is effective in ameliorating some of the physical signs of street heroin withdrawal syndrome (Subhan et al., 2009). In continuation to our previous work, this study was designed to probe further the usefulness of H. perforatum in the management of opium-induced withdrawal syndrome in an animal model of dependence.

Materials and methods

Plant material, extraction and administration

Hypericum perforatum was collected in May 2002 from Galliyat, Khyber Pakhtunkhwa, Pakistan. The specimen was authenticated by a taxonomist at the Department of Botany, University of Peshawar. A voucher bearing number PUP-012529 was obtained, and the specimen was submitted to the herbarium of the Botany Department of the University of Peshawar for future record. Aerial parts including flowers, leaves and stems of the plant were cut into small pieces and soaked in a glass jar with 70% ethanol for 24 h and then filtered via muslin, the method of soaking and filtration was repeated three-times (Williamson et al., 1998). All the filtrates were combined and evaporated to a thick semisolid mass on a waterbath at 40 °C. For oral administration of extracts, a feeding tube was used. The rats were held close to the body with the head firmly held between thumb and fingers while slowly inserting the tube along the side of mouth through the esophagus into the stomach. Great care was exercised to avoid suffocation and sliding the tube into the trachea.

Chemicals

Naloxone (Samchundang Pharm., Seoul, Korea), crude opium (Anti-narcotic Force, Pakistan), chloroform (BDH, Poole, England) and ethanol (Khazana Sugar Mills, Khyber Pakhtunkhwa, Pakistan) were used.

Animals

Sprague-Dawley rats of either sex, weighing 150–200 g grouped in cages with solid bases and sawdust bedding were used in this study. In opium dependence schedules, animals were transferred to grid floor cages to prevent suffocation due to possible cataleptic episodes associated with opium administration. Animals were allowed free access to water and food, maintained at 22.0 ± 2.0 °C and 12/12 h light/dark cycle. The experimental protocols were approved by the ethical committee of the Department of Pharmacy, University of Peshawar, Peshawar, Pakistan, and all experiments were performed in compliance with rulings of the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (1996).

Purification of crude opium

About 20 g of crude opium was crushed to powder, dissolved in sufficient distilled water, shaking frequently at 60 °C and filtered through muslin. The residue was re-dissolved in a mixture of equal amounts of ethanol and chloroform, shaken vigorously and left overnight to facilitate extraction. The mixture was then filtered through muslin, and the filtrate was combined with an aqueous filtrate and evaporated to a thick liquid in a rotary evaporator at 40 °C under reduced pressure and then to a semisolid material on a waterbath maintained at 60 °C. The yield was 93.30%.

Opium dependence induction

Opium dependence was induced using an 8-day modified schedule as previously described (Subhan et al., 2000). Briefly, animals received opium twice daily orally at 09:00 h and 19:00 h, starting at a dose of 80 and increasing to 650 mg/kg. Doses on each day were as follows: First day (80, 100 mg/kg), second day (150, 200 mg/kg), third day (250, 300 mg/kg), fourth day (350, 400 mg/kg), fifth day (450, 500 mg/kg), sixth day (550, 600 mg/kg), seventh day (650, 650 mg/kg) and eighth day (650, 650 mg/kg). Animals also received a morning dose of 650 mg/kg on the test day (day nine). Treatment groups were divided as: (I) Opium treatment group: this group received opium dependence inducing dosing schedule along with saline and served as a saline control to compare with the effects of the other treatments on the withdrawal syndrome. (II) Hypericum perforatum acute treatment group: in addition to the opium dependence schedule, this group also received a single dose of 20 mg/kg H. perforatum extract 1 h before precipitation of the withdrawal syndrome. (III) Hypericum perforatum chronic treatment group: this treatment group received 20 mg/kg H. perforatum twice a day along with opium dependence protocol.

Physical signs of withdrawal

Physical signs of withdrawal including jumps, wet-dog shakes, diarrhea vocalization and salivation were monitored, scored and recorded during the naloxone-induced withdrawal phase. Jumps and wet-dog shakes were scored on incidence while diarrhea, vocalization and salivation were ranked 1, 2 and 3 equating to mild, moderate and severe, respectively. Naloxone-induced hypothermia was also measured at 0, 30, 60 and 120 min using rectal probes.

