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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 2  |  Issue : 1  |  Page : 38-43

Anti-inflammatory and analgesic activity of Punarnavashtak Kwath, an ayurvedic formulation


Department of Rasa-Shastra, Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India

Date of Submission27-Nov-2016
Date of Acceptance16-Apr-2017
Date of Web Publication1-Jun-2017

Correspondence Address:
Vineet Sharma
Department of Rasa-Shastra, Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bjhs.bjhs_45_16

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  Abstract 

Context: Punarnavashtak Kwath (PNK) is a polyherbal formulation assumed to have the potential for curing all types of inflammation, hepatic disorders, and asthma.
Aims: This study was conducted to establish the anti-inflammatory and analgesic activity of ethanol extract of PNK (EEPNK) in albino rats and mice.
Subjects and Methods: The parts of the plant were air -dried in shade and pulverized coarsely and sieved by 20# sieve. Powdered material of plant was defatted with pet ether for 1 day followed by Soxhlet extraction with ethanol (95% v/v) for 7 days. The anti-inflammatory activity of the EEPNK was evaluated by egg albumin-induced paw edema and cotton pellet-induced granuloma tests to establish its effect on acute and chronic stage of inflammation models in albino rats. Whereas analgesic effects of EEPNK were performed in mice of either sex by acetic acid-induced writhing and hot plate methods. Statistics was expressed as a mean ± standard error of mean and statistical analysis was carried out by one-way ANOVA with Tukey's test.
Results: The percent inhibition in rat paw volume was dose dependent (79.14%, 68.51%, and 61.70% at doses of 400, 200, and 100 mg/kg, p.o., respectively) at 240 min readings. Cotton pellet granuloma in rats (chronic study) model EEPNK (400, 200 and 100 mg/kg, p.o.) extensively decreased the granuloma weight as compared to the negative control animals. Pretreatment with EEPNK was (400, 200, and 100 mg/kg p.o.) extensively (P < 0.001) inhibited of writhings in mice, and also significantly (P < 0.001) increased pain latency in hot plate test.
Conclusions: Distinguish with the standard drug (indomethacin), the elevated dose of EEPNK (400 mg/kg b.w.) has a comparable effect on the anti-inflammatory and analgesic activities.

Keywords: Analgesic, anti-inflammatory, Indomethacin, Punarnavashtak Kwath


How to cite this article:
Sharma V. Anti-inflammatory and analgesic activity of Punarnavashtak Kwath, an ayurvedic formulation. BLDE Univ J Health Sci 2017;2:38-43

How to cite this URL:
Sharma V. Anti-inflammatory and analgesic activity of Punarnavashtak Kwath, an ayurvedic formulation. BLDE Univ J Health Sci [serial online] 2017 [cited 2019 Mar 18];2:38-43. Available from: http://www.bldeujournalhs.in/text.asp?2017/2/1/38/207424

In the conventional system of Indian medicine, plant formulation of different plant parts and its extracts are used as the drug of alternative quite than single plant extracts. These herbal formulations are used for the healing of a broad range of ailments.[1] Medicinal plants used in folkloric healing of pain-related disorders have a long and accepted usage [2] mainly in developing countries.[3] Inflammation is naturally differentiated by redness, puffiness, pain, along with heat, which is produced by the body's reaction against mugging pathogens.[4] Nonsteroidal anti-inflammatory drugs, such as indomethacin and diclofenac is approved globally for the management of anti-inflammatory, analgesic as well as cardio disease. Although its use is partial due to gastrointestinal damage.[5],[6] In India, China and Sri Lanka for the treatment of inflammation in ayurvedic as well as other traditional medical practitioners use many herbal preparations,[7] yet the claimed pharmacological activities of many polyherbal formulations have not been established nor refuted by controlled studies. According to Indian classical literature (Bhaisajya Ratnavali and Sarangdhar Samhita, Chakradatta Udarroga), Punarnavashtak Kwath (PNK) is a polyherbal formulation assumed to contain the potential for curing all types of inflammation. PNK is prepared from parts of eight different plants which are used in conventional ayurvedic drug for a variety of function such as reduction of pain, inflammation Azadirachta indica,[8]Picrorhiza kurroa,[9]Zingiber officinale Roscoe,[10],[11]Trichosanthes dioica Roxb.,[12]Terminalia chebula Retz.,[13] and other many disorders such as Pandu (anemia), Kasa (cough), Udar roga (abdominal pain), Swasa (dyspnea), and also to able to improve appetite and digestion (act as immunostimulants (Tinospora cordifolia acts as diuretic and laxative). The anti-inflammatory and analgesic activity of PNK have not been subjected till time so it has been carried out using in vivo experimental models to calculate the anti-inflammatory and analgesic activity of ethanol extract of PNK (EEPNK).


