| Abstract|| |
To evaluate anti-asthmatic, anti-inflammatory, and anti-allergic activity of various subfractions of ethyl acetate fraction of leaves of Vitex negundo. Ethyl acetate fraction (EAF) of leaves of V. negundo was further subfractionated into NaOH fraction, acetone fraction, and chloroform fraction to separate chemical constituents present in EAF using thin layer chromatography technique and different solvent systems. The stabilisation potential of the various subfractions of EAF of leaves of V. negundo against mast cell degranulation was studied on isolated mesentery of rat by exposure to compound 48/80. Dexamethasone (5 mg/kg) was used as a reference standard. AF of EAF of leaves of V. negundo (500 μg/ml) showed significant protection of rat mesenteric mast cells from disruption caused by compound 48/80. This study concluded that AF of EAF of leaves of V. negundo shows mast cell stabilising activity. It may be useful as anti-asthmatic, anti-inflammatory, and anti-allergic agent.
Keywords: Asthma, compound 48/80, mast cell, Vitex negundo
|How to cite this article:|
Patel JI, Deshpande SS. Mast cell stabilising activity of various subfractions of leaves of Vitex Negundo. Int J Green Pharm 2011;5:273-5
| Introduction|| |
The mechanism of the inflammatory response resulting in asthma is complex and involves numerous cell types, including mast cells. The mast cell has long been associated with asthma, because it releases a variety of preformed (histamine, leukotrienes, prostaglandins, and platelet activating factor) and newly synthesised mediators [eosinophils and neutrophils chemotactic factors, tumor necrosis factor-a, and cytokines (IL-4, IL-8, and IL-13)] that could account for several features of asthma-like bronchoconstriction and airway inflammation, respectively. Among the mediators released from mast cells, histamine is a well-characterised and the most potent vasoactive mediator in acute bronchoconstriction.  In this study, compound 48/80 is used as a mast cell degranulating agent. It was described as a histamine liberator. 
Vitex negundo is a species reported under the genus Vitex. Family: Verbenaceae. It is distributed throughout India, Ceylon-Afghanistan, tropical Africa, Madagascar, China, and Philippines.  Ethyl acetate fraction (EAF) of leaves of V. negundo shows anti-asthmatic activity.  Ethanolic extract of V. negundo leaves showed inhibitory effect of degranulation of rat peritoneal mast cells induced by compound 48/80 and egg albumin.  Aqueous extract of mature leaves of V. negundo shows anti-inflammatory analgesic and anti-histaminic activity.  In this study, the various subfractions of EAF of leaves of V. negundo were screened for mast cell degranulation properties.
| Materials and Methods|| |
The leaves of plant V. negundo were collected from Agriculture Research Centre, Jagudan, Mehsana, North Gujarat, India. Its botanical identification was confirmed by the department of Pharmacognosy, KBIPER.
Preparation of Various Subfractions
- Leaves of plant were dried under shade for a week. Then it is powdered and successively fractionated with petroleum ether, ethyl acetate, and distilled water as solvent. Among these, EAF has anti-asthmatic activity. 
- EAF was further subfractionated into NaOH fraction (AQF), acetone fraction (AF), and chloroform fraction (CF) to separate chemical constituents present in EAF using thin layer chromatography technique and different solvent systems.
Protocol for this project was approved by IAEC, K.B. Institute of Pharmaceutical Education and Research, Gandhinagar. Protocol approval no. is KBIPER 08/102. Albino rats of Wistar strain were used.
The method used in this study was described by Norton S. 
The pieces of mesentry were collected in following Petri dish More Detailses containing Ringer Locke solution (NaCl 0.9%, KCI 0.042%, CaCl 2 0.024%, NaHCO 3 0.015%, and dextrose 0.1%) in after the animals were killed.
- Petri dish no. 1 Ringer Locke solution (vehicle control)
- Petri dish no. 2 Ringer Locke solution (positive control)
- Petri dish no. 3 Dexamethasone (10 μg/ml)
- Petri dish no. 4 AQF of EAF of V. negundo leaf (500 μg/ml)
- Petri dish no. 5 AF of EAF of V. negundo leaf (500 μg/ml)
- Petri dish no. 6 CF of EAF of V. negundo leaf (500 μg/ml)
Each Petri dish was incubated for 10 min at 37°C.
Later 0.2 ml of compound 48/80 having concentration 10 μg/ ml was added and again incubated for 10 min at 37°C. After that all pieces were transferred to 4% formaldehyde solution containing 0.1% touidine blue and kept a side for 20-25 min. After staining and fixation of mast cells, mesentry pieces were transferred through acetone and xylene two times and mounted on slides.
All the pieces were examined under light microscope with ×450 magnification. A minimum of 100 cells were counted for intact and disrupted mast cells and from that % protection from degranulation was calculated.
Results were analyzed statistically using one-way analysis of variance followed by Turkey's test. Data were considered statistically significant at P<0.001.
| Results|| |
Compound 48/80 produced significant disruption of mast cells. AQF of EAF of V. negundo leaves produced lesser degranulation of mast cell [Figure 1]. The effect was comparable to dexamethasone-induced protection.
|Figure 1: Effect of various subfractions of Vitex negundo leaves on mast cell degranulation induced by compound 48/80. *Significantly different from control at P<0.001. Each bar in the graph represents mean±SEM of six observations|
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AQF of EAF of V. negundo leaves (500 μg/ml) showed significant protection (80.99±0.7231; P<0.001) when compared with positive control group (50.76±2.1597; P<0.001). Dexamethasone produced significant protection (83.50±0.4355; P<0.001).
| Discussion|| |
It is now well known that mast cells are extensively involved in the pathophysiology of bronchial asthma.  To evaluate the effectiveness of drug on mast cell stability, rat mesentric mast cells have long been considered useful tool. In this study, this model is used to evaluate the effectiveness of the test drug on mast cell stability.
When a piece of mesentery is placed in a solution of 48/80, the cell swells and becomes balloon-like, sometimes twisting and turning as it swells, possibly in response to the mechanical forces of the surrounding tissue.
Because living cells swell before they fragment, and because stained intact cells appear swollen, it was assumed that at least one of the effects of 48/80 was to increase the osmotic pressure within the mast cell and hence cause an inflow of water.
This increase in osmotic pressure might come about in several ways:
- The mast cells might preferentially concentrate 48/80
- A substance that was previously not available for osmotic effect might be liberated or formed in the cell
- The cell might take up or become more permeable to ions present in the extracellular fluid.
But the evidence is in favour of the possibility that 48/80 may make the cell more permeable to extracellular ions and thereby cause disruption of the cell. 
Here in our study a significant protection of rat mesentric mast cells from disruption caused by compound 48/80 was observed with AQF of EAF of V. negundo leaves. Therefore, AQF of EAF of V. negundo leaves may make the mast cell very less permeable to extracellular ions as compared with control group and give protection to mast cell degranulation.
| Conclusion|| |
From the results of this study, it can be concluded that AQF of EAF of V. negundo leaves shows mast cell stabilising activity. It may be useful as anti-asthmatic, anti-inflammatory, and anti-allergic agent.
| Acknowledgment|| |
The authors are thankful to K.B. Institute of Pharmaceutical Education and Research, Gandhinagar, Gujarat, for providing facility for this research work.
| References|| |
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Jignesh I Patel
Department of Pharmacology, B.K. Mody Government Pharmacy College, Polytechnic Campus, Near Aji-dam Chokdi, Rajkot - 360 003, Gujarat