AN EXPERIMENTAL STUDY TO EVALUATE ANTI-INFLAMMATORY ACTIVITY OF MURIVENNA IN WISTAR ALBINO RATS

AN EXPERIMENTAL STUDY TO EVALUATE ANTI-INFLAMMATORY ACTIVITY OF MURIVENNA IN WISTAR ALBINO RATS

Vaishali V. Nair, Arun Mohanan and Abhaya Kumar Mishra ^*

Department of Rasashastra and Bhaishajya Kalpana (Medicinal Chemistry and Pharmacy), Amrita School of Ayurveda, Amritapuri, Amrita Vishwa Vidyapeetham, Clappana, Vallickavu, Kollam - 690525, Kerala, India.

ABSTRACT: Murivenna is a taila (oil) which is widely used in South-Indian practices for external application in many of the localized conditions such as trauma, inflammation of joint, fracture, joint dislocation, wound, etc. Taila (Oil) is considered best for vatahara action. Vata is the main dosha involved in shopha (inflammation); this is a suitable taila (oil) that works in the treatment of shopha (inflammation). Materials and Methods: Murivenna was prepared as per the guidelines of Trivandrum Pharmacopeia and Ayurveda Formulary of India with genuine drugs. A later experimental study was conducted in acute and chronic inflammation using Carrageenan-induced paw edema and cotton pellet granuloma pouch method in acute and chronic inflammatory study, respectively. The data were analyzed statistically using a one-way analysis of variance (ANOVA) followed by multiple comparison Dunnett’s test as a post hoc test. Result: The results indicate the presence of significant anti-inflammatory activity in the experimental model representing acute inflammation and non-significant effect in chronic inflammation. But there is a decrease in the granuloma formation, which suggests that Murivenna can be given in granuloma condition. Conclusion: Murivenna retains anti-inflammatory activity in acute inflammation than in chronic.

Murivenna, Acute Inflammatory Study, Chronic Inflammatory study, Plethysmography technique, Cotton pellet granuloma pouch method

INTRODUCTION: Inflammation is a local defense response. The cardinal signs are - Rubor (redness), Tumor (Swelling), Calor (Heat), Dolor (Pain), function laesa (loss of function). It is of 2 types - acute inflammation and chronic inflammation. Sprain, physical injury, suppression of marmas, or going against the dietic regimen may result in the disturbances of physical phenomenon and thus induce Shopha. Muṛivenna is a taila (oil) which is used for external application in many of the localized condition such as trauma, inflammation of joint, fracture, joint dislocation, wound, etc.

Here an attempt is made to understand an externally used medicine known as Muriveṇṇa for its shophahara property (Anti-inflammatory action).

Aims and Objectives: To evaluate the Anti-inflammatory property of Murivenna in Wistar albino rats

MATERIALS AND METHODS:

Preparation (5/3/2018 to 10/3/2018): The ingredients are Narikela (Cocos nucifera), Tambula (Piper betle), Sigru (Moringa oleifera), Paribhadra (Erythrina indica), Kumari (Aloe barbadensis), Karanja (Pongamia pinnata), Buka (Spermacocea hispida), Palandu (Allium cepa), Tambula (Oryza sativa), Satavari (Asparagus racemosus). These were freshly collected and authenticated. Murivenna was prepared as per Ayurveda Formulary of India ¹ / Trivandrum Ayurveda Pharmacopeia ² in the Lab attached to the Department of Rasashastra and Bhaishajya Kalpana, Amrita School Of Ayurveda. Oil was prepared in khara paka (3^rd stage of processing) and was later filtered and stored in a container. It took 3 days for the completion of preparation.

Experimental Study -7/5/2018 to 2/7/2018:

Test Drug Used:

Murivenna: Prepared and was stored in clean air tight container.

Standard Drug Used:

Diclofenac Sodium Gel:

Brand Name: Voveran Emulgel

Diclofenac belongs to the non-steroidal anti-inflammatory drug (NSAID), which works on pain and inflammation in the body. Diclofenac is used to treat mild to moderate pain and signs and symptoms of osteoarthritis, rheumatoid arthritis etc.