  Subjects and Methods Top


Chemicals and reagents

Indomethacin was buying from Cipla, India. Glacial acetic acid, ethanol, and carboxymethylcellulose (CMC) sodium were purchased from S D Fine-Chem Limited; double distilled water was used in preparation of all solutions. Morphine (Martindale Pharmas, Essex, United Kingdom), the other chemicals as well as reagents were of analytical grade and were procured from HiMedia Research Laboratories Pvt. Ltd. and S D Fine-Chem Limited, Mumbai, India.

Plant material and extraction

A. indica bark, T. dioica leaves, T. cordifolia stem, and Boerhavia diffusa whole plant were collected in the month of April (2016) from medicinal garden of Rajiv Gandhi South Campus, Banaras Hindu University, Varanasi, India. While other plants, P. kurroa stem, Berberis aristata stem, T. chebula fruit, and Z. officinale rhizome, were procured from authentic shop at Gola Dinanath, Varanasi. The identification of the plant material was done by Prof. A. K. Singh at Department of Dravyaguna, Banaras Hindu University, Varanasi, India. The voucher specimen (APRL/HERB/16-17/18-25) of the plant has been deposited in Department of Ayurvedic Pharmacy Research Laboratory, Rajiv Gandhi South Campus, Banaras Hindu University, Mirzapur (Uttar Pradesh), India, for the further reference. All parts of the plant were air-dried in shadow and pulverized coarsely and sieved by 20# sieve. Powdered material of plant was defatted with pet ether for 1 day followed by Soxhlet extraction with ethanol (95% v/v) for 7 days. The extract was concentrated and also evaporated under reduced pressure in a rotary evaporator to reduce the residual toxicity and was then set aside in desiccators until use.

Acute toxicity studies

The acute oral toxicity study of EEPNK was performed according to the Organization for Economic Co-operation and Development-425 guidelines. A single dose of EEPNK 2000 mg/kg, p.o., was administered in 24 h fasted rats (n = 5), and rats were observed at 0, 30, 60, 120, 180, and 240 min and then once a day for the next 2 weeks for any sign or indication of toxicity. The number of rats to stay living at the end of the study time was recorded.[14]

Animals

Adult Charles Fosters albino rats (160 ± 25 g) and Swiss albino mice (25–30 g) of either sex were used for this study. Animals were obtained from the Central Animal House, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. The animals were housed in big polypropylene cages at a controlled room temperature (22°C ± 2°C) and humidity (55% ± 10%). 12 h light and 12 h dark cycle were also followed. The animals were provided with standardized pelleted feed (Amrut Pvt. Ltd.) and fresh drinking water. The rats were acclimatized to the standard laboratory condition for at least 1 week before using the experiment. Body weight of rats was measured regularly. The principle of laboratory animal care guidelines (NIH Publication Number 85-23, revised 1923) was followed. Prior authorization has been taken from the Institutional Animal Ethical Committee (Reg. No. Dean/15-16/CAEC/730).

Preparation of dosage form

The EEPNK was prepared by triturating the accurately weighed quantity of the extract against each subject body weight, with 0.5% solution of CMC for oral route of administration to test group. Indomethacin was dissolved in CMC for oral route administration.

Anti-inflammatory activity

Egg white-induced hind paw edema

The anti-inflammatory activity of PNK was evaluated by egg albumin-induced rat paw edema model.[15] Animals were indiscriminately divided into five groups containing six animals in each group (n = 6). In the subplantar area of right hind paw of rats, 0.1 mL carrageenan suspension (1% w/w prepared in normal saline) was injected. One hour before egg albumin injection, Group I (normal control) received only vehicle (0.5% CMC, p.o.), Group II received indomethacin (10 mg/kg) and served as standard treatment group, and Groups III, IV, and V (served as test treatment groups) received EEPNK 100, 200, and 400 mg/kg, respectively. Increase in the volume of right hind paw was calculated by plethysmometer following the injection of egg albumin at 0 h and after 1 h interval up to 6 h. Anti-inflammatory activity of EEPNK was considered as their capability to diminish paw edema volume through respect to control.

