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ORIGINAL ARTICLE
Year : 2013  |  Volume : 5  |  Issue : 4  |  Page : 306-308  

Mast cell stabilization potential of Sitopaladi churna: An ayurvedic formulation


Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences, Manipal University, Karnataka, India

Date of Submission25-Jun-2012
Date of Decision25-Apr-2013
Date of Web Publication24-Sep-2013

Correspondence Address:
H.N. Aswatha Ram
Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences, Manipal University, Karnataka
India
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Source of Support: Department of pharmacognosy, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, Conflict of Interest: The author(s) declare that they have no competing interests


DOI: 10.4103/0974-8490.118824

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   Abstract 

Background: Sitopaladi churna (SPC) is a popular polyherbal ayurvedic formulation used in the treatment of allergy and respiratory diseases. Objective: The present study was aimed to justify the classical use of antiallergic claim by performing the mast cell stabilizing activity of extracts of SPC. Materials and Methods: The protective effect of aqueous extract and methanolic extract - of SPC against compound 48/80-induced mast cell degranulation model was carried out. Results: Sitopaladi churna aqueous extract (SPCA) at the dose of 300 mg/kg and Sitopaladi churna methanolic extract (SPCM) at the doses of 150 and 300 mg/kg showed better protection of mast cell degranulation (65%-74%) and were comparable to the standard drug ketotifen (79%), when peritoneal mast cells were treated with compound 48/80. The protection against mast cell degranulation was significant (P < 0.0001). Conclusion: From the above results, it has been justified that SPC can be used to treat allergic disorders.

Keywords: Compound 48/80, mast cell stabilizing, Sitopaladi churna


How to cite this article:
Makhija IK, Shreedhara CS, Ram HA. Mast cell stabilization potential of Sitopaladi churna: An ayurvedic formulation. Phcog Res 2013;5:306-8

How to cite this URL:
Makhija IK, Shreedhara CS, Ram HA. Mast cell stabilization potential of Sitopaladi churna: An ayurvedic formulation. Phcog Res [serial online] 2013 [cited 2020 Oct 26];5:306-8. Available from: http://www.phcogres.com/text.asp?2013/5/4/306/118824


   Introduction Top


Mast cells are the constituents of virtually all organs and tissues and are important mediators of inflammatory responses such as allergy and anaphylaxis. [1] Sitopaladi churna (SPC) is a polyherbal ayurvedic medicine traditionally used as a supportive agent for allergy and in the treatment of cold, cough associated with bronchitis, pneumonia, and tuberculosis. [2] With this background, the study was carried out to justify the traditional claims by investigating its use in the treatment of allergy using a validated model.


   Materials and Methods Top


Chemicals

Compound 48/80, ketotifen fumarate and Roswell Park Memorial Institute (RPMI) 1640 medium was purchased from Sigma Aldrich, Bangalore, India. Toluidine blue was purchased from Sisco Research Laboratory, Mumbai, India. All other chemicals used were of analytical grade.

Plant material

SPC consists of five ingredients viz., Saccharum officinarum (sugar candy), Vamshalochana (siliceous concretion), Piper longum (dried fruit), Elettaria cardamomum (dried seed), and Cinnamonum zeylanicumm (stem bark). All these ingredients were procured from the local market of Udupi, Karnataka, India, and were authenticated by botanist Dr. Gopal Krishna Bhat, Professor, Department of Botany, Poorna Prajna College, Udupi, Karnataka, India. Voucher specimen of the same was deposited in the museum of Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences, for future reference.

Preparation of SPC

The churna was prepared according to the procedure given in Ayurvedic Formulary of India. All the ingredients were powdered separately, passed through 80 # sieve and then mixed together in specified proportions to get uniformity blended churna.

Preparation of SPC extracts

SPCA was prepared by maceration of 100 g of the SPC in chloroform water for 7 days with intermittent shaking. The resulting extract was concentrated and lyophilized to obtain a brownish residue (yield 35.98% w/w). The SPCM was prepared by extracting SPC (100 g) exhaustively with methanol using a soxhlet apparatus for 48 h. The methanolic extract was concentrated under reduced pressure at 40°C using a rotary evaporator and lyophilized at −40°C to obtain a reddish-brown syrupy residue (yield 28.34% w/w). Both the residues were stored in a dessicator until its use.