Selection of Animals: Healthy Wistar albino rats, which weighs Fig. 1 in the range of 140 - 330 g were taken from the animal house of S.D.M. Centre for Research in Ayurveda and Allied Sciences, Udupi. The animals were housed in standard laboratory condition by exposure to natural day and night cycle with temperature of 25 ºC and 50-70% relative humidity in well-ventilated conditions kept in polypropylene cages. Animals were provided with normal mouse chow (Sai Durga Food and Feeds or Scientist Choice Laboratory Animal Feed, Bangalore, India) and water ad libitum Fig. 3.

The animals were randomly selected, marked with picric acid to permit individual identification, and kept in respective cages Fig. 2 for 7 days prior to the start of the application of medicine to allow for acclimatization to the laboratory conditions. The experimental study was conducted after obtaining permission from the institutional ethics committee in accordance with the guideline formulated by CPCSEA. Approval No: SDMCRA/IAEC/AM-R-25,IAEC 29- 05-2017

Acute Inflammatory Study: The effect of test drug on carrageenan-induced paw edema in rats was studied as described by Winter, et al., 1962. Pregnant female rats were excluded. Healthy Wistar albino rats of either sex were weighed between 140-330 g, and it is randomly divided into four groups, Table 1, each consisting of 6 rats.

TABLE 1: GROUPING OF ANIMALS IN ACUTE INFLAMMATORY STUDY

Total 24 rats, 6 each in the separate cage was maintained separately at the institute’s animal house and were exposed to natural day and night cycles. Only tap water and food were administered to the first group so as to serve as a control. The second group, third group, and the fourth group was treated externally with castor oil, diclofenac sodium gel, and Murivenna, respectively. The drug was applied once daily for 3 consecutive days, and on the 4^th day, the main procedure is done using the plethysmography technique where basal paw volume reading, 1st hour, 3^rd hour, and 6^th hour reading was taken, and again the drug is applied on the 5^th day and 24^th-hour reading was taken.

Steps Involved while using Digital Plethysmometer (Fig. 7) Filling of Water Cell: Plethysmometer consists of 2 glass stoppers where one is used for a water bath with the soap solution and the other one is used for dipping the rat paw in the soap solution drained from water bath for getting the reading. Here soap solution is used for conductivity. The water bath is filled with the soap solution and distilled water till the conductivity meter shows 240 µL and later, it is drained to the water cell, where 1ml of water is removed using a syringe after the filling of the cell.

Turning on the Unit: Main power was switched on. Every time when the unit is turned on, the unit will program by itself. The number will be displayed, which will be changed after a few seconds automatically to zero. To zero the display after each reading, press the reset button so that the reading will display as 0.0 or 0.00.

Calibration: Calibration was done by dipping 0.5ml, 1ml, and 2ml nobe. If we get the correct reading on the dipping of the nobe, the instrument is said to be calibrated and can be used for further reading of rat paw.

Reading: The reading will show the volume displaced when the paw is dipped into the soap solution. Zero may vary between 0 and 0.1 or 0 and 0.02 max. The minor deviation has to be tolerated.

The reading of rat paw is noted when it stops blinking or changing for a few seconds in the plethysmometer. Later again reset button is pressed to return to the normal reading, where the paw is removed from the glass stopper.

Experimental Procedure for Acute Inflammation: To the first group, tap water was administered to serve as a control. Second, third and fourth groups were treated with castor oil, diclofenac sodium gel, and Murivenna, respectively. The test drug was applied twice daily for three consecutive days, whereas the 2^nd and 3^rd group was applied only once a day for three consecutive days before the main procedure.