Statistical analysis

Results were expressed as mean ± SEM. For calculating significance among the groups, analysis of variance (ANOVA) with Dennett’s post-hoc test was employed using the statistical package of Graph Instate. Student t-test was applied to date where appropriate. p < 0.05 was noted a significant difference.

Results

Effect on jumps

Figure 1 shows the effect of H. perforatum extract on the naloxone-induced jumping behavior of rats. ANOVA with Dennett’s post-hoc test revealed that the chronically treated group with the plant extracts significantly reduced the number of stereotype jumps as compared to the saline control group (F(2, 24) = 3.968, p < 0.05). However, the acute treatment with H. perforatum extract failed to show any significant effect, though a trend toward reduction was clearly observed.

Figure 1. Effect of H. perforatum extract (Hp Ext) on naloxone (NLX)-induced opium withdrawal jumps in rats. Each column represents the mean ± SEM (n = 9). *p < 0.05, values significantly different as compared to saline (ANOVA followed by Dunnett’s post-hoc test). SAL = Saline.

Effect on wet-dog shakes

Figure 2 demonstrates the effect of H. perforatum extract on the body shakes of rats (wet-dog shakes). Statistical analysis showed that the chronically treated group with plant extract significantly reduced the number of wet-dog shakes as compared to the saline control group (ANOVA with Dennett’s post-hoc test (F(2, 24) = 3.689, p < 0.05). However, the acute treatment failed to show any significant effect.

Figure 2. Effect of H. perforatum extract (Hp Ext) on naloxone (NLX)-induced opium withdrawal wet dog shakes in rats. Each column represents the mean ± SEM (n = 9). *p < 0.05, values significantly different as compared to saline (ANOVA followed by Dunnett’s post-hoc test). SAL = Saline.

Effect on diarrhea

The Figure 3 depicts the effect of H. perforatum on the naloxone-induced diarrhea. ANOVA followed by Dennett’s post-hoc analysis revealed that the acutely treated group with plant extract significantly reduced diarrhea as compared to the saline control group (F(2, 24) = 4.850, p < 0.05). Interestingly, the chronically treated group does not show any significant response.

Figure 3. Effect of H. perforatum extract (Hp Ext) on naloxone (NLX)-induced opium withdrawal diarrhea in rats. Each column represents the mean ± SEM (n = 9). *p < 0.05, values significantly different as compared to saline (ANOVA followed by Dunnett’s post-hoc test). SAL = Saline.

Effect on hypothermia

As shown in the Figure 4, H. perforatum extract potentiated naloxone-induced hypothermia. The decrease in the rectal temperature was significant by the chronic treatment at 30 min (ANOVA, F(2, 24) = 4.88, p < 0.05), 60 min (ANOVA, F(2, 240 = 5.364, p < 0.01) and 120 min (ANOVA, F(2, 24) = 4.907, p < 0.05). The acute treatment does not achieve any significance in this study, when compared with saline control.

Figure 4. Effect of H. perforatum extract (Hp Ext) on naloxone (NLX)-induced opium withdrawal hypothermia. Values shown are mean ± SEM (n = 9). *p < 0.05, **p < 0.01, values significantly different as compared to saline (ANOVA followed by Student’s t test). SAL = Saline.

Effect on salivation, squeal-on-touch, lying-painting and ptosis

The extract failed to produce any significant effect either in the chronic or acute treatment group on the salivation, squeal on touch, lying painting and ptosis behaviors of rats undergoing naloxone-induced opium withdrawal (Figure 5a–d).

Figure 5. Effect of H. perforatum extract (Hp Ext) on naloxone (NLX)-induced opium withdrawal salivation (a), squeal on touch (b), lying painting (c) and ptosis (d). Values shown are mean ± SEM (n = 9). p > 0.05, values not significantly different as compared to saline (ANOVA followed by Dunnett’s post-hoc test). SAL = Saline.