Animals

Healthy adult Wistar albino rats weighing about 200-250 g were used for the study. The animals were housed in polypropylene cages, maintained under the standard conditions. (12 h light: 12 h dark cycle; 25 ± 3°C; 35%-60% humidity). They were fed with standard rat pellet diet (Hindustan Lever Ltd., Mumbai, India) and water ad libitum. The Institutional Animal Ethical Committee of Kasturba Medical College, Manipal, India (IAEC/KMC/15/2010), approved the study.

Acute toxicity studies

Graded doses ( 50, 300, and 2000 mg/kg) of SPC were tested for oral acute toxicity studies according to Organization for Economic Co-operation and Development (OECD) 420 guidelines. In brief, after drug administration the animals were evaluated every 10 min for 4 h followed by 24, 48, and 72 h for any changes in spontaneous motor activity, gait, respiration, writhing, piloerection, and so on. Animals were followed up to 30 days for mortality. [3] Doses selected for further studies were between 1/60 th , 1/20 th , and 1/12 th of the maximum tolerated dose.

Compound 48/80-induced allergy test

Compound 48/80-induced allergy test [4],[5] was carried out using healthy adult albino rats. The rats were sensitized by subcutaneous injection of compound 48/80, 1 mg/kg body weight. The sensitized animals were divided into eight groups (n = 6). The first control group received 2% gum acacia solution (2 mL/kg, p.o.). The second group received standard drug ketotifen fumarate (1 mg/kg, p.o.). The other groups received different doses (50, 150, and 300 mg/kg) of SPCA and SPCM, respectively for 14 days. On the 14 th day, 2 h after the assigned treatment, 10 mL of normal saline was injected into the peritoneal cavity of rats, after a gentle massage, the peritoneal fluid was collected and transferred into the siliconized test tubes containing 7-10 mL of RPMI 1640 medium (pH: 7.2-7.4). Purification of the peritoneal mast cells was done as explained by Percoll. [6] Crude peritoneal cell suspensions contained 3% mast cells, and the purity of the mast cells after gradient centrifugation was more than 90%. Purified mast cells (cell density of 2 × 10 6 /mL) incubated with compound 48/80 (5 μg/mL) at 37°C for 10 min. Cells were stained metachromatically with toluidine blue (0.1% w/v, pH 1.0) and quantified by using a neubauer hemocytometer under a Olympus BX 41 microscope (magnification ×400). The viability of the mast cells was determined by their ability to exclude trypan blue. The trypan blue exclusion test indicated a viability of greater than 95%. The percentage of intact cells (granulated) and disrupted (degranulated cells) in different treated groups were calculated.

Statistical analysis

All results are expressed as mean ± SD, n = 6. Results were analyzed by one-way analysis of variance followed by Bonferroni's multiple comparison test to compare between control and test groups.


   Results Top


Acute toxicity studies

SPC extracts did not produce any death till 72 h at 2000 mg/kg, p.o. The animals in all the groups survived up to 30 days without any apparent adverse symptoms.

Compound 48/80-induced mast cell degranulation

SPCA and SPCM in the lower doses have showed least activity with P < 0.01 and P < 0.001, respectively. Whereas, SPCA at the dose of 300 mg/kg and SPCM at the doses of 150 and 300 mg/kg showed better protection of mast cell degranulation (65%-74%) was comparable to the standard drug ketotifen (79%), when peritoneal mast cells are treated with compound 48/80. The protection against mast cell degranulation was significant (P < 0.0001) as shown in [Figure 1].
Figure 1: Effect of the SPC extracts on inhibition of mast cell degranulation in compound 48/80-induced degranulation in rats