The study started with the trial group on 7/5/2018, where the application of Murivenna was done twice daily (morning 9.00 am and evening 3.30 pm) for 3 consecutive days till 9/5/2018 Fig. 4. 250 ml bottle was filled with tap water and was administered to the rats to ensure uniform hydration. This was supposed to minimize the variation in edema formation during the course of study. On 10/5/2018 the main procedure was conducted using a plethysmographic technique where the procedure started after calibration of the instrument, and then the basal paw volume was noted. Later by 10.00am paw oedema was induced by injecting Fig. 5 0.1 ml freshly prepared 1% Carrageenan in distil water i.e., 50mg carrageenan in 5 ml distil water was prepared and 0.1 ml was then subjected to the sub-plantar aponeurosis of the left hind paw Fig. 6. Oil was applied every one hour and reading was noted 1^st hour, 3^rd hour, 6^th hour and 24^th hour Fig. 8.

This procedure was also done for castor control, standard group, and control group. The result was expressed as a percentage increase in paw volume in comparison to the initial value. The percentage increase in paw volumes was calculated by subtracting the initial paw volumes from the paw volumes obtained after the injection of the phlogistic agent. The figure was divided by initial paw volume and then multiplied by a hundred.

% Change = (Hour reading – Basal reading) × 100 / Basal reading

Statistical analysis was done to evaluate the efficacy of Murivenna in comparison to castor control and standard group.

Chronic Inflammation:

TABLE 2: GROUPING OF ANIMALS IN CHRONIC INFLAMMATORY STUDY

The rats were anesthetized with ether Fig. 11. The Dorsum of neck was shaved Fig. 10 and swabbed with 70% (v/v) alcohol. In the intracapsular region, midline incision of 1cm was made. A small tunnel was made on either side of the incision with small blunt forceps. One sterile cotton pellet Fig. 9 weighing 100 mg (prepared by cutting rolled cotton into pieces of 100 mg and sterilized by autoclaving for 30 min under 15 lbs pressure) was inserted into the tunnel on either side of the incision Fig. 12 and the incision was closed with interrupted sutures Fig. 13 after expelling the air from the tunnel.

Group, I was treated with tap water and taken as a control group. Group II and III Table 2 was taken as standard and trial group which is treated with Diclofenac sodium gel and Murivenna Fig. 14, respectively, for 7 consecutive days starting from the day of implantation. The rats were sacrificed on 8^th day after taking blood from the orbital plexus of rat and dissected for collection of the spleen, adrenal glands, thymus, and cotton pellets Fig. 15 along with the clean extraneous tissue Fig. 16. Later the collected organs Fig. 19 i.e., spleen, adrenal gland, and thymus were preserved in a clean glass bottle containing 10% formaldehyde solution after proper weighing and send to the pathology laboratory for histopathological investigations. Also, the cotton pellet, along with extraneous tissue, was weighed (initial weight) and kept in a hot air oven overnight at 80 ºC for drying. Again the pellet was weighed after drying (final weight). The difference between the initial weight and final weight of the pellet was taken as the granuloma tissue weight. The result was expressed as mg granulation tissue formation per 100 g of body weight. The blood which was collected from the orbital plexus Fig. 17 of rat separated from serum was analyzed for the determination of C-reactive protein. After sacrificing the animal's Fig. 18, the tissues were excised out immediately and were transferred into 10% Formaldehyde solution. The tissues were held in it till they are taken up of further processing (histopathological examination).

 Acute Inflammatory Study:

FIG. 1: WEIGHING OF RATS                                                

FIG. 2: GROUPING OF RATS

FIG. 3: PROVIDING FOOD & WATER                    

FIG. 4: APPLICATION OF OIL BEFORE EXP.

FIG. 5: INJECTING INFLAMMATION INDUCING AGENT              

FIG 6. INFLAMMATION OF HIND PAW

FIG. 7: DIGITAL PLETHYSMOMETER                                   

FIG. 8: READING OF PAW

Chronic Inflammatory Study:

FIG. 9: COTTON PELLET PREPARATION             

FIG. 10: PREPARATION FOR IMPLANTATION

FIG. 11: LOCAL ANAESTHESIA                              

FIG. 12: COTTON PELLET IMPLANTATION

FIG. 13: SUTURING AFTER IMPLANTATION          

FIG. 14: OIL FOR APPLICATION OVER SUTURED AREA

FIG. 15: REMOVAL OF COTTON PELLET                

FIG. 16: COTTON PELLET GRANULOMATOUS TISSUE

FIG. 17: ORBITAL BLOOD FOR INVESTIGATION                         

FIG. 18: EUTHANASIA OF RAT

FIG. 19: ORGANS FOR HISTOPATHOLOGY

 RESULT: The data were analyzed statistically using a one-way analysis of variance (ANOVA) followed by multiple comparison Dunnett’s test as post hoc test.

