Procurement and authentication of plant samples
Dried fruits of Solanum xanthocarpum
and fruit pulp of Cassia fistula
were procured from the local market in Mumbai and were authenticated at Nicholas Piramal Life Sciences, Mumbai. Fruits were powdered with a mechanical grinder and stored in an airtight container. The powdered drugs were tested for routine quality parameters like total ash, moisture content, sulfated ash, insoluble ash, etc.[5
Preparation of plant extract[6
Aqueous extraction of the powdered dried fruits of both the plants was carried out separately in a Soxhelet apparatus for 18 h. The extract was dried to a constant weight and kept under refrigeration. The extract obtained was standardized for parameters like %yield, color and consistency and used for further study.
Phytochemical screening of the extracts
Both the extracts were subjected to various tests to check the phytochemical constituents, viz. carbohydrates, glycosides, tannins, alkaloids, saponins, inorganic matter, etc.[5
Wistar Albino rats of either sex with body weight 150–300 g were used for the study. The animals were housed in cages under standard laboratory conditions (12-h light/dark cycle at 25° ± 2°C). They were allowed free access to a standard dry pellet diet (Hindustan Lever Ltd. Mumbai, India) and water ad libitum. All procedures described were reviewed and approved by the SNDT Women’s University Animal Ethics Committee.
Animals were fasted overnight before administering the study drugs. Animals were divided into a total of six groups, as positive control, negative control, Solanum xanthocarpum group, Cassia fistula group, combination 1 and combination 2.
Diclofenac sodium at a dose of 150 mg/kg was administered to the positive control group animals. Oral single doses of individual plant extracts ranged from 100 to 500 mg/kg and a combination of both plant extracts were administered to the other group of animals, as shown in .
Doses administered to animals
After 30 min of the dose administration as shown in , rats were challenged by subcutaneous injection of 0.1 ml of 1% solution of carrageenen into the planter side of the right hind paw. The paw volume was measured plethysmographically immediately after injection and again 1, 2, 3 and 4 h after challenge.
The increase of paw volume from 0 to 4 h was calculated as a percentage. The difference of average values between treated animals and control groups was calculated for each time interval and compared statistically.
The percentage inhibition was calculated according to the following formula:[9
Percentage inhibition = 100 (1 - [A - X/B - Y])
where, A- mean paw volume of treated group after carrageenan injection.
B- mean paw volume of control group after carrageenan injection.
X- mean paw volume of treated group before carrageenan injection.
Y- mean paw volume of control group before carrageenan injection.
Isobolographic analysis of the response
The assessment of synergism is most often made from experiments in which an effect level is chosen and doses of drug A alone, drug B alone and the combination (a,b) that give this effect are determined experimentally. Doses that give the same effect are called isoboles and the method of analysis is an isobolographic method.
A measure of the synergism is made from the value of the interaction index. The index, denoted by γ, is defined by the isobolar relation.
Interaction index: a/A + b/B = γ
where, A = ED50 of Solanum xanthocarpum Schrad and Wendl
B = ED50 of Cassia fistula Linn
(a,b)= Dose of combination that shows ED50
The quantities in the above equation are obtained from the dose-response curves of drugs A, B and the combination.
If γ = 1, the interaction is additive,
if γ <1, the interaction is synergistic and
if γ >1, the interaction is antagonistic in nature.
Isobologram is plotted between dose of drug A and drug B. Doses that give a specified effect are joined, which is called as additivity line. Combinations are plotted on an isobologram: if that combination lies below the additivity line, it shows a synergistic activity and if it lies on the additivity line, it shows an additive activity. If it is beyond the additivity line, an antagonistic activity is indicated.[11