To study Anti-inflammatory activity of 70% Methanolic Extract of  Triumfetta rhomboidea : In Vitro Study

 

B. Thawkar*, M. Kale, M. Oswal, K. Maniyar, K. Kadam, S. Kamat

Department of Pharmacology, Konkan Gyanpeeth Rahul Dharkar College of Pharmacy and Research Institute, Karjat:410201 Dist: Raigad, Maharashtra, India. University of Mumbai.

 

ABSTRACT:

AIM: To investigate and  evaluate anti-inflammatory activity of 70% methanolic extract of Triumfetta rhomboidea by in vitro study.

METHOD: Antiinflammatory activity was studied by membrane stabilizing effect on human red blood cells (HRBC) and protein denaturation activity using 70% of methanolic extract of  TR.

RESULT : The methanolic extract of TR having concentration 400µg/ml shows 34% of  membrane stabilizing activity whereas same concentration shows 208% of protein denaturation activity by protecting from lysis.

CONCLUSION:  From the study it can be said to  be the  anti-inflammatory activity of Triumfetta rhomboidea  is due protein denaturation activity and membrane stabilizing activity.

 

 

 

 

 

 

 

 

INTRODUCTION:

Inflammation   is a complicated series of biological reactions, which takes place only in vascularised tissue^[12]. It generally works by the principle of removing, dilute or barrier the injurious/pathogenic agent or tissue. And secondarily it works by inducing the healing and  the repair of the damaged tissue. Which results into the an accumulation of leukocytes and fluid in the vascularised tissue. Nonspecific, defensive response of the body to tissue damage is called as inflammation or it can be also defined as the response of living tissue to injury. Inflammatory conditions may produce by the pathogens, irritations caused by the chemicals, distortion or disturbances of the cells. It can be also produce by the abrasion of cells (a wound caused by superficial damage to the skin) and extreme temperatures ^[1]. 

 

Inflammation shows five main symptoms or signs which are togetherly called as “PRISH".1) Pain - the inflamed area is painful when touched. 2) Redness–which is  occur because the capillaries are filled up with more blood than normal blood volume. 3) Immobility–In this condition there may be some loss of function. 4) Swelling–which is caused the by an  accumulation of the fluid. 5) Heat– It is due to the more blood in the affected area which makes it feel hot to touch. If  inflammation is unchecked, it can leads to diseases like vasomotor  rhinnorrhoea, rheumatoid arthritis and atherosclerosis.^[15]

 

Ginger Black Pepper, Cardamom, Cayenne, Aloe, Chamomile, Turmeric, Celery seed, Green tea, Pomegranate, Bromelain , Devil's Claw, White Willow Bark, Egg Membrane are used to treat inflammatory action. But there is another drug is used to treat inflammatory action which is Triumfetta rhomboidea Jacq. The plant Triumfetta rhomboidea Jacq (Family : Tiliaceae) is commonly known as Chinese bur or Zinjurdi ^[10], is a shrub generally found in tropical and subtropical regions of India, Ceylon, Malay Peninsula, China, Africa and in America. TR contains carbohydrate glycosides, alkaloid glycosides, phytosterol, steroids, flavonoids, tannin and phenolic compounds^[2][11]. But  methanolic extract of TR may show  negative test for  glycosides and steroids. Various Parts of the plant used for medicinal purpose which are fruit, flower, leaves, bark and root. Root is used as  tonic styptic, galactogogue, aphrodisiac, cooling, useful in dysentery and as diuretic^[7]. TR is also used as anti-snake bite ^[11]. Pounded roots are used to treat Intestinal ulcer. Leaves, Flowers and Fruit are mucilaginous, demulcent, astringent, and also used in gonorrhea and against leprosy. Fruits, flowers and leaves of this plant is used as demulcent and astringent. Bark and fresh leaves are used to treat diarrhoea, dysentery  like disease. Flowers are rubbed with sugar and water if it is given to the patient suffering from gonorrhoea it helps in the  reduce burning. Fruits of the plant Triumfetta rhomboidea Jacq promote parturition.  Ethanolic extract and water extract of Triumfetta rhomboidea Jacq shows good antibacterial activity ^[2][3], Antioxidant activity ^[4]. Powdered leaf of the plant used to treat anemia. The methanolic extract of Triumfetta rhomboidea leaves shows Antimicrobial activity against Staphylococcus aureus, Salmonella typhi and Klebsiella pneumonia ^[7]. It shows the analgesic activity and Anti-inflammatory effect. It also shows Antioxidant and Antitumor activity ^[4]-[6].