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   Discussion Top


In type-I allergy, pathological mechanism involves the degranulation of mast cells, followed by the release of mediators such as histamine, leukotrienes, and prostaglandins from these cells. [7] The mast cells undergo degranulation in response to the immunological stimuli in which the antigen-antibody reaction on the cell surface predominates. Effect of SPC on mast cells degranulation was studied by administering the churna in rats is reported. [8] Antihistaminic and anti-inflammatory activity of SPC extract in rats is also reported. [9] In the present investigation, the extracts of SPC have been studied and are active in the type-I allergic condition because of their ability to inhibit the release of mediators from mast cells [Figure 2] and thereby preventing the harmful effects of the released mediators. The mast cell stabilizing activity can be attributed to a great extent due to the presence of piperine of the formulation, as this principle has been reported to possess this activity. [10] The piperine content was found to be 9.51 ± 0.421 by high performance liquid chromatography. The preliminary phytochemical tests showed the presence of flavone glycosides in the extracts of SPC. Flavonoids have been reported to possess antihistaminic and antiallergic activity. [11],[12],[13] These results fully justify the use of SPC to treat allergic disorders. However, further studies are required to find the actual mechanism of antiallergic effects of constituents of churna.
Figure 2: Microscopic photographs of rat peritoneal mast cells. Compound 48/80 (5 μg/mL), extensive degranulation processes (disrupted cells) and the presence of multiple granules extruded from the mast cells. Standard + 48/80, mast cell contains closely packed secretory granules (intact cells). Mast cells showed evident decrease of the degranulation in extracts treated in dose-dependent manner

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   References Top

1.Church MK, Levi-Schaffer F. The human mast cell. J Allergy Clin Immunol 1997;99:155-60.  Back to cited text no. 1
[PUBMED]    
2.Central Council for Research for Ayurveda and Siddha. Ayurvedic formulary of India. Part 1. 2 nd ed. India: Ministry of Health and Family Welfare, Government of India; 2003. p. 116.  Back to cited text no. 2
    
3.Prabhakar KR, Veerapur VP, Bansal P, Vipan KP, Reddy KM, Barik A, et al. Identification and evaluation of antioxidant, analgesic/anti-inflammatory activity of the most active ninhydrin - phenol adducts synthesized. Bioorg Med Chem 2006;14:7113-20.   Back to cited text no. 3
[PUBMED]    
4.Lee YM, Kim DK, Kim SH, Shin TY, Kim HM. Anti anaphylactic activity of Poncirus trifoliate fruit extract. J Ethnopharmacol 1990;54:77-84.  Back to cited text no. 4
    
5.Loeffler LJ, Lovenberg W, Sjoerdsma A. Effects of dibutyryl-1-3,5-cyclic adenosine monophosphate, phosphodiestarases inhibitors and prostaglandin E1 on compound 48/80 induced histamine release from rat peritoneal mast cells in vitro. Biochem Pharmacol 1971;20:2278-97.  Back to cited text no. 5
    
6.Yurt RW, Leid RW, Austen KR. Native heparin from rat peritoneal mast cells. J Biol Chem 1977;252:518-21.  Back to cited text no. 6
    
7.Bellanti JA. Mechanism of tissue injury produced by immunologic reactions. Immunology. In: Asian ed. Tokyo: W.B. Saunders Co; 1971. p. 184.  Back to cited text no. 7
    
8.Vadnere GP, Gaud RS, Singhai AK, Pathan E. Inhibition of immediate allergic reaction by sitopaladi churna: An experimental study. Pharmacologyonline 2011;2:514-21.   Back to cited text no. 8
    
9.Ahirwar B, Ahirwar D, Ram A. Antihistaminic effect of Sitopaladi churna extract. Res J Pharm Technol 2008;2:89-92.   Back to cited text no. 9
    
10.Kraithep S, Oungbho K, Tewtrakul S. Anti-allergic activity of Thai medicinal plants used in longevity formulation. Songklanakarin J Sci Technol 2008;5:621-25.  Back to cited text no. 10
    
11.Tripathi RM, Sen PC, Das PK. Studies on the mechanism of action of Albizia lebbeck, an Indian indigenous drug used in the treatment of atopic allergy. J Ethnopharmacol 1979;1:385-96.  Back to cited text no. 11
[PUBMED]    
12.Havsteen B. Flavonoids: A class of natural products of high pharmacological activity. Biochem Pharmacol 1983;32:1141-8.  Back to cited text no. 12
[PUBMED]    
13.Pathak D, Pathak K, Singla AK. Flavonoids as medicinal agents-recent advances. Fitoterapia 1991;62:371-89.  Back to cited text no. 13
    


    Figures

  [Figure 1], [Figure 2]



 

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