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 Table of Contents  
Year : 2021  |  Volume : 15  |  Issue : 3  |  Page : 193-197

Pharmacological evaluation of processed Cannabis leaves as a non sedative analgesic: The novel approach

1 Department of Dravyaguna, G.J. Patel Institute of Ayurvedic Studies & Research, New Vallabhvidyanagar, Anand, Gujarat, India
2 Pharmacology laboratory, Institute of Training and Research in Ayurveda, Jamnagar, Gujarat, India
3 Department of Dravyaguna, Institute of Training and Research in Ayurveda, Jamnagar, Gujarat, India

Date of Submission29-Dec-2020
Date of Decision15-Jan-2021
Date of Acceptance26-Jan-2021
Date of Web Publication25-Sep-2021

Correspondence Address:
Swagata Dilip Tavhare
Room 422, First Floor, Department of Dravyaguna, G. J. Patel Institute of Ayurvedic Studies and Research, New Vallabh Vidyanagar, Anand, Gujarat
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/joa.joa_330_20

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Background: Ayurvedic pharmacopoeia records vivid indications of Cannabis (Bhanga) including its analgesic, sedative and intoxicant actions. For inhibition of intoxication, Ayurveda recommends certain processing (Shodhana) techniques for Cannabis. The present study is planned to evaluate the analgesic potential and sedative effects of water washed (Jalaprakshalana)–processed Cannabis sativa leaves through pharmacological experimentation. Methods: Wistar strain albino rats weighing 200 ± 20 g and Swiss albino mice (25–35 g) of either sex were used in the study. Pharmacologically validated models were used to evaluate the analgesic effects (by formalin test and tail flick method) and neuromuscular coordination by rotarod experiment. water-washed–processed Cannabis leaves powder (test drug [TD]) was administered at dose of 22.5 and 45 mg/kg for rat and mouse, respectively, against reference standard morphine sulfate (MS) 5 mg/kg and the results were evaluated statistically. Results: TD at 22.5 and 45 mg/kg showed significant decrease in paw licking response at early phase in formalin test. TD at 22.5 mg/kg showed significant increase in tail flick latency after 3 h, and in 45 mg/kg dose, it was significant up to 4 h. In rotarod experiment, TD did not show any decrease in latency of fall-off time after 1 and 3 h. Conclusion: Water-washed processed Cannabis leaves powder possesses significant analgesic effect at 22.5 mg/kg (250 mg human dose) and 45 mg/kg (500 mg human dose) comparable to MS 5 mg while nonsignificant effect in neuromuscular in-coordination, thus devoid of sedative effect.

Keywords: Analgesic, Bhanga, Cannabis, Jalaprakshalana, Processing, Sedative, Shodhana, Vijaya

How to cite this article:
Tavhare SD, Nariya MB, Acharya R. Pharmacological evaluation of processed Cannabis leaves as a non sedative analgesic: The novel approach. J Ayurveda 2021;15:193-7

How to cite this URL:
Tavhare SD, Nariya MB, Acharya R. Pharmacological evaluation of processed Cannabis leaves as a non sedative analgesic: The novel approach. J Ayurveda [serial online] 2021 [cited 2022 May 19];15:193-7. Available from: http://www.journayu.in/text.asp?2021/15/3/193/326715

  Introduction Top

Pain is common and challenging to manage symptom in cases of patients affected with cancer, chemotherapy induced peripheral neuropathy and central nervous system diseases (CNS) etc.[1],[2] In view of reported opioids crisis[3] cost and side effects of analgesics,[4] there is need of alternative, cheaper and safe analgesic(s). Cannabis and its derived medicines offer a novel approach in pain pharmacotherapy.[5],[6],[7] but legal concern restricts its use.[8],[9] In order to mollify an intoxicant (Madakari)[10] adverse effect of Cannabis; Ayurveda advised certain processing (Shodhana) techniques.[11]

Present study conducted for evaluation of water-washed (Jalaprakshalana) processed Cannabis leaves through pharmacological evaluation

  Materials and Methods Top

Legal concern

Permission for conducting Cannabis related research work was taken from Excise department, Jamnagar.

Ethical clearance

Approval for experimentation was taken from the Institutional Animal Ethics Committee (No: IAEC/19/2015/36) in accordance with the guideline formulated by the Committee for the Purpose of Control and Supervision on Experiments on Animals, India.

Collection, identification, and preparation of test reference standard drug

Collection: Test drug (TD), i.e., Cannabis sativa L. dry leaves were procured from Haridwar, Uttarakhand, India, after obtaining legal permissions from appropriate authorities. It was verified by macroscopic and microscopic examination from the pharmacognosy experts.

TD was tied in muslin cloth and washed under running water till greenish color stops oozing out of cloth. Washed leaves later shade-dried and made into powder from and preserved in an airtight container.[12],[13]

Morphine sulfate (MS) 5 mg of four tablets were received from the Oncology Department, MP Shah Medical College, Jamnagar, for animal study purpose. Vehicle (Anupana) used, i.e., cow milk (Godugdha) and sugar were collected from the local cow-shade and grocery shop, respectively.


