Herbal medicines are those contain active ingredients from plant parts, such as leaves, roots, or flowers. Like conventional medicines herbal medicine has effect on body, and can be harmful if not used correctly.1 Inflammation is a normal response of the body to protect the tissues from infection, injury or diseases. The  response begins with the production and release of chemical agents from cells in infected, injured or  diseased tissues. The inflammatory response can either acute or chronic. Acute inflammation lasts only for  few days. If the wound turns red, hot, hurts or swells. Inflammation is a beneficial process serving to  immobilize the area of injury as the rest of the immune system mobilizes to heal. Chronic inflammation become problem when the solution to infection, injury or disease. Chronically inflamed tissues continue to generate signals that attract leukocytes from the bloodstream.2 The present work is to develop polyherbal capsule containing phytopharmaceuticals applied in the treatment  of inflammation. Polyherbal formulations are effective for anti-inflammatory process. They can stimulate a  variety of physiological functions that accelerates the process of inflammatory action.  Papaya is an herbaceous perennial plant usually has a single, semi woody, hollow, erect stem, which  terminates with a cluster of large, palmately lobed leaves with long petioles and latex vessels in all tissues. A  crown shaped leaves are found in spiral manner, palmate lobes, and each lobe again pinnately lobed. Leaves  are dark green colored.  It also contains active constituents like saponin, flavonoids, flavones, steroids, tannins, glycosides, reducing  sugars. It has Anti-inflammatory action, improve blood sugar control, support skin and hair health, prevent  cancer.3 The Hill Glory Bower is a flowering, semi-herbaceous, and gregarious shrub, which naturally forms groups  around the parent plant. These are erect, quadrangular branches covered with a dense, short layer of yellow  hairs. It contains active ingredients such as Clerodolone, Clerodone, Clerodol, and a steroid named  Clerosterol, glycosides, alkaloids, terpenoids, tannins. Antimicrobial, anthelmintic, hepatoprotective, anti- inflammatory, antioxidant, anticonvulsant activity.4 Heliotropium indicum is an erect, thick fetid, annual or perennial herb with hirsute ascending branches. The  leaves are opposite or sub-opposite, alternate or sub-alternate and straight forward, sheet-shaped from ovate

to elliptical, hairy, and sharp. The margins of the leaves are undulate; the nerves present on both sides are  serrulate or cordate and clearly visible under the leaves. It contains active constituents like alkaloids,  flavonoids, saponins, glycosides, oils, steroids. It has wound healing, anti-inflammatory, anti-dote, cure eye  infection, antiseptic, treat kidney problem.6 Pineapple leaves considered an herbaceous, tropical, and monocot perennial plant. Its leaves are spiral in

arrangement and on the terminal ends have flowers which then produce edible fruit. It contains active

 constituents such as alkaloids, tannins, flavonoids, glycosides. It helps to fight cough and cold, anti- inflammatory, antioxidant activity, monitor nervous system function.5

MATERIALS AND METHODS

Collection of plant material

The fresh leaves of Clerodendrom infortunatum, Carica papaya, Heliotropium indicum, Ananas comosus  were collected from Kasargod and Kozhikode district, Kerala(India) in the month of April 2024.

Authentication of plant material

The plant material was collected from Kasaragod and Kozhikode district, Kerala(India) in the month of  April 2024. The plant material was identified and authenticated by Dr.K.M. Sreekumar, Professor  (Entomology) College of Agriculture Kerala, Agricultural University, Padanakkad, Kasargod District,  Kerala.

Extraction of plant material

Extraction of   leaves of Clerodendruminfortunatum, Caricapapaya, Heliotropium indicum, Ananas comosus was carried out by using maceration method. Heliotropium indicum was washed and shade dried. For maceration the dried plant material was macerated  in 95% Ethanol for 3 days, filtered and dried to obtain ethanolic extract. Caricapapaya was washed and dried. For maceration the dried plant material was macerated in methanol for  24 hours and filtered and dried to obtain methanolic extract. Ananas comosus was washed, dried and powdered. The powdered plant material was macerated in methanol  for 72 hours, filtered and dried to obtain extract. Clerodendrom infortunatum was washed and dried. The extract sample was soaked in 200ml of methanol for  32 hours and filtered and dried to obtain the extract.  Preliminary phytochemical screening The extracts of selected plants were subjected to phytochemical screening to detect the various  phytoconstituents present such as alkaloids, carbohydrate, proteins, glycosides, tannins, saponins,  flavonoids, terpenoids, steroids, amino acids, phytosterols