 

MATERIALS AND METHODS:

Plant material :

The leaves of Triumfetta rhomboidea (Family: Tiliaceae) were collected in the month of Dec 2013 from the outskirt of  college, Karjat, Maharashtra, India. The plant material was taxonomically identified by Dr. R.V. Bhagat, Anatrao Pawar College, Pirangut, Pune, India and the Authentication No. was APCP/27/2013-14.

 

Preparation of extract:

The dried powdered  leaves were extracted by  70% Methanol in a Soxhlet extraction apparatus then after defatted with petroleum ether. The solvent was removed under reduced pressure and semi-solid mass was obtained and vacuum dried to yield a solid residue. The extract showed positive test for steroids, saponins, tannins and flavonoids. The extracts were dissolved in water  and  Diclofenac sodium  was used as standard.

 

Drug and chemicals:

Diclofenac sodium, Double distilled water. All other chemicals were of analytical grade obtained   commercially. Double distilled water was used throughout the study.

 

Evaluation of anti-inflammatory effect by membrane stabilizing property ^[8,12,13,16]:

Alsver solution was prepared by dissolving 2% dextrose, 08% sodium citrate, 0.05% citric acid and 0.042% sodium chloride in distilled water, which was later sterilized. Blood was collected from median cubital vein of the first author .the collected blood was mixed with equal volume of sterilized  alsever solution. the blood was centrifuged at 1000-2000 rpm and packed cells were washed with isosaline . And a suspension in 10% (v/v) isosaline was made. Various concentrations of the Triumfetta rhomboidea extracts was prepared  in a mixture of 1ml phosphate buffer, 2ml  hypo saline and 0.5 ml HRBC suspension. Diclofenac sodium was used as the reference drug. Instead of  hypo saline 2ml distilled water was used  in control. The assay mixtures was incubated at 37^oc for 30 minutes and centrifuged. The hemoglobin content in the supernatant solution was estimated using UV spectrometer at 560nm. The percentage haemolysis was calculated by the assuming the haemolysis  produced in presence of distilled water as 100%.   The Percentage of HRBC membrane stabilization or protection was calculated using this equation

 

Percentage inhibition of haemolysis =100 X [OD₁-OD₂/OD₁]

 

Where, OD₁=optical density of hypotonic buffer saline solution alone and OD₂=optical density of test sample in hypotonic medium. 

 

Evaluation of in-vitro anti-inflammatory activity by protein denaturation method ^[8,12-15] :

The10 ml reaction mixture consists of 0.4ml of egg  albumin which is obtained from the fresh hen’s egg, 5.6 ml of phosphate buffer saline (PBS, pH 6.4)and 4 ml of varying concentration of Triumfetta rhomboidea extract which makes the final concentrations 25, 50, 100, 200, and 400 µg/ml. Similar quantity of double distilled water is used as control. Then the mixture is incubated at 37^oc ± 2 in incubator for 15 min. After this mixture is heated at70^oc for 5 min. After cooling of the mixture absorbance was measured at 660 nm. Vehicle of the mixture is used as blank. Viscosity was measured with the help of  Ostwald viscometer. Diclofenac sodium at the final concentration of (25, 50, 100 and 200 µg/ml) was used as a reference drug and treated similarly for the determination of absorbance and viscosity. For the determination of  percent inhibition of protein denaturation following formula was used,

 

% Inhibition = 100 x (Vt / Vc - 1)

Where, Vt = absorbance of test sample

               Vc = absorbance of control

 

The extract concentration for 50% inhibition (IC50) was determined by plotting graph of percentage inhibition with respect to control against treatment concentration.

 

RESULTS:

Membrane stabilizing activity:

Since HRBC membranes are similar to liposomal membrane components, the prevention of hypo tonicity induce HRBC membrane lysis was taken as a measure of ant- inflammatory activity of drugs. The result obtains demonstrated that  TR can significantly and dose dependently inhibit RBC hemolysis (Table no.1).In this study TR extract at conc. range of  25 to 400 microgram/ml protect significantly  the erythrocyte membrane against lysis induced hypotonic solution. Also diclofenac sodium offered significant protection of  RBC’s against the damaging effect. At concentration of 400µg/ml, the TR extract produce 34.7% inhibition of RBC hemolysis as compare with 68.66% produced by diclofenac sodium.

