Anti-inflammatory Activity of Herbal Tablet of Phyllanthus emblica on Carrageenan-induced Paw Edema in Wistar Rats

Aim: In the current investigation, tablet dosage form of hydroalcohol extract of leaves of P. emblica was developed and evaluated foranti-inflammatory activities. Methodology: Hydroalcohol extract of leaves of P. Emblica (HPE) was prepared and formulated tablet dosage form. The mechanical properties e.g. hardness, friability, disintegration, and dissolution of tablet were evaluated. Anti-inflammatory activities of HPE (150 mg/kg) and HPE (650 mg) tablets were performed in carrageenan induced hind paw edema in wistar rats. Results: The anti-inflammatory activity was found to be significant in carrageenan inducedpaw edema. HPE (150 mg/kg) and HPE tablets (650 mg) were significantly (p < 0.05) reduced the carrageenan induced paw edemaby 66.41% and 69.43% respectively as compared to Original Research Article Behera et al.; JPRI, 33(54B): 155-167, 2021; Article no.JPRI.70206 156 carragenan control. The % inhibition of standard drug (dichlorofenac sodium) was recorded to be 70.18% after 5 h. Conclusion: The study standardised the development of HPE tablet and its anti-inflammatory


INTRODUCTION
Inflammation is a biological protective response of vasculartissues against external agents such as irritants, pathogens or damaged cells. It is characterised as increased movement of plasmaand innate immune system cells such as neutrophils andmacrophages from the blood into the injured tissues. Inflammatory symptoms such as increased bloodflow, vasodilatation, and enhanced cellular metabolism, extravasation of fluids and release ofsoluble mediators are noticed [1][2]. In the presence of inflammatory substances, phospholipase A2 get activated and release of inflammatory mediators such asprostaglandin, histamine, cytokines, leukotrienes, and serotonin occurs. As a result vascular permeability increases and excess accumulation of leukocytes takes place at the site of inflammation [3][4][5]. In light of these findings, studies on plants with proven folkloric anti-inflammatory properties are seen as a fruitful and reasonable research strategy in the search for new anti-inflammatory medications [6].
Phyllanthus emblica L. (family-Phyllanthaceae, synonym-Emblica officinalis) is commonly known as Indian gooseberry or amlaan indigenous species to India. In traditional medicinal system this plant is used in the treatment in various diseases [7]. Every part of P. emblicais useful owing to its medicinal and pharmaceutical properties. This plant has been scientifically validated for anti-inflammatory, adaptogenic, anticancer, antioxidant, nootropic, antidiabetic, immunomodulatory, osteoporosis antimicrobial and hyperlipidemiaactivities [8]. Various parts of this plant possess phenolics, flavonoid, vitamin, amino acid, minerals and tannins which are responsible to elicit pharmacological activities [9]. In previous studies, anti-inflammatory activities of different extracts of fruit and leaf of this species have been performed in carrageenan induced edema. So, the present investigation is based on evaluation of anti-inflammatory activities of hydroalcohol extract of leaf of P. emblicaand its tablet formulation in carrageenan induced paw edema in wistar rats.

Material and Reagents
All the solvents, chemicals and reagents were of analytical grade and purchased from Qualigenes Pvt. Ltd., India, Rankem Pvt. Ltd., India, S.D. Fine-Chem Pvt. Ltd., India.

Preparation of Plant Extract
The collected leaves of P. Emblica were shade dried. The dried plant material (700 g) was grounded to coarse powder. The material was defatted with petroleum ether to remove oil, fats and then extracted with hydroalcohol (80:20) by percolation for 72 h. The extract was evaporated under reduced pressure in rotavapor (R-100, Buchi, Switzerland) to collect dried semisolid mass and it was kept in desiccator for further investigations [10].

Excipients selection
Excipients are important in the design of a drug delivery system as it directly affects quality and efficiency. In the selection of excipients, the following parameterssuch as compendial ingredients, compatibility, stability drug release and availability were considered [11].