Cotton pellet-induced granuloma

Diethyl ether was employed to anesthetize the rats after shaving off their fur. Implant of preweighed, sterile cotton pellets (10 mg) was made in the axilla area of rat. Group I (normal control) received only vehicle (0.5% CMC, p.o.), Group II received indomethacin (10 mg/kg) and provided as standard treatment group and also Groups III, IV, and V (served as test treatment groups) received EEPNK 100, 200, and 400 mg/kg, respectively, for 1 week, start from day of cotton pellet insert. On 8th day, cotton pellets were separated and removed from adjacent tissues following diethyl ether anesthesia. The pellets were dried at 60°C temperature for 1 day and signify weight of granuloma tissue produced in control group. The treatment group was obtained from the variation between the initial and final weights of both cotton pellet with its fastened granulomatous tissue.[16] Anti-inflammatory activity of EEPNK was evaluated by determining the percentage inhibition of granuloma formation with esteem to control group with following formula:



Analgesic activity

Writhing test

Animals were separated into five groups of six animals in each group (n = 6). Group I (negative control) served as untreated group and administered only CMC (0.5% CMC, p.o.). Group II (standard treated group) received a single dosage of standard (indomethacin 10 mg/kg, p.o.), whereas Groups III, IV, and V (EEPNK treated groups) received the single dose of the EEPNK 100, 200, and 400 mg/kg, p.o., respectively. After 30 min, writhing was tempted in mice by intraperitoneal injection of 0.6% acetic acid (10 mL/kg, i.p.). The number of writhing was calculated over a time of 20 min, as previously reported.[17]

Hot plate method

The hot-plate test was conducted based on earlier described techniques [18] where alignment of animals was kept comparable to that of writhing test. The paws of mice and rats are extremely susceptible to heat at temperatures which are not injurious to the skin. The temperature of the hot plate was set at 55°C ± 1°C to observe its pain responses (hind paw licking or jumping).[19] The animals were alone placed on the hot plate and the moment in time until either licking or jumping happened was recorded by a stopwatch, 30, 60, 90, and 120 min after treatment. The mice exhibiting latent response time shorter than 5 s or longer than 30 s were excluded from the study.

Statistical analysis

Statistics was expressed as a mean ± standard error of mean and statistical analysis was carried out by one-way ANOVA with Tukey's test performed using GraphPad InStat version 3.00, GraphPad Software, San Diego, California, USA, www.graphpad.com. P value was considered statistically significant when P < 0.05.


  Results Top


Acute toxicity test

In acute toxicity study, no toxic signs or mortality were observed at any of the tested doses up to 5000 mg/kg during the study period of 14 days.

Inflammatory model

The result showed that EEPNK exhibited anti-inflammatory activities in this model at all three dose levels. At 400 mg/kg, EEPNK showed better therapeutic effect as compared with standard drug indomethacin [Table 1]. Further, this test result can be confirmed after cotton pellet-induced granuloma model test, which has been shown same therapeutic effect [Table 2].
Table 1: Ingredients of Punarnavashtak Kwath

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Table 2: Anti-inflammatory activity of the ethanol extract of Punarnavashtak Kwath in egg white-induced paw edema method

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Analgesic activity

In acetic acid-induced writhing, EEPNK significantly inhibited the harmful stimulus induced by acetic acid [Table 3] and gave better effect when compared with control. Whereas the results of hot plate test are shown in [Table 4]. In this test, dose-dependent enhance in time of reaction (latency) to thermal stimulus in the mice. Pretreatment of the mice with 100, 200, or 400 mg/kg EEPNK significantly increased pain latency by 5.47, 11.20, and 16.23 s, respectively, whereas indomethacin (10 mg/kg. p.o.) significantly increased the pain latency 20.25 s [Table 4].
Table 3: Effect of ethanol extract of Punarnavashtak Kwath on cotton pellet-induced granuloma

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Table 4: Effect of ethanol extract of Punarnavashtak Kwath on acetic acid-induced writhing in mice

