Antihistaminic Effect of Sitopaladi Churna Extract

 

Bharti Ahirwar1*, Dheeraj Ahirwar2 and Alpana Ram1

1SLT Institute of Pharmaceutical Sciences, Guru Ghasidas University, Bilaspur (C.G.)-495 009, India

2School of Pharmacy, Chouksey Engineering College, Bilaspur (C.G.)

*Corresponding Author E-mail: ah_bharti@yahoo.com

 

ABSTRACT

Anti-inflammatory and mast cell stabilizing activity of sitopaladi churna extract was studied on the egg albumin-induced edema, carrageenin-induced edema in hind paw, cotton pellet implantation, and degranulation of mast cells by compound 48/80. Sitopaladi churna extract exhibited good anti-inflammatory effects in rats, causing a dose-related inhibition of the increase in the paw circumference (acute inflammation) induced by subplantar injection of fresh egg albumin and carrageenin. It also significantly decreased the weight of cotton pellet (chronic inflammation). It also protected mast cell disruption induced by compound 48/80. These findings reveal that antihistaminic and anti-inflammatory activity of sitopaladi churna extract may be due to inhibition of release of inflammatory mediators and mast cell stabilizing potential. 

 

KEY WORDS                 Histamine, Carrageenin, Anti-inflammatory,

 


INTRODUCTION:

Popularity of Ayurvedic medicines in society, technological advances in manufacturing processes and encouragement from government has resulted in flooding of Indian markets with Ayurvedic agents. Sitopaladi churna is an Ayurvedic herbal remedy composed of a mixture of four plants ingredients including Vanshlochan (Bambusa arundinacea), Pippali, (Piper longum) Ela (Cardamum zeylanicum), Twak (Cinnamomum zeylanicum) and Mishri. Out of these ingredients of sitopaladi churna Piper longum has been shown to reduce the passive cutaneous anaphylaxis in rats and protect guinea pigs against antigen-induced bronchospasm. Piperine, the main bioactive component of Piper longum decreased the rate and amplitude of respiration and nonspecific blockage of acetylcholine, histamine and 5-HT induced spasm on isolated guinea pig and rabbit intestine1. Vanslochan is the essence of the white earthy concretion formed in hollow of bamboos, bamboos mamma can help to cure cough, fever, T.B. and thirst. Muniappan et al. (2003) reported anti-inflammatory and antiulcer activity of Bambusa arundinacea2. Cardamom used as appetizer, carminative, diaphoretic, digestive stimulant, expectorant, stomachic and in asthma3, 4

 

 

Singh et al. (1995) reported that cinnamon bark oil, a potent fungitoxicant against fungi causing respiratory tract mycoses. In this research Singh identified cinnamic aldehyde as the active fungitoxic constituent of cinnamon bark oil5. Nagai et al. (1982) investigated antiallergic reaction of aqueous extract of Cinnamon cassia and they found that aqueous extract has an anti-complement action and inhibits the complement dependent allergic reaction6.

Some of these findings have been supported by clinical trials. The application of sitopaladi churna has undoubtedly a folkloric background. This has enjoyed a wide reputation and demand scientific verification. Hence every marketed preparation must confirm to the test of safety and efficacy. Keeping this perspective in mind, an attempt has been made here to develop a general method for standardization of Ayurvedic formulation on the basis of pharmacological evaluation and to assess the effect of these plant ingredients together for any synergistic or antagonistic effect.

 

MATERIALS AND METHODS:

Sitopaladi churna was procured from the local market. The voucher specimen is preserved in departmental laboratory for further reference. Drugs used were histamine (Sigma chemicals) Salbutamol and Prednisolone (Glaxo India Ltd) toluidine blue (Loba chemie), Carrageenin and other chemicals from Hi-media laboratories limited, Mumbai.

 

Preparation of Extract:

To prepare aqueous extract 500 g of churna was mixed with 1000 ml distilled water in a flask and boiled for half hour. Following cooling at room temperature, the brew was filtered using Whatman No.1 filter paper. The filtrate was then concentrated in a vacuum rotary evaporator (yield 10.2 % w/w) and the extract stored at 4˚C until required. The extract was suspended in 1% carboxy methyl cellulose (CMC). Phytochemical screening gave positive tests for alkaloids and volatile oils.

Animals:

Inbred Wistar rats (170-190 g) and guinea pigs (375-550 g) of either sex housed in standard conditions (Temperature 22 ± 2º C, relative humidity 60 ± 5º C and 12 h light/dark cycle) were used. They were fed with standard pellet diet and water ad libitum. The Institutional Animal Ethics Committee approved the experimental protocol (Registration No. CPCSEA/06/2007).