Granulation
Hydroalcoholextract of leaves of P. emblica (HPE) was blended with lactose (I.P.) dry starch (I.P.) sodium starch glycolate (I.P.) and microcrystalline cellulose (I.P.) were individually passed through mesh no.40 sieve and blended in a double cone blender for 15 minutes. The powder blend was granulated with starch paste and passed through mesh no 12 sieve and dried at 50 ± 2 ˚C in a hot air oven for 15 h. The dried granules were checked for LOD (NMT 4%) and lubricated with the mixture of talc (I.P.) and magnesium state (I.P.) and compressed [12].

Compression
Each tablet (650 mg) was compressed by using tablet compression machine eight station (Emtech, Ahmedabad, India). Theoretical average wt. 650 mg were taken for compression into flat round tablets. Nine batches of 250 no. of tablets were prepared by wet granulation method using starch paste as granulating agent. The granules were dried after passing through 12 no. sieve for 18 h at 50 ˚C and finally passed through 16 no. mesh sieve, blended and lubricated [12].

Drug excipient compatibility study
The interaction of extract and excipient was analysed with KBrby using FT-IR (Perkin Elmer ® , Model Spectrum 1, USA). IR spectrum of drug wasmeasured at a modest scanning speed of 4000-400 cm -1 [11].

Test for disintegration
The disintegration times (DT) of the tablets were determined in distilled water at 37± 0.5 ˚C using a digital disintegration testing apparatus (Electrolab, India) by following the standard method [12].

Dissolution test
In vitro release study of the tablets was determined at 37 ± 0.5 ˚C in 900ml of 0.1M HCl using a dissolution test apparatus (Electrolab, India) with the paddle rotated at 100rpm. Samples (5ml) were withdrawn and replaced with fresh medium at fixed time (5, 10, 15, 30, 60, 90 and 120 min) intervals. The amount of drug released was determined at 314.8nmin UV-Vis (UV-1800 Shimadzu) spectrophotometer [13].

Stability
In accordance to ICH guidelines, the optimized batch no. B1 was subjected to three months of stability testing at 30 ± 2º C/65 ± 5% RH and 40 ± 2º C/75± 5% RH. Physicochemical parameters and active content were considered for stability of the drug [14][15].

Optimization through Experimental Design
To study the effect of variables, we applied the 32 factorial designs. The amount of SSG (X1) and MCC (X2) were kept as an independent variables and hardness, disintegration time, % drug release (Y1) were selected as dependent variables. The data analysis of values obtained from various batches for drug release and swelling were subjected to multiple regression analysis using statistical software (Design Expert, 8.0.7.1).The quadratic model fitted by carrying out multiple regression analysis as shown below: Y= β0+ β1X1 + β2X2 + β11X1 2 + β22X2 2 + β12X1X2 Where Y: measured response; X: levels of factors; β: coefficient computed from the responses of the formulations. The levels of the two factors were selected on the basis of the preliminary studies carried out before implementing the experimental design. All other formulation and processing variable were kept constant throughout the study. The coefficients corresponding quadratic effects (b11and b22) are determined from the results of the experiment to identify statistically significant terms. Equation containing only statistically significant terms is then used to draw response surface plots (Design Expert, 8.0.7.1) to visualize the impact of changing variables at a glance [16].

Experimental animals
Swiss albino mice (50-100 g) of both sexes purchased from Imgenex India Pvt. Ltd., Bhubaneswar, Odisha. All of the animals were kept in conventional laboratory conditions for a week at temp of 25-30° C, relative humidity of 60-70%, and 12 h day-night period. Animals were fed with normal pellet diet (Pranav Agro Industries Ltd., Pune) and given free access to water. The ethical clearance for animal handling was approved by Institutional Animal Ethical Committee (IAEC) of Centurion University of Technology and Management (Regd. No.2024/PO/Re/S/18/CPCSEA).

Acute toxicity study
Acute oral toxicity tests were carried out in accordance with the OECD-423 guidelines (Acute toxic class method). In this analysis, Swiss albino rat(both sex, 100-200 g) (n=3/each dose) were chosen using a random sampling technique. The animals were fasted for 4 h and only had access to water. The extract (suspended with 0.5% w/v, CMC) was given orally to different groups of mice at doses of 5mg/kg, 50mg/kg, 300mg/kg, and 2000 mg/kg, and any behavioural changes were observed every hour for 24 h, and then mortality was observed for three days.If mortality was observed in 2/3 or 3/3 animals, then the dose administered was considered as toxic dose. However, if mortality was observed in only one mouse out of 3 no. of animals then the same dose was repeated again to confirm the toxic effect. If mortality was not observed, the procedure was then repeated with higher doses such as 5, 50, 300, 2000 mg/kg. The general behaviours were observed for first one hour and after 24 h of test drug administration [17].

