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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright  © 2024 The  Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access   Full Text Article                                                                                                                                            Research Article

To Evaluate and Compare the Analgesic Activity of Piperine and Extracts of Piper Nigrum L. Fruit 

Monika Jain , Prakhar Nema , Sakshi Jain , Rubeena Khan , Shweta Dixena, Prateek Kumar Jain, Harshita Jain *

Adina College of Pharmacy, ADINA Campus Rd, Lahdara, Sagar, MP, 470001, India

 

 

INTRODUCTION

According to the International Association for the Study of Pain (IASP), pain is defined as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage” ¹.  Common drugs for pain relief such as aspirin and morphine have been widely used in recent decades. In most instances, these analgesic drugs, particularly opioids and nonsteroidal anti-inflammatory drugs (NSAIDs), can only relieve 50% of the pain in about 30% of patients².In addition, many of these drugs cause serious side effects. Studies have shown that opiates cause physical dependency, tolerance and addiction while NSAIDs usually cause gastrointestinal disorders³. As such, research to discover other alternatives to treat pain is crucial. Medicinal herbs have been used for centuries for therapeutic purposes. Many of these herbs with analgesic activity had been used without any adverse effects. Secondary metabolites are utilized by plants as shielding molecules and accountable for their biological efficacy and are distinctly effective for human beings because of their great influence in healthcare system⁴. Raw materials produced by plants are the absolute onset of curative radical and their chemical structures can be used for the exploration of new compounds⁵. Piper nigrum L. (P. nigrum) is one of the best-known species of Piperaceae family. It is cultivated in Pakistan, the hills of southwestern India as well as in South America and Africa. It is robust-woody climbing perennial plant. Piperine is a major alkaloid constituent of P. nigrum. The pungency of the black pepper is because of its alkaloidal constituents present in the fruit. It is commonly used as a spice all over the world and also possesses pharmacological properties and thus used in traditional system of medicine, such as Ayruvedic and Unani medicine for the treatment of various diseases such as fever, pain and inflammation⁶. In view of this, exploration of new analgesic and anti-inflammatory drugs to restrict these side effects is still a difficult project and researches are being carried out in order to find alternatives to NSAID and opiates. Plant-based drugs provide a lot of clues for finding new drugs. The aim of the present study is to evaluate the analgesic activities of the major constituents of P. nigrum fruit, piperine as well as hexane and ethanol extracts. Diclofenac sodium and acetylsalicylic acid were used as standard drugs for comparison. In view of the earlier studies and references available, it is evident that not many studies have been reported on analgesic activity of piperine and its hexane and ethanol extracts. Therefore the present study was undertaken to evaluate its analgesic activity on animal model, so as to support the local herbal drug manufacturing industry and to develop some topical preparations as an economical way to manage pain and inflammation of moderate severity. To ascertain the safety profile of piperine and the hexane and ethanol extract acute toxicity studies have also been performed. Acute toxicity refers to the adverse effects that occur on first exposure to a single dose of a substance, which can be manifested by the abnormal behaviors and other physiological activities like sedation, hyperactivity, hypo activity, pupil shrinkage, paralytic effect on hind limbs, writhes, erection of tail and body hairs, shivering of body, increase or decrease heart beat and furthermore the rate of mortality^7,8.

MATERIALS AND METHODS 

Plant material

The fruit of P. nigrum was purchased from local market of Sagar, MP, India. The taxonomical identification and authentication of the plant material was done by Dr. Zia Ul Hasan, Department of Botany, SAFIA College Bhopal (M.P). The specimens of voucher have been submitted and preserved in the herbarium of SAFIA College Bhopal (M.P).

Chemical reagents

Diclofenac sodium, piperine and acetylsalicylic acid were purchased from Sigma Aldrich Chemical Co. (Milwaukee, WI, USA). Dimethyl sulfoxide as a suspending agent was purchased from HiMedia Laboratories Pvt. Ltd. (Mumbai, India), India. All other chemical used in this study purchased from SD Fine-Chem Chem. Ltd. (Mumbai, India) and SRL Pvt. Ltd. (Mumbai, India).All the chemicals used in this study were of analytical grade. The pure piperine, hexane and ethanol extract of P. nigrum fruit were solubilized in 10% dimethyl sulfoxide for intra-peritoneal and oral administration to the test group at different doses according to the body weight.

Extraction 

Defatting of plant material

Powdered material of P. nigrum was shadow dried at room temperature. The shadow dried plant material was crudely powdered and subjected to extraction with petroleum ether (60-80°C) in a soxhlet apparatus. The extraction was incessant till the defatting of the material had taken place. 