 

Dose fixation:

Dose fixation was done by staircase method7 on mice (20-25 g). Sitopaladi churna extract was homogenized in 1% carboxy methyl cellulose in distilled water. The homogenate was administered orally by means of intragastric catherter. It was observed that sitopaladi churna extract was found to be lethal at the dose of 2000 mg/kg body weight. Hence one-tenth and one-twentieth of 2000 mg/kg, i.e. 100 mg/kg and 200mg/kg body weight of sitopaladi churna extract were fixed as different doses.

 

Anti-inflammatory study:

This test was performed as described by Akah and Nwambie, 1994 using fresh egg albumin induced hind paw edema as a model of acute inflammation. The extract was administered intraperitoneally (i.p.) in two doses 1 hour before inducing inflammation. Control rats received equivalent amounts of normal saline. Average edema percent inflammation and percent inhibition of edema were calculated8.

 

Acute inflammations (Carrageenin-induced hind paw edema):

Twenty-four rats were divided into four groups of six each. Group I received vehicle (0.2 ml saline) and served as control. Group II and III received churna extract at the dose of 100 and 200 mg/kg body weight respectively. The drugs were administered 1 h prior to experimentation. Group V was administered with standard drug, pheylbutazone at the dose of 100 mg/kg. All the animals were injected with 0.1 ml of 1% (w/v) carrageenin in the subcutaneous region of the hind paw. During the early first hour histamine release induced swelling in the rat paw. The swelling was measured by mercury displacement method at the end of the first hour. The reduction in the paw swelling

 

volume compared with control was taken as the determination of histamine activity9

 

TABLE 1. EFFECT OF SITOPALADI CHURNA EXTRACT ON EGG ALBUMIN-INDUCED EDEMA, n= 6

Treatment

                 Average edema in ml after (min)

30

60

90

120

Control (0.2ml) vehicle

9.18±1.10

13.14±1.16

15.21±2.19

11.12±1.17

SCE 100mg/kg

8.19±0.08

10.11±1.23

7.17±1.13*

6.11±1.61

SCE 200mg/kg

7.18±1.01

7.11±0.15

5.13±0.01*

6.31±0.15

* = p<0.05, Values are Mean ± SE, SCE = Sitopaladi churna extract

 

Chronic inflammations (Cotton pellet implantation):

Sterile cotton pellets (10 mg) were implanted subcutaneously (SC) in rats under light ether anesthesia. The animals were treated with two different doses of extract for seven days. On the eighth day the animals were sacrificed and the cotton pellets along with the granulomatous tissue were removed, dried at 50˚C for 12 h and weighed. The increase in weight over the initial weight was recorded9. A separate group of animals was treated with phenylbutazone (100mg/kg).

 

Effect on mast cell degranulation

Twenty four rats were divided in four groups of 6 animals each. Rat in group I received 5 ml/kg of saline (vehicle) and served as control. Group II and III rats were administered Sitopaladi churn extract at the dose of 100 and 200 mg/kg once a day for 7 days. Group IV received prednisolone a standard drug. On day 7, 2 hours after the respective assigned treatments, the rats were injected 10 ml of normal saline into peritoneal cavity. After gentle massage, the peritoneal fluid was collected and transferred into siliconized test tubes containing 7-10 ml RPMI-1640 medium pH7.2-7.4). Mast cells were then washed thrice by centrifuged at a low speed (400-500 rpm) by discarding the supernatant and taking the pellet of the mast cells into the medium. Mast cell suspension (1×106 cells/ml) was challenged with 0.5 µg/ml of compound 48/80 and stained with 10% toulidine blue to examine the percent protection against degranulation14.

 

Statistical analysis:

The results of various studies were expressed as Mean ± S.E. and analyzed statistically using Students “t” test. The minimum level of significance was fixed at p<0.05.

 

RESULTS:

Effect on egg albumin-induced edema:

In control rats, fresh egg albumin induced a progressive increase in paw circumference with maximum swelling occurring at 90 min. The sitopaladi churna extract (100 and 200mg/kg) had remarkable anti-inflammatory activity against acute inflammation suppressing the paw edema in a dose dependent manner. The reduction in edema became significant (p<0.05) 60 min after egg albumin administration.