Experimental design for antiinflammatory activity in carrageenaninduced paw oedema model
Anti-inflammatory activity was studied in carrageenan-induced edemaanimal model. The experimental animals were divided into 5 groups (n=6). In this model, acute inflammation was induced by sub-plantar injection of carrageenan suspension in normal saline in rat hind paw of groups II-V. Carrageenan was administered after 30 min of administration of test drugs and standard drug. The thickness of hind paw was measured by plethysmometric method. After 1, 3, and 5 h of carrageenan injection, inflammation was measured in animal. The % inhibition of inflammatory edema in test and standard groups animals was calculated by the formula, i = 100{1-(a-x)/(b-y)}where a = mean hind paw volume of test/standard group animals after carageenan injection, b = mean hind paw volume of positive control animals after carageenan injection, x = mean hind paw volume of test/standard group animals before carageenan injection, y = mean hind paw volume of positive control animals before carageenan injection [18].

Statistical analysis
The results of anti-inflammatory activity were expressed as mean ± SD. One-way analysis of variance (One-way ANOVA) was used to compare the groups at p<0.05, followed by Tukey's multiple comparison test using GraphPad software (Prism 7).

Drug Excipient Compatibility Study
The peak values (wave number) and the possibility of the functional group are recorded (Fig. 1). The FTIR result shows (Tables 1 and 2) that there is no significant change in the peak values of the extract when tested individually or collectively. Hence, the ingredients selected as an excipient are compatible with the extract.

Physicochemical Tests
All the batches of the formulated tablet were subjected to evaluation concerning hardness (kg/cm 2 ), thickness (mm), diameter (cm), friability (%), disintegration time (sec), and dissolution time (%). The obtained result of the evaluation parameters are given below (Table 3). All the fabricated batches displayed the necessary hardness of more than 5 kg/cm 2 along with friability value of less than 1%, ultimately representing the required strength and resistibility of the formulations. The tablet fabrication was done employing the punch to produce dimensions of 5 mm × 1.3 cm. The results confirmed that the produced tablets were detected to be free from any problems like capping, picking, and chipping.

Dissolution Study
At  Table 4. In the present study, the dissolution rate of batch no. B1 was greater than other batches. Also, the drug released from nine batches of formulations adopted zero-order kinetics (Fig. 2). The results identified tablets of batch no. B1 as compatible formulation and investigated for further studies.

Stability Studies
Sample tablets of the optimized formulation i.e. batch no. B1 was kept for stability studies. Stability studies for the tablet revealed good physical stability and organoleptic properties of tablets. The results suggested that the developed tablet formulation is stable for duration of three months with no loss of properties (Table 5).

Optimization through Factorial Design
Optimization of tablet formulations of HPEwas performed by using 3 2 full factorial designs. The comparison of the experimental and expected values of the responses is performed to determine the model's reliability. To determine simplicity of the experiment, two-variable analysis at three experimental levels were performed. A total of 9 no. of batches of tablet were prepared and these batches were evaluated for hardness, disintegration time and % drug release ( Table 6). The dependent variables values of the formulations were calculated to generate polynomial equations for the dependent variable in Design expert software.

Response 1: Hardness
For Hardness, the following equation was obtained from the design model, The positive coefficient of X1 indicated an increase in hardness with an increase in SSG, in the same way, the negative coefficient of X2 indicates a decrease in (Y1) i.e. hardness with an increase in MCC concentration. The mean hardness of tablet was calculated as 3.10.The surface responses plot for hardness shows that SSG (X1) effect was positive on hardness. As the concentration of SSG increased (from -1 to +1) i.e. from 32-52 then increase in hardness of tablet was observed.MCC also shows a negative effect on response Y1. As we increased MCC concentration from 28-48 then decrease in hardness was noticed but to a slight extent. Contour plot and Response surface plot showing the effect of factorial variables on hardness is depicted in Fig. 3.  (Fig. 4).

