In-vitro Anti-In lammatory activity of Digera muricata Extracts

In lammation is a process that is generally termed for pain and redness, which includes swelling. But it is de ined as a heterogeneous group of processes which result in the pain and increase in vascular circulation and denaturation of protein that results in tissue degeneration. There are many synthetic drugs available to treat and relieve the in lammation. They arepotent andhavemany side effects too. So there is an increased search for alternative means of treatment option. Thereby there was a high focus on the medicinal plants and herbs. Digera muricate, which is used to treat many diseases like analgesic activity, antidiabetic activity and anticancer activity too, this plant has proven to possess the antioxidant potential also. So there is no enough work done to verify the in lammatory potential of the plant. This was the primary consideration for this research to select the plant to prove it has anti-in lammatory potential. Having in the account that the ethical problems in the usage of lab animals, the anti-in lammatory potential of the plant was evaluated in-vitro using enzyme inhibition assays. The in-vitromodels of the in lammatory activity are used for testing the anti-in lammatory activity, and the results were not different from invivo models. The extracts showed a dose based rise in the action, and the plant process a better anti-in lammatory activity than the standard drugs.


Invitro,
In lammation, Digera muricate ABSTRACT In lammation is a process that is generally termed for pain and redness, which includes swelling. But it is de ined as a heterogeneous group of processes which result in the pain and increase in vascular circulation and denaturation of protein that results in tissue degeneration. There are many synthetic drugs available to treat and relieve the in lammation. They are potent and have many side effects too. So there is an increased search for alternative means of treatment option. Thereby there was a high focus on the medicinal plants and herbs. Digera muricate, which is used to treat many diseases like analgesic activity, antidiabetic activity and anticancer activity too, this plant has proven to possess the antioxidant potential also. So there is no enough work done to verify the in lammatory potential of the plant. This was the primary consideration for this research to select the plant to prove it has anti-in lammatory potential. Having in the account that the ethical problems in the usage of lab animals, the anti-in lammatory potential of the plant was evaluated in-vitro using enzyme inhibition assays. The in-vitro models of the in lammatory activity are used for testing the anti-in lammatory activity, and the results were not different from invivo models. The extracts showed a dose based rise in the action, and the plant process a better anti-in lammatory activity than the standard drugs.

INTRODUCTION
In lammation is a process that is generally termed for pain and redness, which includes swelling. But it is de ined as a heterogeneous group of processes which result in the pain and increase in vascular circulation and denaturation of protein that results in tissue degeneration. This in lammation may be due to micro-organisms, physiological stress, oxidative stress etc. and result in pain, swelling and redness. There may be seen as an inability to do the normal functioning of the body and its organs (Jain and Bari, 2010). When there is an in lammation in the body, tissue releases cytokines and Pg's and histamines which are called as pain mediators. This will increase blood low to the site of the injury, which results in the production of chemotaxis. Therefore, tissue repair starts to work (Koster et al., 1959).
There are many synthetic drugs available to treat and relieve the in lammation. They are potent and   (Shiddamallayya et al., 2010). They are serving humans as an alternative treatment option in many diseases like diabetes, hypertension, CV disorders etc. Many of those plants were used to treat in lammations also (Soudahmini et al., 2005).
One of those plants is Digera muricate, which is used to treat many diseases like analgesic activity, antidiabetic activity and anticancer activity too (Koster et al., 1959). This plant had been proven to possess the antioxidant potential also. So there is no enough work done to verify the in lammatory potential of the plant. This was the primary consideration for this research to select the plant to prove it has anti-in lammatory potential. There were many folklore claims of the plant had used to treat many other diseases like diarrhoea, and it as has a cooling effect on the body. Having in the account that the ethical problems in the usage of lab animals, the anti-in lammatory potential of the plant was evaluated in-vitro using enzyme inhibition assays.