Chemicals and solvents of laboratory and analytical grade were used throughout this work. Pellet feed for rat and mice was procured of VRK brand supplied by Keval Sales Corporation, Vadodara.

Experimental animals

The analgesic and rotarod experiments were performed on an equal number of male and female in house bred Wistar albino rats (weighed 200 ± 20 g) and Swiss albino mice (weighed 25–35 g) of age of 6–8 weeks, respectively. Selected animals were kept under acclimatization for a week before experiment initiation, were housed in a cage made of polypropylene with stainless steel top grill and sterile paddy husk as bedding, were exposed to light and dark cycles for 12 h each with the relative humidity of 50%–70% and ambient temperature of 23°C ± 3°C, and were allowed free access to the standard pellet diet and water ad libitum.

Dose calculation

Dose calculation for animals was done by referring to the table of Paget and Barnes.[14] Ayurvedic Pharmacopoeia of India[11] recommends human dose of processed Cannabis leaf powder as 250 mg which is equivalent to animal dose 22.5 mg/kg body weight (BW) of rat and 32.5 mg/kg BW of mice.

Grouping of rats for analgesic experiment

Group I receiving distilled water (DW, 4.5 mL/kg, po) was assigned as the negative control (NC) while Group II rats receiving vehicle at a dose of 4.5 mL/kg, po served as vehicle control (VC). Group III and Group IV were given intervention of TD mixed in vehicle at a dose of 22.5 mg/kg (TD1) and 45 mg/kg (TD2). Fifth group receiving MS (5 mg/kg, po) served as positive control. TD was administered to animals by oral route with the help of feeding cannula.

Experimental design

Analgesic activity

Before experimentation, animals were fasted overnight. Before starting the experiment, mice in each group were allowed 20-min acclimatization in a transparent observation cage.[15] After acclimatization, the overnight-fasted mice were randomly selected and assigned into groups of five, each group with six mice. Analgesic activity was performed by formalin-induced lick test and tail flick method.

Formalin test

Rats that fasted overnight with the provision of water were used. Overnight-fasted mice were randomly selected and assigned into groups of five (as explained in grouping of rats), each group with six rats. Before starting the experiment, rats in each group were allowed 20-min acclimatization in a transparent observation cage.[15] After acclimatization, pain response was induced by injecting 0.1 ml of 2% v/v formalin in the subplantar region of the left hind paw by subcutaneous route. Considering that the number of paw lickings in specific duration was noted as an index of nociception, two phases of nociception, namely the early and late phase, were observed during the course of the experiment. The first phase (caused by direct motivation of the nociceptors) was recorded by taking the time of the animals spent licking their paw for 0–5 min after the injection of formalin. The second phase (inflammatory pain produced by release of inflammatory mediators) was recorded by taking the time the animal spent licking its paw for 11–30 min after formalin injection. The percentage inhibition of nociception for the two phases was calculated using the following formula:[16]

% inhibition = Control mean − test mean control mean × 100.

Tail flick test

Rats were placed on the tail flick unit so that constant heat intensity was applied to the lower third of the animal's tail. Tail removal from the radiant warmth was taken as end point. When the animal flicked its tail in response to the noxious stimulus, both the heat source and timer were stopped. Cut off time of 15 s was considered to avoid tail injury by heat. Basal reaction time of each animal to radiant heat was recorded on radiant heat source, and those having tail flick latency (TFL) <10 s were selected. Selected animals were randomly divided into five groups (n = 6), and vehicle, test drugs, and reference standard were administered to the respective groups 1 h before the experiment. The TFL was recorded at 30, 60, 120, 180, and 240 min after drug administration.[17]

Neuropharmacologic screening: Effect on motor coordination

Rotarod test

Effect on motor coordination was examined by the rotarod apparatus.[18] During pretest screening of training session, only mice that demonstrated their ability to remain on the revolving rod (20–25 rpm) for 5 min were selected for studies.

Group I receiving DW (4.5 mL/kg, po) was assigned as the NC while Group II receiving vehicle at a dose of 4.5 mL/kg, po served as VC. Group III was given intervention of TD mixed in vehicle at a dose of 22.5 mg/kg (TD1) while Group IV receiving reference standard MS (5 mg/kg, po) served as positive control.

Rotarod consists of a circular rod turning at a constant or increasing speed. Selected albino mice of one group simultaneously were placed on a horizontal rod having a diameter of 32 mm, separated by vertical barriers. Fall-off time was recorded at initial, after 1 h, and after 3 h of drug administration for each animal.[19] Time until the fall is the index of motor capability. The difference in the fall-off time from the rotating rod between the control and treated groups is taken as an index of muscle relaxation. Decrease in fall-off time is suggestive of depression of CNS.[18]

Statistical analysis

The data obtained in the study were tabulated and expressed as mean ± standard errors of the mean. Statistical analysis was carried out by one-way analysis of variance followed by Tukey's multiple “t” test and Student's “t” test for paired and unpaired data. Results were considered significant when P < 0.05.