Response 3: Percent drug release
For % drug release, the following equation was obtained from the design model, The positive coefficient of X1 indicated an increase in % drug release with an increase in SSG, in the same way, a negative coefficient of X2 indicated a decrease in (Y1) i.e. % drug release with an increase in MCC concentration. The mean % drug release of the tablet was found to be 84.51.
Surface responses plot for % drug release showed that SSG (X1) effect was positive on % drug release. As the concentration of SSG increases (from -1 to +1) i.e. from 32-52 then % drug release was increased. MCC showed a negative effect on response Y1. As we increase the MCC concentration from 28-48 then % drug release was decreased but to slight extent. The effect of factorial variables on drug release is depicted in the contour plot and response surface plot (Fig. 5).
After optimization of drug composition in factorial design only 6 no. of combination formulations were selected and the best result was shown in combination of SSG (43.97 mg) and MCC (28 mg) ( Table 7).

Acute Toxicity Study
Acute oral toxicity tests were carried out in accordance with the OECD-423 guidelines (Acute toxic class method). Since there is no known dosage for the extract, it was subjected to an acute toxicity study using Swiss albino mice in accordance with OECD-423 guidelines, and it was found to be safe up to a dose level of 2000mg/kg.The following were the general behaviour found within the first hour and after 24 h of test drug administration (Table 8). According to the findings of the above acute toxicity report, there is no mortality after 72 h and no improvement in the animal's general behaviour [19].

Anti-inflammatory Activity
Inflammation is a part of vascular tissues' complex biological reaction to adverse stimuli like pathogens, damaged cells, or irritants. Redness, swollen joints, joint discomfort, stiffness, and loss of joint function are all symptoms. NSAIDs are currently used to treat inflammation. Unfortunately, these medications raise the risk of blood clots, which can lead to heart attacks and strokes. As a result, the development of potent anti-inflammatory medications from natural ingredients is currently being considered. The carrageenan-induced acute inflammation test is one of the best ways to find anti-inflammatory agents [20].  A2 A3 A1  A2  A3 A1 A2  A3  A1  A2  A3  1  Tremor  ------------2  Convulsion  ------------3  Straub Reaction  ------------4 Piloreaction + + + + + + + + + + + + 5 Catatonia Loss of Righting reflux  ------------12  Ptosis  ------------13  Lacrimation  +  +  +  +  +  +  +  +  +  +  +  +  14 Salivation Values are given in mean ± SD (n=6). Statistical significance differences were represented as * p< 0.05, ** p< 0.01, and *** p< 0.001 ED-edema volume, EI-edema inhibition, HPE-hydroalcohol extract of leaves of P. emblica On the basis of the results of acute toxicity studies, HPO (150 mg/kg) was selected and effective dose for treatment of edemaand tablet (650 mg/kg) of batch no. B1 wereopted for evaluation of anti-inflammatory activities in carrageenan induced animal model. The animals were observed for 5 h for change in their paw edema. In negative control group, administration of carrageenan caused inflammation and after 5 h, the degree of edema was observed to be maximum (2.65±0.02). However, on administration of HPE (150 mg/kg) and tablet (650 mg) caused significant reduction in edema (HPE, 66.41% and tablet, 69.43%) after 5 h and the results were found to be similar to that of standard drug (diclofenac sodium, 70.18%). The anti-inflammatory effect of HPE was attributed to the presence of bioactive compounds in it which may inhibit the secretion of inflammatory markers such as COX-1 and COX-2 and inhibit synthesis of prostaglandins (Table 9).

CONCLUSION
In the present study, the results and discussions proved the potential anti-inflammatory activity of P. emblica in tablet formulation. The selection of excipients and methodologies adopted to manufacture tablet formulation of hydroalcohol extraction of P. emblica (HPO) were found to be result-oriented as it is evidenced by various investigation of the current research work. Currently, the regular used NSAIDs are associated with many unwanted complications. Henceforth, the developed formulation of HPO warrants further investigations on antiinflammatory mechanism of action of drug at molecular level in various inflammatory diseases. Furthermore, extensive research work is needed to develop different dosage forms of HPO with reduced toxicity and value-added clinical utility.

ETHICAL APPROVAL
Animal Ethic committee approval has been taken to carry out this study.