Extraction
The aerial plant parts were collected in November and dried under shade and then ground into a ine powder with a blender. This powder is stored in room temperature for a day to use for further extraction. This powder was used for extraction using methanol using Soxhlet extractor, and the iltrate is reduced to get the desired consistency of extract, which is like paste consistency using an evaporator and stored the extract in a desiccator. The plant extract was dissolved in distilled water, that the concentration of extracts was maintained at 100, 200, 300, 400 and 500 microgram/ml. This was used for further Assay.

Activity
The anti-in lammatory activity of the plant was assayed invitro using enzyme analysis as follows

Protein denaturation inhibition assay
In this method, the heat was used to denaturate the protein as per the method described by Mizushima and Kobayashi (1968). The UV absorption was measured for all the concentrations at 660nm.

Antagonizing proteinase
This method was adopted to test the activity according to the method described by Oyedepo and Femurewa (1995). The UV absorption was measured at 210nm. The percentage inhibition and absorbance were noted according to the method.

Lipoxygenase inhibiting activity
In this method, the extracts were tested from antiin lammatory activity in different concentrations as planned above. The process was followed as per the procedure by Shinde et al. (1999). Lipoxidase enzyme was used as a marker for the in lammatory mediator, and the activity was measured in UV at 234 nm. The percentage of inhibition and absorbance was noted and tabulated.

Membrane stabilization assay
In this method, RBC cells are used to study the stabilization of membrane of cells as there is a similarity of membranes of RBC and cytokines. In this method, a healthy human volunteer was selected who did not use any NSAID in near history for seven days, and his blood sample was withdrawn and centrifuged to separate RBC. The cells were collected and suspended in saline solution.
In this method, hemolysis was measured in two ways.

Haemolysis induced by heating.
The method was adopted to estimate the amount of anti-in lammatory activity by preventing the haemolysis of the RBC invitro, which is induced by heat according to the process detailed by Sakat et al. (2010). The standard drug used in this method is aspirin.

Haemolysis induced by hypotonicity.
In this method, the extracts were tested by different concentrations, and the haemolysis was induced using a hypotonic saline solution. The technique was adopted as given by procured by Azeem et al. (2010). The percentage inhibition was measured and compared with the standard, Diclofenac sodium.

RESULTS AND DISCUSSION
The anti-in lammatory activity of the plant was investigated in-vitro using various models, and the results were tabulated in Table 1 & Table 2. In protein denaturation assay, the extracts showed better activity than the standard drug, aspirin with almost 3-4 per cent better activity. During the in lammation process, the denaturation of protein occurs by the application of heat. It is the most notable causes of in lammation, and the extract showed an increase in activity in a dose based manner (Kim et al., 2004).
In the lipoxygenase method, the extracts at different doses showed a dose-dependent increase in the activity so at 500microgm extracts showed the highest activity, which is less than that of the standard. Lipoxygenase induces the oxidative free radicals and initiates the stress-based in lammation in the body. The extracts, when successfully inhibited the enzymes, means the antioxidant activity of the extract was an added advantage (Sokol et al., 2008).
Proteinase is an enzyme responsible for the in lammation mediation at the lysosomal level. Neutrophils had rich content of the enzyme. Therefore the enzyme inhibition is said to lower the in lammation via the mechanism of the enzyme. The extracts a better activity compared to the standard drug Table 1. The antioxidant activity of the extract might have added advantage in the antiin lammatory activity in this Assay.
As discussed earlier, membrane stabilization assay was used on RBC as there is a similarity between the lysosome membrane and the RBC. These lysosome breakdowns and releases corrosive enzymes that digest the tissue and cell, which leads to in lammation. The extracts were used to prevent the breakage of the membrane and to stabilize it. So RBC hemolysis was assayed in two methods. In the heat-induced method and hypotonicity method, there was a significant reduction in membrane breakage. The activity was similar and higher than the standard drugs, Aspirin and Diclofenac sodium Table 2 ( Alhakmani et al., 2013).

CONCLUSION
The in-vitro models of the in lammatory activity are used for testing the anti-in lammatory action, and the results were not different from invivo models.
The extracts showed a dose based rise in the activity, and the plant possess a better anti-in lammatory activity than the standard drugs.