  Results Top

Analgesic activity

Formalin test

TD significantly diminished the mean time of animals spent licking the injected paw in early phase. At dose 22.5 mg/kg dose, TD shortened the time of licking by 37.14% in early phase and 43.23% in late phase. At 45 mg/kg, it was shortened by 51.5% in early phase and 21.10% in late phase. Thus, statistically significant decrease in paw licking response was observed at TD1 and TD2 levels in early phase while nonsignificant decrease in paw licking response was observed in late phase intervals when compared to VC group. Response shown by TD2 and MS in decreasing paw licking activity was statistically equivalent and significant [Table 1].
Table 1: Effect formaldehyde-induced paw edema

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Tail flick method

In comparison to initial readings, NC group showed nonsignificant decrease in TFL up to 1 h while significant decrease after 2 h (P < 0.05) and 4 h (P < 0.02); VC group showed significant (P < 0.05) increase in TFL after 30 min, TD1 and TD2 showed statistically significant increase in TFL after 3 h, TD2 group showed statistically significant increase in TFL after 4 h, and MS group showed statistically significant increase in TFL response after 30 min, 2 h, 3 h, and 4 h [Table 2] and [Table 3].
Table 2: Effect on tail flick latency (initial to 3 h)

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Table 3: Effect on tail flick latency (3-4 h)

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Rotarod test

TD1 did not show any decrease in latency of fall-off time after 1 and 3 h in comparison to NC and VC. MS group showed statistically significant increase in latency of fall-off time after 1 and 3 h when compared to initial and NC [Table 4].
Table 4: Mean fall-off time in rotarod method

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  Discussion Top

Analgesic activity

Formalin test

Analgesic potential of TD is significant in early phase of formalin-induced pain while morphine potential is observed in early as well as late phase [Table 1]. Early phase, i.e., neurogenic phase, involves stimulation of nociceptors directly by a chemical and detected by Aδ fibers of the central nociceptive primary afferent terminals. Analgesic effects of Cannabis is due to antinociceptive nature of cannabinoids.[5] Tetrahydro-cannabinol (THC), an active component of Cannabis, has been proved to produce analgesic and antihyperalgesic effects in animal models.[20],[21] Processed Cannabis used in this study had 65% THC strength.[22]

Tail flick method

Water-washed processed Cannabis at 22.5 and 45 mg/kg produces significant analgesic activity in comparison to water (NC) [Table 2] and [Table 3].

The peak effect of morphine usually occurred 30 min after drug administration and continued up to 4 h, whereas peak effects of TDs appeared after 3 h, and in TD2, it continued thereafter and declined slowly. Although different dose levels of processed Cannabis and morphine were used for comparison, it is found that at 3 h, the effect of TD1, TD2, and MS is superimposable. Following processed Cannabis in dose 22.5 and 45 mg/kg and morphine at 5 mg/kg, essentially all animals reached maximum reaction time at 3 h. Morphine retained its antinociceptive efficacy throughout the study, as compared to NC group.

As thermal-induced nociception indicates narcotic involvement, an ability of drug to prolong the reaction latency to thermally induced pain by tail flick analgesiometer in rats is suggestive of central analgesic activity.

Rotarod test

Morphine showed significant muscle relaxant activity which was absent in processed Cannabis, suggestive of lack of neuromuscular impairment due to it[23] [Table 4]. Locomotor activity and muscle coordination are the indices of alertness and muscle relaxation.[24] Decrease in locomotor activity may lead to calming and sedation as a result of reduced excitability of the CNS.[25],[26] At 22.5 mg/kg, TD did not produce any significant muscle-relaxant activity, suggestive of non sedative effect [Table 4].

  Conclusion Top

The study concludes that Water-washed processed Cannabis leaves powder possesses significant analgesic effect at 22.5 mg/kg (250 mg human dose) and 45 mg/kg (500 mg human dose) comparable to MS 5 mg while nonsignificant effect in neuromuscular in-coordination, thus devoid of sedative effect.


Authors are thankful to Director IPGT&RA, Gujarat Ayurved University, for providing necessary permission to carry out research work. We acknowledge cooperation received from Excise Department, Jamnagar, for legal permission, legal drug dealers from Haridwar, Uttarakhand, and Dr. Agrawal, Professor and Head Oncology Department, MP Shah Medical College, Jamnagar, for help in procuring test drugs of experiment was acknowledged.

Financial support and sponsorship

Grant for the research work was provided by IPGT&RA, Ministry of AYUSH, Government of India, as a part of Ph.D. research project registration number 463.

Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2], [Table 3], [Table 4]


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