 

 

 

Table 1 : Effect of Triumfetta rhomboidea on hypotonic solution induced heamolysis of erythrocyte membrane

Values are Mean ±SD;N=3 in each group *P<0.05;when compare to control (one way ANOVA followed by Dunnett multiple comparison test)

 

 

Inhibition of protein denaturation:

Different concentration of  Methanolic extract of  TR plant shows inhibitory effect on protein denaturation. These results are shown  in table no:2. Cocentration between 25 – 400 µg/ml TR shows significant effect on protein denaturation by protecting membrane against lysis. Methanolic extract of plant Triumfetta rhomboidea shows 208% of inhibitory activity where as Diclofenac sodium shows 259% protein denaturating effect.It is again established by comparing their IC 50 values. The TR plant shows IC 50 values 52.5 µg/ml where as that of Diclofenac sodium was found to be 27.5 µg/ml.

 

Table 2 : Effect of Triumfetta rhomboidea on protein denaturation

 

 

DISCUSSION:

There are certain difficulties in using animals in experimental pharmacological research, such as ethical issues and the lack of rationale for their use when other suitable methods are available or could be investigated. Hence, in the present study Membrane stabilizing activity and the protein denaturation bioassay was selected for in vitro assessment of anti-inflammatory property of  methanolic extract of TR.

 

Membrane stabilizing activity of Triumfetta rhomboidea:

Anti-inflammatory drugs are generally used to treat nociceptive pain^[9]. The extract of plant Triumfetta rhomboidea shows good effect in prevention of Polymorphonuclear leucocytes which are the  granulocytes , category of white blood cells which contains granules in their cytoplasm. It also shows effect on platelet function. Vital power of the cell is depends upon the integrity of membrane,. When RBC exposes to the injurious of harmfull substances it causes lysis of cell membrane with by the oxidation of haemoglobin and haemolysis. This haemolytic effect is due to the excessive collection of fluid in cells which leads to rupturing of membrane. The ruptured of membrane results in the cells becomes more sensitive to the secondary injury which may cause cellular damage. Extract may have membrane stabilizing property which interfere with the release of  inflammatory mediators phase and release of phospholipase which act as trigger for formation of inflammatory mediators.

 

Inhibition of Protein Denaturation:

Denaturation of tissue proteins is one of the well-documented causes of inflammatory ^[13][14] and arthritic diseases^[14] construction of auto antigens in certain arthritic diseases may be due to denaturation of proteins in vivo^[8]. Agents that can prevent protein denaturation therefore, likely worthwhile for development of anti-inflammatory drug . The increase in  absorbances of  test samples relating to control indicated stabilization of protein i.e. inhibition of heat-induced protein (albumin) denaturation by  methanolic extract of TR and reference drug diclofenac sodium^. This anti-denaturation effect was further sustained by the change in viscosities. It has been reported that the viscosities of protein solutions increase on denaturation ^[6]. In the present study, the relatively high viscosity of control dispersion validated this fact. Presence of methanolic extract of TR prevented this, implying inhibition of protein denaturation. Here, the viscosities decreased when compared with control. Although, the viscosities of the test samples (extract/drug), of all concentrations were always less than that of control. ^[15]

 

Phytoconstituent and anti-inflammatory activity:

The preliminary phytochemical investigation revealed Triumfetta rhomboidea leaves consists of alkaloids, saponin, tannins, gums, phenolic compounds and flavonoids have been reported to be associated with antioxidative action in biological systems, acting as scavengers of singlet oxygen and free radicals.^[6] such candidate is best suitable for studying anti-inflammatory activity.

 

CONCLUSION:

From the above result and it is observed that the Methanolic extract of Triumfetta rhomboidea plant shows anti-inflammatory activity by the inhibition of protein denaturation and  membrane stabilizing activity. This activity might be due to presence of flavonoids, phenolic compounds in the extract. It is necessary that full structural detection of active components and their toxicological properties has to be explored further.

 

ACKNOWLEDGEMENT:

The authors would be thankful to management Konkan Gyanpeeth College of Pharmacy and Research Institute, Karjat for providing facilities to carry out research activity and Dr. R.V. Bhagat, Anatrao Pawar College, Pirangut, Pune for the identification of plant material.

 

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Received on 01.01.2016          Modified on 23.01.2016

Accepted on 14.02.2016        © RJPT All right reserved