The data shows there was a decrease in paw volume in the 24^th hour in the standard group and test group when compared to the control group; the observed decrease was found to be statistically non-significant. Thus in any acute condition, it can be used as an anti-inflammatory medicine as it is tridoshahara in nature and simultaneously pacify the signs and symptoms.

Acute Inflammatory Study: Data related to the effect of Murivenna on paw edema of carrageenan-induced paw edema at different time intervals is shown in Table 3.

The data shows there was a very significant increase in paw volume in the control group of 1^st hour, 3^rd hour and 6^th hour and a non-significant increase in 24^th hour when compared to the basal volume of the same group.

The data shows there was a very significant increase in paw volume in the castor control group of 1^st hour, 3^rd hour, 6^th hour, and 24^th hour when compared to the basal volume of the same group.

The data shows there was a very significant increase in paw volume in the standard group of 1^st hour, 3^rd hour, and 6^th hour and a non-significant increase in 24^th hour when compared to the basal volume of the same group.

 TABLE 3: THE EFFECT OF MURIVENNA ON PERCENTAGE INCREASE OF PAW OEDEMA AT DIFFERENT TIME INTERVAL

Data: MEAN ± SEM. **p < 0.01 in comparison to control group.

The data shows there was a very significant increase in paw volume in a test group of 1^st hour, 3^rd hour, and 6^th hour and a non-significant increase in 24^th hour when compared to the basal volume of the same group.

 Acute Inflammatory Study Graph:

Chronic Inflammatory Study: The cotton pellet implanted granuloma method was used to evaluate chronic inflammation.

 TABLE 4: EFFECT OF MURIVENNA ON COTTON PELLET IMPLANTED GRANULOMA FORMATION

Data: MEAN ± SEM

Data related to the effect of test drug on the weight of cotton pellet has been shown in Table 4.

The data shows there was a decrease in cotton pellet implanted granuloma formation in the standard group and test group when compared to the control group; the observed decrease was found to be statistically non-significant.

TABLE 5: THE EFFECT OF MURIVENNA ON WEIGHT OF SPLEEN IN COTTON PELLET IMPLANTED RATS

Data: MEAN ± SEM

Data related to the effect of test drug on the weight of Spleen has been shown in Table 5.

The data shows there was an increase in weight of the spleen in the control group and standard when compared to the normal control group; the observed increase was found to be statistically non- significant.

The data shows there was a decrease in the weight of Spleen in the test group when compared to the normal control group; the observed decrease was found to be statistically non-significant.

TABLE 6: THE EFFECT OF MURIVENNA ON WEIGHT OF THYMUS IN COTTON PELLET IMPLANTED RATS

Data: MEAN ± SEM

Data related to the effect of test drug on the weight of Thymus has been shown in Table 6. The data shows there was a decrease in the weight of Thymus in the control group, standard group, and test group when compared to the normal control group; the observed decrease was found to be statistically non-significant.

TABLE 7: THE EFFECT OF MURIVENNA ON WEIGHT OF ADRENAL GLAND IN COTTON PELLET IMPLANTED RATS

Data: MEAN ± SEM, *p < 0.05 in comparison to normal control.

Data related to the effect of test drug on the weight of the Adrenal gland has been shown in Table 7.

The data shows there was an increase in the weight of the Adrenal gland in the control group and test drug when compared to the normal control group; the observed increase was found to be statistically non-significant.

The data shows there was an increase in the weight of the Adrenal gland in the standard group when compared to normal control group; the observed increase was found to be statistically significant.

