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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 13  |  Issue : 2  |  Page : 120-126

Preparation and development of polyherbal formulation of medicinal plants for antiarthritic activity


1 Department of Pharmacognosy and Phytochemistry, KLE College of Pharmacy, KLE Academy of Higher Education and Research, Belagavi, Karnataka, India
2 Department of Pharmacognosy and Phytochemistry, KLE College of Pharmacy; Dr. Prabhakar Kore Basic Science Research Center, KLE Academy of Higher Education and Research, Belagavi, Karnataka, India

Date of Submission22-Jul-2019
Date of Acceptance16-Mar-2020
Date of Web Publication23-Jun-2020

Correspondence Address:
Dr. Sunil S Jalalpure
Dr. Prabhakar Kore Basic Science Research Center, KLE Academy of Higher Education and Research, Nehru Nagar, Belagavi . 590 010, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/kleuhsj.kleuhsj_163_19

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  Abstract 


AIM AND OBJECTIVES: The main objective of the present research was to prepare and develop polyherbal formulation of medicinal plants such as seed coat and kernel of Terminalia chebula and Terminalia bellirica and dried fruits of Emblica officinalisand evaluate the antiarthritic activity using animal models.
MATERIALS AND METHODS: Using seed coat and kernel of T. chebula and T. bellirica and dried fruits of E. officinalis, Polyherbal formulation was prepared. Further antiarthritic activity of the prepared formulation was tested in female Wistar rats, using Freund's complete adjuvant as an inducing agent.
RESULTS: The antiarthritic activity of polyherbal formulation against Freund's complete adjuvant-induced paw edema shows that all the three doses 250, 500, and 750 mg/kg p.o have significant effects and markedly reduced the swelling of paw.
CONCLUSION: Polyherbal formulation exerts a significant protective effect against Freund's complete adjuvant-induced paw edema in rats.

Keywords: Antiarthritic activity, Freund's complete adjuvant, polyherbal formulation


How to cite this article:
Chimagave SS, Jalalpure SS, Kurangi BK. Preparation and development of polyherbal formulation of medicinal plants for antiarthritic activity. Indian J Health Sci Biomed Res 2020;13:120-6

How to cite this URL:
Chimagave SS, Jalalpure SS, Kurangi BK. Preparation and development of polyherbal formulation of medicinal plants for antiarthritic activity. Indian J Health Sci Biomed Res [serial online] 2020 [cited 2020 Nov 26];13:120-6. Available from: https://www.ijournalhs.org/text.asp?2020/13/2/120/287412




  Introduction Top


The medicinal plants have been considered as sacred and used by early civilization to treat illness of human being.[1] Polyherbal formulation containing seed coat and kernel of Terminalia bellirica and Terminalia chebula and fruits of Emblica officinalis is a more important and powerful medicine of the Rasayana group, and it is mostly applicable for promoting health and immunization against diseases.[2],[3],[4]

Arthritis is a joint disorder that contains inflammation including one or more joints of the body and is an autoimmune type systemic disorder with severe chronic type of inflammation in synovial joints and continuous breaking up of bone and cartilage. Arthritis mainly attacks on the joints rather than organs and muscles that often progress to destruction of the cartilage and abnormal stiffening of the joints due to fusion of the bones. In arthritis, over 100 different types are involved depending upon the stages of chronic inflammation of the synovial joints; it is the oldest known disease of humankind and affects a more population of the universe; no major progress has been made in developing a permanent cure. The main disadvantage in the available potent synthetic drugs lies in their toxicity and side effects and also shows reappearance of symptoms after discontinuation.[1] Polyherbal formulation is the formulation consisting single or multiple herbs applicable for the treatment of sickness of human beings and immunity. The ayurvedic literature “Saranghar Samhita” emphasized the idea of polyherbalism to achieve the desirable therapeutic efficacy. The active phytoconstituents of single plants are not sufficient to obtain the desired therapeutic activity; however, upon mixing the selected multiple medicinal plants in a desired ratio, it will give a greater therapeutic activity and reduce the toxicity.[5] Oral formulations that allow for individual dosing are easy to swallow and these formulations are applicable for geriatric patients.[6],[7]