Discussion

Results of this study demonstrate that the crude hydro-ethanolic extract of H. perforatum decreases some of the naloxone-induced opium withdrawal physical signs at the 20 mg/kg body weight dose. A significant decrease was observed in jumps and wet-dog shakes in chronically treated groups only and diarrhea in the acutely treated group. However, acute treatment with H. perforatum extract did not show any significant effect on either naloxone-induced withdrawal jumps or wet-dog shakes behavior. Interestingly, the extract maintained a significant fall in temperature in the chronic treatment group. Furthermore, the extract treatment also failed to show any notable effect on the salivation, squeal on touch, lying painting and ptosis behaviors.

It has been reported in the literature that binding of [³H]-naloxone to the mu (μ) and kappa (κ)-opioid receptor was inhibited in the presence of H. perforatum extract, showing IC₅₀ values of approximately 25 and 90 µg/ml, respectively (Simmen et al., 1998). Furthermore, a direct binding of hypericin, one of the main active constituents of H. perforatum, to sigma (σ) opioid receptors has been demonstrated in an in vitro study model (Raffa, 1998). These observations suggest that the constituents of the plant extract exhibit opioid receptor binding properties, which may lead to displacement of naloxone from the opioid-binding site. It has been also suggested that the agent having the ability to displace naloxone from the opioid receptors binding sites either directly or indirectly ameliorates opioid withdrawal symptoms in rats (Subhan et al., 2000). It is, therefore, possible that H. perforatum extract may have reduced the signs of opium withdrawal by the displacement of naloxone from their binding sites.

In a separate study, we have also observed that the plant extract produced dose-dependent anti-nociceptive effect in mouse abdominal constriction assay. This effect was antagonized significantly by opioid antagonist, naloxone, thus indicating the interaction of Hypericum constituents with opioid receptors (Subhan et al., 2007). It is also well authenticated that Hypericum preparations has antidepressant properties mediated through increased neurotransmission of monoamines, including 5-hydroxytryptamine (5-HT), noradrenaline (Misane & Ogren, 2001), dopamine (Franklin & Cowen, 2001), gamma amino butyric acid and l-glutamate (Calapai et al., 1999; Chatterjee et al., 1998; Nathan, 1999). It is also known that 5-HT plays a significant role in the mechanisms of analgesia (Sewell, 1991) and opioid dependence (Akaoka & Aston-Jones, 1993). Moreover, 5-HT favors the development of physical dependence to morphine, and agents that facilitate 5-HT transmission should mimic the effects of morphine and prevent opioid withdrawal syndrome precipitated by naloxone (Samanin et al., 1980).

Moreover, this study corroborates previous findings of our laboratory (Subhan et al., 2009) and other laboratories (Feily & Abbasi, 2009), where H. perforatum extract has been reported to attenuate heroin and morphine withdrawal syndromes, respectively. This study is unique of its kind in the sense that crude opium is used as a dependence-inducing agent, which simulates common practice in rural communities where opium is used as a remedy for cough and pain conditions, and a natural remedy of H. perforatum extract is suggested for people to manage the withdrawal symptoms of opium dependence.

Conclusions

This study reveals that the hydro-ethanolic extract of H. perforatum attenuates some of the naloxone-precipitated opium withdrawal physical signs either through opioid receptor interactions or enhanced 5-HT transmission pathways. As there are also reports that enhanced 5-HT level increases the messengers of opioid peptides (Rossby et al., 1996), it is conceivable that the plant extract may have increased opioidergic activity indirectly through enhanced 5-HT transmission. However, further study of interaction between opioid receptors and the extract of H. perforatum and its effect on the neurotransmitter levels, particularly 5-HT, is warranted to clarify the mode of action of H. perforatum in terms of drug dependence.

Declaration of interest

The authors report no declaration of interest. We also acknowledge the financial support of Peshawar University.

Acknowledgements

We gratefully acknowledge the help and cooperation for supply of opium by the Anti-narcotic force, Peshawar Division.