Extraction by hot continuous percolation process

10 gm of P. nigrum dried material were exhaustively extracted indivisibly with 80% ethanol and hexane using hot continuous percolation for 24 hrs look of colorless solvent in the siphon tube was taken as the ending point of extraction. The extracts were concentrated to ¾ of its inventive volume by distillation. The concentrated extracts were taken in a china dish and evaporated on a thermostat controlled water bath till it forms a thick paste and stored in an air tight container free from any contamination until it was used. Finally the percentage yields were calculated of the dried extracts⁹.

Experimental animals

Swiss albino male mice (20-25 gm) were group housed (n= 6) under a standard 12 hr light/dark cycle and controlled conditions of temperature and humidity (25±2°C, 55-65%). Mice received standard rodent chow and water ad libitum. Animas were acclimatized to laboratory conditions for 7 days before carrying out the experiments. All the experiments were carried in a noise-free room between 08.00 to 15.00hr. Separate group (n=6) of mice was used for each set of experiments. The animal studies were approved by the Institutional Animal Ethics Committee (IAEC), constituted for the purpose of control and supervision of experimental animals by Ministry of Environment and Forests, Government of India, New Delhi, India.

Acute toxicity studies

Acute toxicity studies were conducted on Swiss albino mice as per the Organization for Economic Co-operation and Development (OECD) 423 guidelines¹⁰. Four different doses of piperine, hexane and ethanol extracts, i.e. 75, 50, 25 and 15 mg/kg body weight were administered intraperitoneally to the mice of each tested group. The mice in both test and control groups were allowed for free access to water and feed. The animals were observed continuously just after drug administration at 30 min, 60 min and 120 min interval of time for changes in general behaviors and physiological activities. Furthermore, the occurrence of mortality was noted up to 72 h. The maximum non-lethal dose was found to be 25 mg/kg body weight; hence 5, 10,20mg/kg dose was taken as effective dose for piperine and both extracts to evaluate analgesic activity.

Animals were divided into three major groups. Each group contain six animals (n=6).

Group-1: treated with saline water served as control group.

Group-2: drug treated group 

Group-3: further divided in three sub groups

Analgesic activity

Analgesic activity was investigated using following methods: (1) tail immersion method based on thermal radiant heat as a source of pain; (2) hot plate method based on jumping from hot plate at 55°C; (3) acetic acid induced writhing based on chemical radiant as a source of pain^11-13.

Tail immersion method

After administration of designed drugs as above mentioned, the base line latency was measured before and after drug treatment in a regular interval of 30 min, 60 min and 120 min by immersing the tail tips (1-2 cm) of the mice in water bath thermostatically maintained at temperature of (45±1) °C. The actual flick response of mice was measured by stop watch and results were compared with control and standard group. The maximum cutoff time for immersion was 180 seconds to avoid the injury of the tissues of tails.

Hot plate

Animals in all groups were individually exposed to the hot plate. The time taken in seconds for fore paw licking or jumping was taken as reaction time and was measured in a regular time interval and the reaction strength of each rat was determined before and after drug treatment in a regular interval of 30 min, 60 min and 120 min. A cutoff period of 15 seconds was set up to avoid damage to the paws. The groups administered with tested extracts and pure compound were compared to control and standard drug groups.

Acetic acid induced writhing

Tested extracts and pure compound were administered intraperitoneally using different doses as defined in study design, 30 min prior to administration of 0.1 mL acetic acid (1%). Animals were observed individually and the number of writhes was counted for 20 min commencing 5 min after injection of acetic acid. The significant reduction in number of writhes of treated groups was compared to that of the control and standard groups. The percentage inhibition of abdominal constrictions was calculated using the following formula.

Inhibition (%) =Mean No. of writhes (Control) - Mean No. of writhes (Test)/ Mean No. of writhes (Control) ×100

Statistical analysis

Data are given as mean ± standard error of mean. Data were analyzed with ANOVA where control group was compared with rest group.

RESULTS

Tail immersion method

Results of the analgesic activity of pure compound piperine, hexane and ethanol extracts measured by tail immersion method are given in Table 1. At a portion of 5 mg/kg and 10 mg/kg the piperine demonstrated great pain relieving impact when contrasted with control and standard medication. Piperine displayed most extreme movement after 120 min at a portion of 5 mg/kg when contrasted with control and standard medication while at a portion of 20 mg/kg the compound didn't indicated any further improvement in pain relieving action. The hexane separate indicated greatest pain relieving action at a portion of 10 mg/kg when contrasted with control and standard medication and the pain relieving impact expanded with entry of time and accomplished pinnacle level after 60 min. The ethanol separate indicated great pain relieving action at the portion of 5, 10 and 20 mg/kg when contrasted with control and less pain relieving action, when contrasted with standard at all portion level. Most extreme pain relieving impact was accomplished at a portion of 20 mg/kg after 120 min when contrasted with control and standard medication. Measurable computation demonstrated that P. nigrum L., has non-huge pain relieving movement by warm boosts or thermal stimuli.