  Materials and Methods Top


Collection of fruits and authentication

The fruits of the plants E. officinalis, T. chebula, and T. bellirica were collected from local area of Belagavi and authenticated by Dr. Harsha Hegde, Indian Council of Medical Research-National Institute of Traditional Medicine (ICMR-NITM), Belagavi. The voucher specimen (RMRC-1418, RMRC-1419, and RMRC-1420) has been deposited in the ICMR Herbaria and Department of Pharmacognosy and Phytochemistry, KLE College of Pharmacy, Belagavi, India, for future reference.

Chemicals

Freund's complete adjuvant (FCA) (HiMedia Laboratories Pvt., Ltd., Bengaluru), mercury and Whatman no. 42, and ashless filter paper (125 mm) (Merck Lab., Mumbai) used were of pharmaceutical grade. Other chemicals such as hydrochloric acid (HCl) and ethanol and reagents used were of analytical grade.

Animals

Wistar rats weighing 150–200 g were used for the study. The animals were kept in clean transparent polypropylene cages maintained at 25°C ± 2°C with 12-h dark and light cycle. The animals were fed with standard diet and water. The experimental protocol was approved by the Institutional Animal Ethics Committee of KLE College of Pharmacy, Belagavi, India.

Processing of plant

The fruits of the plant E. officinalis, T. chebula, and T. bellirica were collected from Belagavi area, and it is authenticated. All powders were passed in mesh no. 85, and sterilization process was carried out in ultraviolet (UV) radiation. Seed coat and kernel of Terminalia chebula and Terminalia bellirica and fruits of Emblica officinalis has been selected for further processing.

Preparation of churna

The fruits of the plants E. officinalis, T. chebula, and T. bellirica were collected and authenticated. In the processing step, first remove seeds of the fruit by breaking the fruits of T. chebula and T. bellirica and then obtain the seed coat and kernel of these two myrobalan powdered in a blender. Fruits of E. officinalis are powdered in the blender making a fine powder. Finally, all three fruits powders were passed through sieve no. 80 to obtain uniformly sized powder particles. UV sterilization process has been performed to eliminate and deactivate all forms of life, such as fungi and bacteria to avoid damage of active constituents. After the UV sterilization process, churna was stored in an air-tight container, for further processing.

Physicochemical parameters

The same powdered plant materials such as seed coat and kernel of T. bellirica and T. chebula and fruits of E. officinalis were taken for investigation of analysis such as determination of total ash, acid in-soluble ash, and water-soluble ash.

Ash values

Determination of total ash

Weight accurately 2.5 gm of powdered drug and add into crucible and incinerated at temperature not exceeding 450° C in a muffle furnace for 6 hours, until it free from carbon, then it was cooled and calculate the percentage of weight loss of powder.

Determination of acid-insoluble ash

Ash was boiled for 5–10 min with 25 ml of dilute HCl; insoluble matter was collected on the ashless filter paper in a crucible. It was washed with hot water and ignited again in a muffle furnace at the temperature not exceeding 450°C for 2 h and weighed the crucible. The percentage of acid-insoluble ash was calculated with reference to the air-dried drug.

Determination water-soluble ash

With 25 ml of water, the ash was boiled for 5 min; then in a crucible, an insoluble matter was collected on an ashless filter paper, washed with hot water, and ignited at the temperature not exceeding 450°C for 2 h. From the total ash weight, the insoluble matter weight was subtracted and this difference in the weights gives the weight of water-soluble ash. The percentage of water-soluble ash was calculated with reference to the air-dried drugs.