Formulation of polyherbal capsule

Powdered mixture along with other excipients were weighed and pass through the sieve no.44. The  Pre-formulation studies were carried out. Three trials were conducted and most suitable is selected (trial 3).  “0” size capsule shells were de dusted transferred into polybags and filled by using hand operated gelatin capsule filling machine to an average net content of weight.7-8

Bulk density, angle of repose, Hausner’sratio, Carr's index, and particle size distributionwas determines for

evaluating the physical characteristics of the powder.9

A. Bulk Density                                                                                                                                                             10g of powder mixture was taken in a graduated measuring cylinder on a wooden surface. Bulk density is calculated using formulated.

B. Angle of repose                                                                                                                                                                                                            

It was determined by using funnel method. The powder was allowed to flow through a funnel fixed on a stand to form a heap.

C. Compressibility /Carr's index

This is calculated using the formula:

Carr's index=(Tapped density - Bulk tapped density) Tapped density X 100

D. Hausner’s ratio

The formula used to determine the Hausner's ratio is:

Hausner’s ratio=Tapped density Bulk density

INVITRO ANTI-INFLAMMATORY ACTIVITY

The HRBC membrane stabilization was used as a method to evaluate the anti-inflammatory activity. The  collected blood was mixed with an equal volume of sterilized Alsever medium (2%w/v dextrose, 0.8%  sodium citrate, 0.5% citric acidand0.42% sodium chloride in water). The blood was further centrifuged at 3000 rpm for 10 min and the packed cells were washed with isosaline (0.85%pH 7.2) and finally 10% v/vsuspension was made with isosaline.The assay mixture contained the secondarymetabolite from the plant  extract,1 ml phosphate buffer (0.15 M pH 7.4),2 ml hypo saline (0.36%) and 0.5 ml HRBC suspension.  Diclophenac was used as a referencedrug.2 ml distilled water was used as the control. The assay mixture  was incubated at 37°C for 30 min and centrifuged at 3000 rpm for 10 min. The hemoglobin content in the supernatant was estimated using a UV - Visible spectrophotometer at 560 nm,

The percentage of hemolysis = (OD of test solution / OD of control) x 100

OD = Optical density

Anti- inflammatory activity is percent of protection is calculated using the formula,

Percent of protection = 100 - Hemolytic percentage

“OD of test” is optical density or the test sample’s absorbance and “OD of control” isoptical density or  absorbance of negative control.10

EVALUATION OF POLYHERBAL CAPSULE11

1. Weight variation test

The variability in the amount of powder contained in each herbal capsule was measured by the weight

 variation test. Twenty herbal capsules were randomly selected and weighed. Then the average weight is calculated and compared with the individual herbal capsuleweight.The percentage variation was calculated  as per USP (2010) specifications.The herbal capsule shell is not less than 90% and not more than 110% of  theoretically calculated weight of each unit.

2. Disintegration Test

The disintegration test is themeasure of the time required under a given set of conditions in which the  selected capsule was disintegrated into the particlesthat pass through 10 mesh screenswithin a specifiedtime.

3. Determination of Moisture content

The loss on the drying test is important when the herbal substance is known to behygroscopic. An excess of  water in medicinal plant material will encourage microbialgrowth, the presence of fungi, and insect  Deterioration. In modern pharmaceutical technology, the moisture content provides information concerning

The shelf life and quality of the drug.

Moisture content (%) = (Final weight of sample / initial weight of sample) x 100

4. Determination of pH

1g of capsule powder was taken and dissolved in 100ml demineralized water. The pHvalue of the solution  was determined by a digital pH meter. The pH meter was calibrated using a buffer of 4.9 and 7 pH .The  electrodes were immersed in the test solution and pH was measured.

RESULTS

Preliminary phytochemical screening

The crude drugs with solvents like water, methanol, ethanol and they were subjected to preliminary

 phytochemical screening to detect the phytochemical constituents.

Evaluation of poly herbal capsule

After filling of powder mixture the capsule is evaluated for weight variation among the capsule, moisture  content, disintegration testing and pH of 1% aqueous solution is determined.

Invitro anti- inflammatory activity by HRBC Membrane stabilization method

The HRBC membrane stabilization was used to evaluate the anti -inflammatory activity. It is done by using  collected goat blood and mixed with Alsever medium prepared. Anti-inflammatory activity is determined by HRBC method to determined the percentage prevention of Hemolysis.

DISCUSSION