TABLE 8: THE EFFECT OF MURIVENNA ON SERUM C – REACTIVE PROTEIN LEVEL IN COTTON PELLET IMPLANTED RATS

Data: MEAN ± SEM

Data related to the effect of test drug on C-reactive protein has been shown in Table 8.

The data shows there was an increase in serum C – reactive protein in the control group when compared to the normal control group; the observed increase was found to be statistically non- significant.

The data shows there was an increase in C – reactive protein in the standard group and test group when compared to the normal control group; the observed increase was found to be statistically non-significant.

Result of Histopathology: Spleen:

TABLE 9: THE RESULT OF HISTOPATHOLOGY OF SPLEEN OF CONTROL GROUP (FIG. 22 & 23)

TABLE 10: THE RESULT OF HISTOPATHOLOGY OF SPLEEN OF STANDARD GROUP (FIG. 24 & 25)

TABLE 11: THE RESULT OF HISTOPATHOLOGY OF SPLEEN OF TEST GROUP (FIG. 26 & 27)

Adrenal Gland:

TABLE 12: THE RESULT OF HISTOPATHOLOGY OF ADRENAL GLAND OF CONTROL GROUP (FIG. 30 & 31)

TABLE 13: THE RESULT OF HISTOPATHOLOGY OF ADRENAL GLAND OF STANDARD GROUP (FIG. 32 & 33)

TABLE 14: THE RESULT OF HISTOPATHOLOGY OF ADRENAL GLAND OF TEST GROUP (FIG. 34 & 35)

Thymus:

TABLE 15: THE RESULT OF HISTOPATHOLOGY OF THYMUS OF CONTROL GROUP (FIG. 38 & 39)

TABLE 16: THE RESULT OF HISTOPATHOLOGY OF THYMUS OF STANDARD GROUP (FIG. 40 & 41)

TABLE 17: THE RESULT OF HISTOPATHOLOGY OF THYMUS OF TEST GROUP (FIG. 42 & 43)

 Photomicrograph of Histopathology Section: Spleen:

Adrenal:

Thymus:

DISCUSSION:

TABLE 18: THE MURIVENNA INGREDIENTS WITH ANTI-INFLAMMATORY CONSTITUENTS

Discussion on Experimental Study: The experimental study of Murivenna has been designed to carry out the anti-inflammatory activity of the drug in an established animal model, as no work has been found reported in this formulation with specific to this activity. The outcome of experimental study has been provided in the form of consolidated tables as follows for easy comparison and discussion.

Discussion on Acute Inflammation:

Effect on Carrageenan Induced Paw Oedema:

TABLE 19: CONSOLIDATED STATEMENT ON PERCENTAGE CHANGE OF PAW OEDEMA AT DIFFERENT TIME INTERVAL ON ADMINISTRATION OF MURIVENNA

Where NSD – Non-Significant Decrease, NSI – Non-Significant Increase, SI – Significant Increase

Carrageenan has been found to give results that are more consistent and is widely used as a standard edema-inducing agent. It is one of the most suitable acute models to screen anti-inflammatory agents. The edema developed in the paw of the rat after injection of carrageenan is a biphasic event. The initial phase of the edema is due to the release of histamine and serotonin, and the edema is maintained during the plateau phase by kinin like substance and the second accelerating phase swelling due to the release of prostaglandin like substances. In the present study, Murivenna is found to have non-significant decrease Table 19 which means it significantly inhibited both the phases of carrageenan-induced paw edema.

Discussion on Chronic Inflammation: In chronic inflammatory model, the test formulation did not showed any significant impact on the majority of the parameters studied.

TABLE 20: CONSOLIDATED STATEMENT ON COTTON PELLET IMPLANTED GRANULOMA FORMATION ON ADMINISTRATION OF MURIVENNA

Where NSD – Non Significant Decrease

Granulomatous tissue formation is related to the chronic inflammatory process, which is an indication for proliferative phases of inflammation. Inflammation involves the proliferation of macrophages, neutrophils, and fibroblast, which are the basic sources for the formation of granuloma. Thus this method is widely used to evaluate the transudative and proliferative components of chronic inflammation. The dry weight of the pellets relates to the amount of granulomatous tissue. Test formulation non-significantly decreased the weight of granulation tissue, and Diclofenac, which was used as a standard anti-inflammatory agent, also non- significantly decreased Table 20 the weight of granulation tissue. This may indicate the ability of test formulation in reducing the synthesis of proteins, collagen, and infiltration of macrophages.