Preparation of the formulation

All powdered drugs were taken in various proportions and sieved using mesh no. 85. Powdered drugs were sterilized using UV radiation at 60°C. Powdered drugs were homogenized using mortar and pestle. The powdered drugs were mixed in a geometrical ratio. Sufficient quantity of gum acacia was added to powdered drugs, and 0.01% butylated hydroxy toluene (BHT) was added to the above mixture with continuous mixing to obtain the desired formulation. The prepared polyherbal formulations are depicted in [Figure 1].
Figure 1: Polyherbal formulation

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Acute toxicity study

The tested powdered drugs did not show any type of toxicity and mortality in each group when given orally at the dose of 3000, >2000, <2000 mg/kg (E. officinalis, T. chebula, and T. bellirica). Hence, 250, 500, and 750 mg/kg p.o. were taken as a therapeutic dose.[8],[9]

Induction of arthritics using complete Freund's adjuvant

All the rats were divided into six groups. Each treatment group consists of six Wistar rats, where arthritis was inducted into the rats by complete Freund's adjuvant [Figure 2]. Polyherbal formulations were given in the dose of 250, 500, and 750 mg/kg p.o for 21 days. FCA injection was given subcutaneously to induce the arthritis into the rats, as per the method provided by Kamalutheen et al. (2009).[10],[11]
Figure 2: Complete Freund's adjuvant induced arthritis in animal

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On the 0th day, the basal paw volume of left hind paw of each animal was measured, using mercury plethysmometer; then, all the animals of the groups were anesthetized; after that, 0.1 ml of FCA injection was given into the ankle joint of left hind paw and allowed to stand for recover. For comparison, the study was performed with the dosing of standard drug indomethacin (10 mg/kg bwt) and polyherbal formulation with three different concentrations such s 250, 500, and 750 mg/kg bwt. On the 0th, 1st, 3rd, 7th, 14th, and 21st day of the study period, the rat paw volume of injected paw was measured using mercury plethysmometer[12],[13] [Figure 3].
Figure 3: Measurement of paw volume using plethysmometer

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Evaluation of arthritis severity

Before injection of FCA into the left hind paw of the rat, the paw volume was measured using mercury plethysmometer. After injection, the paw volume was again measured on the 1st, 3rd, 5th, 7th, 14th, and 21st day. During the period of experiment, the body weights of each rat were measured on every 3rd day after FCA injection (Patil and Suryavanshi, 2007).[14]

Hematological parameters

Hematological parameters such as white blood cell (WBC), red blood cell (RBC), and hemoglobin (Hb) values were evaluated using routine laboratory methods (Patil et al., 2009).[13],[15],[16]

Histopathological examination

All the animals were anesthetized by ether anesthesia and sacrificed by cervical decapitation. After that, right hind limb just distal to the knee had been removed, washed with saline, and kept in 10% formalin solution. Further procedure was done by histopathological department. The arthritis assessment parameters such as joint space, synovial ulceration, synovial granulation tissue, edema, and periarticular inflammation have been studied [Table 1].
Table 1: Physicochemical investigation of fruits of Emblica officinalis and seed coat and kernel of Terminalia bellirica and Terminalia chebula

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  Results Top


Phytochemical investigation

Results of phytochemical investigation of powdered seed coat and kernels of T. bellirica and T. chebula and powdered fruits of E. officinalis showed the presence of carbohydrates, glycoside, fats, tannins, flavonoids, amino acids, and proteins[17],[18] [Table 2].
Table 2: Physicochemical tests for Terminalia bellirica, Terminalia chebula, and Emblica officinalis

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Physicochemical tests

T. bellirica, T. chebula, and fruits of E. officinalis have been tested for physicochemical tests such as total ash, acid-insoluble ash, and water-soluble ash values.[19] The results are mentioned in [Table 3].
Table 3: Histopathological observation of joints

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Pharmacological screening

There was a significant increase in paw volume in disease group compared to normal in the 1st, 2nd, and 3rd weeks. Further, treatment group showed significant decreases in paw volume in the 1st, 2nd, and 3rd week compared to disease control and was found to be compared with indomethacin group.

