|Year : 2015 | Volume
| Issue : 2 | Page : 156-159
Effect of fruits of Opuntia elatior Mill on mast cell degranulation
Sanjay P Chauhan1, NR Sheth2, BN Suhagia1
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Dharmsinh Desai University, Nadiad, Gujarat, India
2 Department of Pharmaceutical Sciences, Saurashtra University, Rajkot, Gujarat, India
|Date of Submission||17-Apr-2014|
|Date of Decision||10-Aug-2014|
|Date of Acceptance||26-Aug-2014|
|Date of Web Publication||1-Apr-2015|
Dr. Sanjay P Chauhan
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Dharmsinh Desai University, Nadiad, Gujarat
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: The presence of potentially active nutrients and their multifunctional properties make prickly pear a perfect candidate for the production of phytopharmaceutical products. Among the numerous Opuntia species, bioactive compounds have been isolated and characterized primarily from Opuntia ficus-indica, Opuntia polycantha, Opuntia stricta, Opuntia dilleni for various medicinal properties. Objective: Based on the traditional use of prickly pear for enhancement of immune function, the objective of the present study to evaluate the effect of prickly pear on mast cell degranulation function. Materials and Methods: The Opuntia fruit juice (OFJ) (10-200 μl/ml) were studied for the effect on sensitized rat peritoneal mast cell degranulation induced by immunological (egg albumin), and nonimmunological (compound 48/80) stimuli and compared with that of the reference standard, sodium cromoglycate and ketotifen (10 μg/ml). Results and Conclusion: The OFJ exhibited significantly (P < 0.001) concentration dependent inhibition of mast cell degranulation. The IC 50 value of OFJ was found 12.24 and 18 μl/ml for immunological and nonimmunological induced mast cell degranulation, respectively. The betacyanin is an active principle compound in prickly pear that may responsible for mast cell stabilizing action.
Keywords: Immunomodulatory, mast cell, Opuntia, prickly pear
|How to cite this article:|
Chauhan SP, Sheth N R, Suhagia B N. Effect of fruits of Opuntia elatior Mill on mast cell degranulation. J Pharm Bioall Sci 2015;7:156-9
The cactus Opuntia (subfamily: Opuntiodae, family: Cactaceae) is a xerophytic plant producing about 200-300 species. In local parlance cactus is called prickly pear, slipper thorn, tuna (English) and has different vernacular names in India like Hathlo Thor, Chorhthlo(Gujarati), Haththathoira, Nagphana, Nagphani (Hindi), Snuhi, Vajrakantaka, Bahushala (Sanskrit), Nagadali, Nagakkali (Tamil), Nagamulla, Nagajemudu (Telugu). It was found that cacti in India did not all belong to one species as an Opuntia dillenii, but 3-4 species distributed over different regions in India. O. dillenii Haw. is found mainly in the southern parts of the India, while Opuntia vulgaris Mill (Syn Opuntia monacantha Haw.) is distributed mainly in the northern parts; Opuntia elatior Mill. is found in western. , The presence of potentially active nutrients and their multifunctional properties make Opuntia spp. fruits and cladodes perfect candidates for the production of phytopharmaceutical products. Although traditionally appreciated for its pharmacological properties by the Native Americans, cactus pear is still hardly recognized because of insufficient scientific information.  The Opuntia species were used as analgesic and antiinflammatory, anticancer, antidiabetic, antihyperlipidemic and - hypercholesterolemic, antioxidant, antiulcer, antiviral, diuretics, immunomodulatory, improve platelet function, neuroprotective, wound healing, monoamino-oxidase inhibitor, and nutritional important. ,
Mast cells are one of the most mysterious and multifunctional cells of the immune system. They play a major role in the expansion of many physiologic changes during allergic responses. Mast cell activation both by IgE-dependent and IgE-independent stimuli, bring about the process of degranulation that results in the fusion of the cytoplasmic granule membranes with the plasma membrane. This is accompanied by the fast external release of granule-associated stored mediators such as histamine, neutral proteases, acid hydrolyses, proteoglycans, chemotactic factors, and cytokines.  Among inflammatory substances released on degranulation of mast cells, histamine is the best-characterized and most potent vasoactive mediator concerned in the acute phase of urgent hypersensitivity. 