 

 

Table 1: Analgesic activity of piperine and extract using tail immersion method in mice

Response time is the time taken by mice to pull back the tail, recorded by stop watch in a moment or two.

 

Hot plate method

Results of analgesic activity of pure compound piperine, hexane and ethanol extracts measured by hot plate method are given in Table 2. The piperine showed great pain relieving impact at a portion of 5 and 10 mg/kg when contrasted with control and standard. Greatest pain relieving impact was noted at a portion of 10 mg/kg after 120 min. The hexane and ethanol removes demonstrated helpless pain relieving impact at all dosages (5, 10 and 20 mg/kg) when contrasted with control and standard medication.

 

 

Table 2: Analgesic activity of piperine and extract using hot plate method in mice

 

Writhing method

The results of analgesic activity of pure compound piperine, hexane and ethanol extracts analyzed by writhing method are depicted in Table 3. Piperine displayed no pain relieving impact at dosages of 5 and 20mg/kg when contrasted with control and standard. The drug demonstrated most extreme security at a portion of 20 mg/kg (100%).hexane and ethanol removes indicated least pain relieving impact at all dosages of 5, 10 and 20mg/kg when contrasted with control and standard. However hexane separate demonstrated noteworthy assurance at a portion of 5 and 10 mg/kg (99.55%) and the ethanol extricate indicated greatest insurance at a portion of 20 mg/kg (100%).

 

 

Table 3: Analgesic activity of piperine and extract using writhing method in mice

 

 

DISCUSSION

In the present study analgesic activity of pure compound piperine, hexane and ethanol extracts of P. nigrum was analyzed by three different methods (tail immersion, hot plate and writhing method). An analgesic activity was determined at a dose of 5, 10 and 20 mg/kg. Acetyl salicylic acid and diclofenac sodium were used as standard reference drugs. Tail drenching technique which was utilized for assessing halfway acting pain relieving impacts of medications indicated no expansion in idleness. Pain relieving impact against warm poisonous boosts might be acquired through narcotic receptors or through tweak of a few synapses associated with important marvels. The general pain relieving impact of piperine, hexane and ethanol removes by warm boundary was expanded with section of time and most extreme impact was accomplished after 60 and 120 min. The oral organization of piperine, hexane and ethanol extricates test included the expanded nociceptive limit in mice and it had expanded up to 60 min and afterward diminished with entry of time. Noteworthy decrease in the creature affectability to torment brought about by the weight showed focal securing impact of piperine, hexane and ethanol separates were equivalent to acetyl salicylic corrosive. The pain relieving impact of piperine, hexane and ethanol concentrates of P. nigrum was researched by using whirlpool's hot plate strategy. This test included stamped focal pain relieving impact as confirm by noteworthy increment in response time. The hexane and ethanol concentrates of P. nigrum have no pain relieving at all dosages while unadulterated compound piperine demonstrated most extreme action at a portion of 10 mg/kg after 120 min by increment in the response time (increment edge capability of torment) might be because of the restraint of prostaglandins union. Squirming test is a concoction technique used to initiate agony of fringe beginning by infusion of aggravation standards like phenylquinone or acidic corrosive in mice. Pain relieving action of the test compound is surmised from decline in the recurrence of squirms. The squirming reaction is considered as a reflexive test. Signs transmitted to focal sensory system because of torment because of bothering, cause arrival of middle people, for example, prostaglandins which adds to the expanded affectability to nociceptors. Piperine, hexane and ethanol concentrates of P. nigrum diminished the quantity of squirms altogether at all dosages contrasted with reference tranquilize diclofenac sodium (NSAID) and control. Diminishes in squirms are commonly considered as a significant boundary of pain relieving action in acidic corrosive instigated squirming test.

CONCLUSION

In conclusion, from the present study, P. nigrum possess potent analgesic activity. In the present study, piperine revealed significant analgesic activity. Acute toxicity assessment results indicated zero percentage of mortality of P. nigrum at a dose of 20mg/kg, however at high doses (75, 50 and 25 mg/kg) 100% mortality were observed in mice after intraperitoneal administration. 

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