It was observed from the graph that, there were significant decreases in paw edema volume in the 1st, 2nd, and 3rd week compared to disease control and indomethacin-treated group.

There was no significant change in body weight in the 0th and 1st week among the groups; however, significant change in body weight was observed in the 2nd and 3rd week in treated animals compared to disease and also observed in disease control compared to normal.

  1. In pharmacological screening the level of significance and its comparison with normal group, further group compared with disease control is needed for understanding the concept
  2. There were significant increases in RBC in disease control group compared to normal. However, no significant change in the RBC was observed in the treatment group compared to negative control
  3. There were significant decreases in %Hb in disease control group compared to normal.


Histopathological observation of joints

Animals were divided into six groups, each containing six animals. The arthritis was induced by injection of FCA at a dose of 0.1 ml into the ankle joint of left hind paw. The suspension is administered orally in rats at the dose of 250, 500, and 750 mg/kg body weight and standard drug indomethacin at the dose of 10 mg/kg orally. According to the present study, it clearly indicates that the polyherbal suspension has promising antiarthritic effect on FCA-induced rats.

In the present study, we observed that treatment with polyherbal formulation significantly reduced FCA-induced arthritis at the 2nd and 3rd week. This observation suggest that these formulations may modify effects of mediators such as kinins and prostaglandins, which are released in the 2nd and 3rd week, and it shows polyherbal formulation effectively reduced the FCA-induced arthritis in female Wistar rats, which suggest antiarthritic activity for polyherbal formulation. Histopathological results showed that joint space and their thickness decreased when the polyherbal formulation administered into the female Wistar rats, and further, it had shown decreased level of synovial inflammation at the 2nd and 3rd weeks (Group No. IV – Test 1) at the dosing of 250 mg/kg body weight orally as compared to Group No. V – Test 2 (500 mg/kg) and Group No. VI – Test 3 (750 mg/kg) [Table 1].

Histopathological results

Results of the histopathological study are shown in [Figure 4], [Figure 5], [Figure 6].
Figure 4: The effect of polyherbal formulation and Indomethacin on Freund's complete adjuvant-induced paw edema in Wistar rats. Group I: Control; Group II: Freund's complete adjuvant-induced rats; Group III: Indomethacin (10 mg/kg body weight) treated Freund's complete adjuvant-induced rats; Comparisons are made with a: Group I versus Group II, III, and IV, b. Group II ***P < 0.001 compared to Normal. #P < 0.005 compared to negative control, ###P < 0.001 compared to negative contro

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Figure 5: The effect of polyherbal formulation and indomethacin on Freund's complete adjuvant-induced paw edema in Wistar rats. Group I: Control; Group II: Freund's complete adjuvant-induced rats; Group III: Indomethacin (10 mg/kg body weight) treated Freund's complete adjuvant- induced rats; Group I versus Group II, III and IV, Group II *P < 0.05 compared to normal, ***P < 0.001 compared to normal #P < 0.05 compared to normal, ###P < 0.001 compared to negative control

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Figure 6: The effect of polyherbal formulation and indomethacin on Freund's complete adjuvant-induced paw edema in Wistar rats. Group I: Control; Group II: Freund's complete adjuvant-induced rats; Group III: Indomethacin (10 mg/kg body weight) treated Freund's complete adjuvant-induced rats