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  Discussion Top


In classical ayurvedic literature, PNK commonly used for the management of inflammation, pain, and fever along with other diseases,[20] but this classical ayurvedic formulation has not been considered scientifically. Earlier phytochemical analysis demonstrated the presence of mainly alkaloid, flavonoid, phenolics, tannin saponins, and carbohydrates in PNK extract. Accessible literature has exposed the function of these phytochemical in healing of inflammation and related problems.[21],[22],[23],[24] Previously, result suggested that PNK has shown better antioxidant and hepatoprotective activity.[25],[26] The result proposed that the EEPNK is nontoxic as no death was calculated at the dose of 5000 mg/kg. The objective of the current study was to examine the anti-inflammatory and analgesic activities effect of EEPNK (400, 200, and 100 mg/kg, p.o.) against physical- and chemical-induced inflammation and pain in experimental rat models. In egg albumin-induced rat hind paw edema (acute study), EEPNK (400, 200, and 100 mg/kg, p.o.) produced a significant (P = 0.05) inhibition in paw edema volume as contrasted to the negative control animals [Table 2]. The percent inhibition in rat paw volume was dose dependent (79.14%, 68.51%, and 61.70% at doses of 400, 200, and 100 mg/kg, p.o., respectively) at 240 min readings. Indomethacin (10 mg/kg, p.o.) significantly (P = 0.05) inhibited the rat paw volume by 80.63% at 240 min reading. EEPNK was found to be similar active as compared with standard drug at a dose of 400 mg/kg. Whereas in cotton pellet granuloma in rats (chronic study) model, EEPNK (400, 200 and 100 mg/kg, p.o.) significantly reduced the weight of granuloma as compared to the negative control animals. The inhibition degree was found in dose-dependent method (43.38%, 34.28%, and 18.99%) at the doses of 400, 200, and 100 mg/kg, p.o., respectively, whereas indomethacin (10 mg/kg, p.o.) degree of inhibition was 53.0% [Table 3]. EEPNK (400, 200, and 100 mg/kg, p.o.) dose dependently reduced the inflammation and pain in treated rats. The anti-inflammatory activity of EEPNK was performed by the egg albumin-induced paw edema and cotton pellet-induced granuloma model in rats. These models are well-known experimental models for the evaluation of acute and chronic anti-inflammatory activity, respectively. Egg albumin used as a phlogistic agent causes edema in rats hind paw. Egg white-induced hind paw edema methods are suitable for screening agents for anti-inflammatory activity which are frequently used to assess the antiedemateous effect of natural product.[27],[28] Cotton pellet-induced granuloma formation is a characteristic aspect of a well-known chronic and subchronic inflammatory test model. This model has been engaged to evaluate the proliferative and transudative mechanism of chronic inflammation. The liquid adsorbed by the pellet notably influence the damp mass of the granuloma, while the parched mass associated well with the quantity of granulomatous tissue produced.[29] The EEPNK showed reduce in the formation of granuloma that reproduced its efficiency to diminish the improved level of fibroblasts and synthesis of collagen during mucopolysaccharide, which are normal proliferative actions of granulation tissue formation.[29] Our results confirm that the small dose of EEPNK (100 mg/kg b.w.) in the anti-inflammatory and analgesic test has a considerable inhibitory outcome compared to the negative control. Distinguish with the standard drug (indomethacin), the high dose of EEPNK (400 mg/kg b.w.) has a comparable effect on the anti-inflammatory and analgesic activities. Analgesic effects of EEPNK were performed in mice of either sex by acetic acid-induced writhing and hot plate methods. In acetic acid-induced writhing, the EEPNK exhibited marked a reduction in the number of abdominal constriction induced by the injection of aqueous solution of acetic acid (0.6%) in a dose-dependent behavior. Pretreatment with EEPNK at a dose of 400, 200, and 100 mg/kg, p.o. was significantly (P = 0.001) inhibited to 77.43%, 70.57%, and 63.67% of writhings, respectively, in mice. Pretreatment of the mice with standard drug (indomethacin) significantly (P = 0.001) writhing inhibited by 78.74%. The analgesic effect was observed by the group that received 400 mg/kg formed a similar result with indomethacin group [Table 4]. Whereas in hot plate method, there was a dose-dependent enhance in latency period to thermal incentive in the mice. Pretreatment of the animals with EEPNK at a dose of 400, 200, and 100 mg/kg, p.o., and indomethacin (10 mg/kg p.o.) significantly (P = 0.001) increased pain latency [Table 5]. The reduce in a number of writhes caused by the EEPNK, proposes that the analgesic effect may peripherally intercede through the release of prostaglandins, inhibition of synthesis as well as other endogenous substances. Hot plate method [18] is the most ordinary thermal nociception model used for performing central analgesic efficiency of drugs or mixture of drugs. This procedure is measured to be selective for the centrally acting analgesics, so the effects of numerous drugs on this pain model proved that it could have centrally acting antinociceptive activity.[30] In this model, sensory nerves sensitize the nociceptors with a minimum contribution of endogenous stuff such as prostaglandins.[31]
Table 5: Analgesic activity of ethanol extract of Punarnavashtak Kwath in the hot plate test

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  Conclusion Top


The acute toxicity test proved that EEPNK is safe for clinical use. The present study showed that EEPNK infatuated significant anti-inflammatory and analgesic activities in a dose-dependent behavior. This can be expected due to the presence of a different class of phytoconstituents (alkaloid, phenolics, terpenoids, and steroids). Therefore, EEPNK could be a potential source in the management of pain and inflammation.

Acknowledgement

The author is very thankful to Mr. Himanshu for his help in statistical analysis of data and also grateful to the IIT (BHU) Varanasi, for providing the facilities to perform this investigation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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