While little is known regarding the medicinal properties of betalains and other phytoconstituents from Opuntia, and essentially nothing is known regarding their potential immunomodulatory properties. Among the numerous Opuntia species, bioactive compounds have been isolated and characterized primarily from Opuntia ficus-indica, Opuntia polycantha, Opuntia stricta, O. dilleni for various medicinal properties.  Although mast cells play an important role in allergy and asthma, the potential immunomodulatory effects of betalains from Opuntia were not studied and reported. Based on previous studies and traditional use showing that prickly pear enhances immune function, we suggest that at least part of the effects of fruits of O. elatior Mill. on mast cell is though inhibition of the degradation function. In the present study, we examined the effects of the fruits of O. elatior Mill. on mast cell-mediated anaphylaxis.
| Materials and Methods|| |
Collection, authentication and preparation of fruit juice (Opuntia fruit juice)
The fruits of O. elatior Mill. were collected from roadside weed near Atkot, Ta: Jasdan, Dist: Rajkot, Gujarat, India at Latitude (22° 1′ 48″ N), Longitude (71° 12′ 0″ E) and Elevation 193 M (633 ft) and authenticated by Raw Materials Herbarium and Museum, National Institute of Science and Communication and Information Resources, New Delhi. The herbarium (specimen voucher no.: Rbpmpc/museum/herbarium/07-08/01) was preserved in the museum of Department of Pharmacognosy, Smt. R. B. Patel Mahila Pharmacy College, Atkot. Mature fruits of O. elatior Mill. were collected and immediately taken to the laboratory. Spines and glochides were removed from fruits by just heating on the wire gauge burner and then washed with water. The peel of the fruits was removed manually, and pulp subjected to homogenization for 5 min using boss portable blender (Boss Appliances, Daman). After homogenization, fruits juice was filtered though glass filter G 4 (Borosil Glass Works Ltd., Mumbai) and filtered Opuntia fruit juice (OFJ) was used for mast cell degranulation test.
Identification of betalains by spectrophotometric, high-performance liquid chromatography (LC) and LC-mass spectroscopy analysis were performed and recently published by Chauhan et al. 
Mast cell degranulation test
A modification of the method described by Jippo-Kanemoto et al.  was used for immunological and nonimmunological mast cell degranulation. Albino rats were sensitized by administering egg albumin (1 ml, 10% w/v) intraperitoneally as well as subcutaneously on the 1 st , 3 rd , 5 th and on the 12 th day of the first egg albumin administration and sacrificed by spinal dislocation under light ether anesthesia. In compound 48/80-induced mast cell degranulation, rats were also scarified by spinal dislocation under light ether anesthesia. In both tests, the peritoneal cavity was lavaged with 10 ml of tyrode solution. The lavaged fluid was collected and centrifuged at 2000 g for 5 min. The pellet was separated, washed with tyrode solution and finally resuspended in 1 ml tyrode solution. A volume of 0.1 ml of this lavage fluid was transferred to 10 test tubes. The lavage fluid was then subjected to following treatment schedule.
- Test tube no. 1 : Negative control
- Test tube no. 2 : Positive control
- Test tube no. 3 : 0.1 ml (10 μg/ml) of sodium cromoglycate
- Test tube no. 4 : 0.1 ml (10 μg/ml) of ketotifen
- Test tube no. 5-11 : 0.1 ml (10, 20, 40, 60, 80, 100, and 200 μl/ml) of OFJ.
Each test tubes were incubated for 10 min at 37°C and then egg albumin (0.1 ml, 1 mg/ml) and compound 48/80 (0.1 ml, 10 μg/ml) was added to each test tube except test tube no. 1. After further incubation for 10 min at 37°C, toluidine blue (0.1 ml, 10% w/v) was added and examined under light microscope with ×450 magnification. A minimum of 100 cells was counted for intact and disrupted mast cells and from it percentage protection from degranulation was calculated.
All the values are expressed as mean ± standard error of the mean. The data were analyzed by one-way ANOVA, followed by Turkey's multiple comparison tests. A level of P < 0.05 was considered to be statistically significant. The level of significance was noted and interpreted accordingly.
| Results|| |
The phytochemical analysis indicated the presence of color pigment betacyanin as an active principle and a good source of sugar content and low acidity of the fruit that make it very sweet and delicious. The total betacyanin content (47.1 mg/100 ml) equivalent to betanin obtained from fruits of O. elatior Mill. was higher when compared to O. ficus-indica and Opuntia undulate Griff. while lower when compared to O. stricta Haw. 
