Antiarthritic efficacy of Clematis orientalis

Materials and Methods

Drugs and chemicals

Formaldehyde (VWR, International Ltd, England), diclofenac sodium (Sigma-Aldrich, USA), aspirin (Uni-Chem, Germany), bovine serum albumin (Sigma-Aldrich, USA), egg albumin from fresh hen’s egg, n-butanol (Sigma-Aldrich, USA),  sodium chloride (Sigma-Aldrich, USA), disodium hydrogen phosphate (Merck, Germany), potassium dihydrogen phosphate (Riedel-de-Haen, USA), sodium hydroxide (Sigma-Aldrich, USA) and hydrochloric acid (Riedel-de-Haen, USA) were used.

Plant material

Flowers of C. orientalis were collected from Gilgit Baltistan and were authenticated by a botanist through macroscopic and microscopic examination. A voucher specimen was deposited in the herbarium of Department of Pharmacology, College of Pharmacy.

Extraction and fractionation

The aqueous ethanolic extract was prepared by cold maceration technique. Air dried flowers were grounded to a fine powder and soaked in aqueous-ethanol (30:70) mixture. Soaking and filtration process was repeated 3 times more. After removal of solvent under pressure in a rotary evaporator, the residue was dried with 12.6% yield from 5 kg powdered plant material (Alamgeer et al., 2015). Different fractions were prepared using extract (120 g) by sequential solvent partition using various organic solvents hexane, dichloromethane, ethyl acetate and butanol so as to concentrate the activities in any one of the resultant composites.  Fractions were obtained with following yields as hexane (10.7%), dichloromethane (0.9%) ethyl acetate (2.3%), butanol (24.2%) and aqueous (60.1%). Among the fractions, hexane, butanol and aqueous fractions got in adequate quantities to conduct all investigational procedures (in vitro and in vivo biological activities).

Animals

Sprague-Dawley rats (200-300 g) had been used in this study. All the rats were kept under standard laboratory conditions of 23 to 25°C, relative humidity 55% with 12 hours light/dark cycle. They were fed with stock rodent diet and tap water. 

Experimental protocol

Inhibition of protein denaturation method using Bovine serum albumin.

The reaction mix comprising of 5% aqueous solution of bovine serum albumin (0.5 mL) and 70% ethanolic extract/fractions of C. orientalis (0.1 mL) and the standard drug aspirin was prepared at different concentrations. 1N HCl was employed to maintain 6.3 pH. Afterwards, samples were incubated at 37°C for 20 min followed by heating at 57°C for 30 min. Subsequent to refrigeration the samples, 2.5 mL buffer (pH 6.3) was added to each test tube. For control test tube dist. H₂O was used rather than aqueous ethanolic extract/fractions of the plant while product control did not contain bovine serum albumin. Optical density was taken using spectrophotometer at 660 nm. Blockade of protein distortion was deliberated as follows:

 Age inhibition =  {100 – (Abs. of Ts – Abs. of PC)   / Abs. of TC }  Ã— 100                                                 

(Alamgeer et al., 2017a)

Inhibition of protein denaturation method using fresh egg albumin

In this method reaction mixture (5 mL) contained 0.2 mL of egg albumin (from fresh hen’s egg), 2.8 mL of phosphate buffered saline (PBS 6.4) and 2 mL of different concentrations of 70% ethanolic extract of plant/fractions. Distilled water served as control. The reaction mixture was incubated at 37 ± 2°C in an incubator for 15 min followed by heating at 70°C for 5 min. After cooling, their absorbance was measured at 660 nm. Standard drug diclofenac sodium at same concentrations served as reference control and treated similarly for the determination of absorbance. Following formula has been used to compute protection against pathological alterations of egg albumin:

%Inhibition = 100 X [Vt /Vc-1]

Where, Vt = absorbance of the test sample, Vc = absorbance of control (Uttra and Alamgeer, 2017).

Human red blood cell (HRBC) membrane stabilization method

Preparation of reagents

Alsever’s solution was prepared by dissolving 2 g dextrose, 0.8 g sodium citrate, 0.05 g citric acid and 0.4 g sodium chloride in distilled water. The final volume was made up to 100 mL with distilled water. Hypotonic saline was prepared by dissolving 0.4 g of sodium chloride in 100 mL of distilled water. While isotonic saline was prepared by dissolving 0.9 g of sodium chloride in 100 mL of distilled water. Buffer of pH 7.4, 0.2 M was prepared using 2.4 g disodium hydrogen phosphate, 0.2 g of potassium dihydrogen phosphate and 8 g of sodium chloride in 100 mL of distilled water (Gautam et al., 2011).

Preparation of suspension (10% v/v) of human red blood cell (HRBC)

10% v/v suspension of RBCs was prepared using blood from a normal healthy participant with isosaline for use in the experiment by following method as described by (Alamgeer et al., 2015).

Assay of membrane stabilizing activity

Assay mixtures containing 1 mL of PBS, hypotonic saline (2 mL) and 0.5 mL of HRBC suspension and 0.5 mL of different concentrations of ethanol extract of the plant, fractions, standard drug were separately mixed. 1 mL of phosphate buffer, 2 mL of hypotonic saline, 0.5 mL of ethanolic (70%) extract at different concentrations and 0.5 mL of 10% erythrocytes suspension were used as a test solution. 1 mL phosphate buffer, 2 mL water and 0.5 mL of RBCs was used as test control. 1 mL phosphate buffer, 2 mL hypotonic saline, 0.5 mL standard drug solution of varying concentration and 0.5 mL of cell suspension were taken as a standard solution (diclofenac sodium). After that resultant solution heated in an incubator at 37°C for half an hour followed by centrifugation at 3,000 rpm. The optical density of upper layer was assessed at 560 nm. Percentage of HRBC membrane stabilization or protection against lysis has been analyzed using a formula as described by Alamgeer et al. (2017b).                                                                           

Formaldehyde-induced arthritis in rats

Animals have been divided into different groups with 5 rats in each group. Group I was given distilled water (3 mL/kg; PO) and labeled as a negative control. Group II was treated with piroxicam at 10 mg/kg. Group III, IV and V were treated with crude extract at 50, 100 and 200 mg/kg dose respectively. Group VI, VII and VIII was administered hexane fraction at 50, 100 and 200 mg/kg in that order. Similarly, Group IX, X and XI were given butanol fraction at same doses as given above.  Likewise, Group XII, XIII and XIV were treated with aqueous fraction at 50, 100 and 200 mg/kg correspondingly. All treatments were given orally for 10 days respectively. On day 1, 30 min after drug administration, formaldehyde (0.1 mL of 2% solution) was administered subcutaneously in rat left hind paw of all groups. The injection was given again on the 3^rd day. The antiarthritic effect was appraised by measuring paw diameter and paw volume of the treated and control rats at alternative days during a period of 10 days by means of digital vernier caliper and digital plethysmometer accordingly and blockade of paw edema was calculated as described by Alamgeer et al. (2017b).

Statistical analysis

Data were analyzed by two-way ANOVA followed by Bonferroni multiple comparison tests using the GraphPad Prism. The minimal level of significance was considered as p<0.05.