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The effect of polyherbal formulation on histopathological changes in the ankle joint (periarticular inflammation) left hind paw on FCA-induced rats is shown in [Figure 7]. The effect has shown between different groups such as periarticular inflammation (control) and negative control [Figure 7]a, periarticular inflammation (positive control) [Figure 7]b, joint space distance (Test 2 and Test 3) [Figure 7]c, and joint space distance (Test 1 and Test 3) [Figure 7]d.
Figure 7: (a) The periarticular inflammation (control) and (negative control) effect of polyherbal formulation on histopathological changes in ankle joint (periarticular inflammation) left hind paw on Freund's complete adjuvant induced rats. (b) The periarticular inflammation (positive control and Test 1) effect of polyherbal formulation on histopathological changes in ankle joint (periarticular inflammation) left hind paw on Freund's complete adjuvant-induced rats. (c) The joint space distance (Test 2, Test 3) effect of polyherbal formulation on histopathological changes in ankle joint (joint space distance) left hind paw on Freund's complete adjuvant-induced rats. (d) Joint space distance (Test 1, Test 3) effect of polyherbal formulation on histopathological changes in ankle joint (joint space distance) left hind paw on Freund's complete adjuvant-induced rats

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  Discussion Top


The present investigation was carried out to formulate and evaluate the well-known Indian medicinal plants for their antiarthritic activity in FCA-induced female Wistar rats, and the results were compared with standard Non steroidal anti-inflammatory drug (NSAID) indomethacin. The polyherbal suspension formulation was prepared by plants using seed coat and kernel of T. chebula and T. bellirica and fruits of E. officinalis as an active medicinal ingredients and ghee as a vehicle. Gum acacia and BHT were added as a suspending agent and a preservative, respectively. The polyherbal suspension was evaluated for their physicochemical parameters and acute toxicity.[20] Animals were divided into six groups, each containing six animals.[21] The arthritis was induced by injection of FCA at a dose of 0.1 ml into the ankle joint of left hind paw. The suspension is administered orally in rats at the dose of 250, 500, and 750 mg/kg body weight and standard drug indomethacin at the dose of 10 mg/kg orally. According to the present study, it clearly indicates that the polyherbal suspension has promising antiarthritic effect on FCA-induced rats.[22],[23]

There were normal microscopic features in normal group including joint space, synovial hyperplasia, edema, granulation time, cartilage, and bone destruction. However, the negative control group showed the occasional periarticular granuloma, moderate periarticular inflammations, and total synovial ulceration. Further, all the total groups showed the reduction in inflammation parameters such as edema, pannus formation, synovial cellular degradation, and synovial microphages. In the present study, we observed that treatment with polyherbal formulation significantly reduced FCA-induced arthritis at the 2nd and 3rd week. This observation suggests that these formulations may modify effects of mediators such as kinins and prostaglandins, which are released in the 2nd and 3rd week. In the present study, we have observed that the treatment with polyherbal formulation effectively reduced the FCA-induced arthritis in female Wistar rats which suggest antiarthritic activity for polyherbal formulation. Injection of FCA into rat's hind paw induces increased paw volume as primary lesions with maximum after 6–7 days. Secondary lesions occur after delay of appropriately 12–15 days, which are characterized by inflammation of noninjected sites. Primary lesion was due to inflammation, whereas secondary inflammation in uninjected paw was due to immune response.

Histopathological results showed that joint space and their thickness decreased when the polyherbal formulation administered into the female Wistar rats, and also, it had shown decreased level of synovial inflammation at the 2nd and 3rd week (Group No. IV – Test 1) at the dosing of 250 mg/kg body weight orally as compared to Group No. V – Test 2 (500 mg/kg) and Group No. VI – Test 3 (750 mg/kg).


  Conclusion Top


A study effort has been made for preparing new polyherbal formulation by seed coat and kernel of T. bellirica and T. chebula and fruits of E. officinalis for pharmacological screening of antiarthritic activity. Finally, the polyherbal formulation was tested for antiarthritic activity and was compared with standard drug indomethacin. The result shows significant antiarthritic potency for polyherbal formulation for its commercial use.

Histopathological results showed that joint space and their thickness decreased when the polyherbal formulation administered into the female Wistar rats, and also, it had shown decreased level of synovial inflammation at the 2nd and 3rd week (Group No. IV – Test 1) at the dosing of 250 mg/kg body weight orally.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
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  [Table 1], [Table 2], [Table 3]



 

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