Discussion on Blood Parameter – CRP: CRP is present in the normal human body in minute amount. It is generally present in hepatocyte cells of the liver. It increases during any kind of inflammation or infection. CRP stimulates the immune system to produce more soldiers (neutrophils, lymphocyte, macrophages etc.) to fight against the condition. When antibodies are built sufficiently, CRP drops down. Thus here in this study, CRP is increased, showing inflammation in both test and standard group.

TABLE 21: CONSOLIDATED STATEMENT ON WEIGHT OF ORGANS ON ADMINISTRATION OF MURIVENNA

Where NSD – Non-Significant Decrease, NSI – Non Significant Increase, SI – Significant Increase

The weight of the spleen and thymus was non- significantly decreased in Table 21 due to degenerative changes and the weight of the adrenal gland non significantly increased due to stimulation of organ activity.

Discussion on Histopathological Results: In a histopathological examination of spleen, thymus and adrenal gland sections, tissue architecture was normal in the normal control group. Mild to moderate degenerative changes with severe inflammation was noticed in the positive control group, and normal to mild changes found in the standard group. It is also noted that there are only mild degenerative changes found in the test group.

This indicates that the test formulation has a better effect in acute inflammation compared to chronic.

CONCLUSION: Shopha is compared to inflammation in contemporary science. As per Ayurveda, application of oil is indicated in śhopha or inflammation. Murivenna containing 10 main ingredients was prepared according to Ayurvedic Formulary of India, and an Experimental study was conducted in chemically and surgically induced inflammation in acute and chronic cases, respectively, in wistar albino rats.

The results indicate the presence of significant anti-inflammatory activity in the experimental model representing acute inflammation and non- significant effect in chronic inflammation. But there is a decrease in the granuloma formation, which suggests that Murivenna can be given in granuloma condition. The effects were statistically compared with the effect observed in control and the standard group, which indicate a significant effect in acute inflammation. Thus the obtained result clearly indicates Murivenna retains anti-inflammatory activity in acute inflammation than in chronic as most of the individual ingredients of the test formulation is proved for its anti-inflammatory action.

ACKNOWLEDGEMENT: I would like to express my sincere gratitude towards Mr. Sudhakar Bhatt - Research Officer, S.D.M Centre for Research in Ayurveda and Allied Science and Dr. Anjana Haridas, Dr. Jyothi K, Dr. Nisha G, Dr. Radhika Panicker, Dr. Sreeja Raj, Dr. Dhanya Krishnan, Dr. Priyada K.V, Dr. AkhilRaj. A.R, Dr. Chitra. S, Dr. Sai Lekshmi, Dr. Sujithra. M, Dr. Aswin T. Das- PG Scholars, Amrita School of Ayurveda, Kollam, Dr. Shruti Kamble – PG Scholar, MIAMS, Manipal, and Maya V. Nair, Venugopalan Nair K, Vaishakh V Nair, Kottayam for their valuable suggestions and support.

CONFLICTS OF INTEREST: We declare no conflicts of interest.

REFERENCES:

1. The Ayurvedic Formulary of India, Part III First Edition, Published by the Controller of Publications Civil Lines, Delhi-110054, 203.
2. Dr. Lalithamma, The Pharmacopeia of Govt. Ayurveda College, Published by Dr. N. Babu Vijayanandhan, Thiruvanthapuram 2002 edition, 174.
   

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   How to cite this article:

   Nair VV, Mohanan A and Mishra AK: An experimental study to evaluate anti-inflammatory activity of Murivenna in Wistar albino rats. Int J Pharm Sci & Res 2020; 11(10): 5091-03. doi: 10.13040/IJPSR.0975-8232.11(10).5091-03.

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