The OFJ (10-200 μl/ml) were studied for the effect on sensitized rat peritoneal mast cell degranulation induced by immunological (egg albumin) and nonimmunological (compound 48/80) stimuli and compared with that of the reference compound (sodium cromoglycate and ketotifen, 10 μg/ml). Egg albumin and compound 48/80 incubated mast cell demonstrated 93.17% and 92.63% of degranulation, respectively. Sodium cromoglycate and ketotifen as a reference standard produced an inhibition of 75.33% and 80.33% in egg albumin and 79.33% and 84.33% in compound 48/80-induced degranulation, respectively. The OFJ exhibited significant (P < 0.001) concentration - dependent inhibition of mast cell degranulation with respect to positive control group [Figure 1] and [Figure 2]. Further, we observed better inhibition with OFJ (200 μl/ml) than sodium cromoglycate in egg albumin induced mast cell degranulation. The IC 50 of fruit juice was 12.24 μl/ml and 18 μl/ml for egg albumin and compound 48/80 induced mast cell degranulation, respectively. [Figure 3] represented the photographs of intact and degranulated mast cells.
|Figure 1: Effect of Opuntia fruit juice on egg albumin induced rat mast cell degranulation. Values are mean ± standard error of mean (n = 6); analyzed by one-way ANOVA followed by Turkey's multiple comparison test, $P < 0.001 for change difference versus positive control group|
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|Figure 2: Effect of Opuntia fruit juice on compound 48/80 induced rat mast cell degranulation. Values are mean ± standard error of mean (n = 6); analyzed by one-way ANOVA followed by Turkey's multiple comparison test, $P < 0.001 for change difference versus positive control group|
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| Discussion|| |
Mast cell degranulation is important in the initiation of immediate responses following exposure to allergens. Degranulated cells liberate mediators of inflammation such as histamine, leukotrienes (LTs), platelet activating factors and chemotactic factors for eosinophils, neutrophils etc., from mast cells. The unique mediator profile of mast cells, elicited upon activation through their high-affinity receptors for IgE, include preformed granule-associated inflammatory mediators (histamine, neutral proteases, preformed cytokines, and proteoglycans) that are released by exocytosis. Finally, activated mast cells synthesize and secrete a host of proinflammatory, chemoattractive, and immunomodulatory cytokines over a period of several hours. ,, The bioactivities of these mediators include brochoconstriction (cysteinyl [cys]-LTs, histamine, PGD 2 ), vasodilation and tissue edema (histamine, cys-LTs), leukocyte infiltration (cys-LTs, PGD 2 , tryptases, cytokines and chemokines), collagen matrix turnover and stromal cell growth (tryptases, cytokines), and hyperplasia of bronchial smooth muscle (tryptases, cys-LTs). These properties of mast cells and their normal residence in bronchi would seem to position them for a potentially relevant role in the pathophysiology of asthma.  Degranulation of mast cells has been taken as the criteria of positive anaphylaxis. Ketotifen fumarate, a well-known mast cell stabilizer, reduces the synthesis of prostaglandins E 2 , thromboxane A 2 , leukotriene C 4 and B 4 . It also inhibits release of histamine, serotonin and other inflammatory mediators from mast cells. Simultaneously it blocks H 1 receptors. Cromolyn sodium, which is developed from the structural modification of Khellin is the mast cell stabilizer used in the treatment of mild to moderate asthma by raising cAMP levels due to inhibition of the enzyme phosphodiesterase. 
In the present study, the fruit juice of O. elatior Mill. was found to inhibit the degranulation of mast cells induced by an immunological and a nonimmunological stimulus. It is known that the physiological stimulus for the release of histamine from mast cells is provided by a combination of antigen with specific antibody fixed on the cell surface. This combination is believed to transiently increase the permeability of the membrane to calcium ions showing an absolute requirement for calcium ions for the secretory process to occur.  Anaphylactic and compound 48/80 induced secretion from mast cells share a common requirement as far as the presence of calcium ions is concerned. However, compound 48/80 can utilize intracellular calcium stores to initiate the release process, even in the absence of calcium in the extracellular medium.  On the other hand, anaphylactic release requires the presence of calcium in the extracellular medium, which moves onto the cell via calcium gates in the membranes. , A significant protection of rat peritoneal mast cells from disruption by antigen and compound 48/80 by fruit juice of O. elatior Mill. points toward its ability to interfere the release and/or synthesis of mediators of inflammation, indicating its mast cell stabilizing activity. Hence, it may be assumed that the cytoprotective effect induced by fruit juice of O. elatior Mill. on mast cell surface could be due to its ability to alter the influx of calcium ions. The betacyanin is an active principle compound in prickly pear that may responsible for mast cell stabilizing action.
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[Figure 1], [Figure 2], [Figure 3]