 

In the sitopaladi churna extract treated animals, maximal edema occurred at 60 min. This was significantly (p<0.05) lower that the maximum swelling of the control rats at 90 min (Table 1).

 

TABLE 2. EFFECT OF SITOPALADI CHURNA EXTRACT ON CARRAGEENIN-INDUCED EDEMA, n = 6

Treatment

Average edema in ml after (min)

30

60

90

120

Control (0.2ml) vehicle

4.10±

0.11

7.11±

0.16

9.01±

1.19

8.21±

0.17

SCE 100mg/kg

4.90±

0.01

6.21±

0.13

5.78±

0.13*

7.19±

1.61

SCE 200mg/kg

4.78±

0.01

6.10±

0.19

5.11±

0.03*

6.01±

0.19

Phenylbutazone 100mg/kg

3.10±

0.01

2.90±

0.17*

2.79±

0.91*

4.01±

0.19

* = p<0.05, Values are Mean ± SE, SCE = Sitopaladi churna extract

 

 

Effect on carrageenin-induced edema:

Sitopaladi churna extract exhibited the effect on carrageenin-induced edema in a dose dependent manner. Treatment with extract at the dose of 100 and 200mg/kg dose showed significantly (p<0.05) reduced the paw volume after 60 and 90 min when compare with control. Maximum effect was observed at 90 min (Table 2).

 

 

TABLE 3. EFFECT OF SITOPALADI CHURNA EXTRACT ON COTTON PELLET GRANULOMA, n = 6

Treatment

Dose

Wt of cotton pellet (mg)

Control

0.2ml

51.6±1.4

SCE

100mg/kg

36.4±2.2*

SCE

200mg/kg

28.2±1.7*

Phenylbutazone

100mg/kg

24.6±1.2*

Values are Mean ± S.E., * = p< 0.05. SCE = Sitopaladi churna extract

 

Effect on cotton pellet:

A dose dependent reduction in the weight of cotton pellets was observed after sitopaladi churna extract treatment (Table 3). Treatment with extract at the dose of 100 and 200mg/kg dose showed significantly (p<0.05) reduced the weight of cotton pellet.

 

Effect of extract on compound 48/80-induced mast cell degranulation

Churna extract treatment showed significant mast cell stabilizing activity following exposure to compound 48/80. The effect was statistical significance (p<0.05) when compared to control (Table 4).

 

DISCUSSION:

Drugs that relax the bronchial tree, especially the β2- adrenoceptor stimulants are employed in the management of asthmatics15. Asthma is a chronic

 

 

inflammatory airway disease characterized by bronchial hyperresponsiveness to various stimuli and increased numbers of both inflammatory cells and inflammatory mediators16. On the other hand, it non-specifically abolished that effect of histamine and isoprenaline on the tissue. Although the blocked of histamine may be beneficial17. Our results showed that in control rats, fresh egg albumin induced a progressive increase in paw swelling. The sitopaladi extract (100 and 200 mg/kg) has remarkable anti-inflammatory activity against acute inflammation suppressing the paw edema in a dose-dependent manner. The reduction in edema became significant (p<0.05) 60 min after egg albumin administration. In sitopaladi extract treated animals, maximal edema occurred at 60 min. this was significant (p<0.05) lower than the maximum swelling of the control rats at 90 min. The anti-inflammatory effect of the extract is noteworthy. There is evidence that inflammation of the airway usually results in airway obstruction and asthma18. In another parameter of anti-inflammatory study extract showed a significant reduction in paw edema volume and decreased weight of cotton pellet granuloma, suggesting a potential anti-inflammatory effect in both acute and chronic inflammation. It is well known that, degranulation of mast cells will result in the release of several mediators like histamine, which are implicated in inflammation and allergy19. Some anti-inflammatory agents modify inflammatory responses by accelerating destruction or antagonizing the action of the mediators of inflammatory reactions (Histamine, 5-HT etc). Hence it may be possible that the anti-inflammatory effect of the extract could result from the blockade of mediators of inflammatory reactions thus relieving bronchial edema17.

 

TABLE 4. EFFECT OF SITOPALADI CHURNA EXTRACT ON MAST CELL DEGRANULATION,   n = 6

                                                        Mast cells %

Group                     Intact                     Disrupted

Control 0.2 ml         18.05±4.76              67.54±2.66

SCE 100mg/kg         36.22±1.64*            41.66±5.45*

SCE  200mg/kg        56.21±4.55*            33.71±4.41*

Prednisolone           60.18V3.81*           21.70±3.80*

10mg/kg 

Values are Mean ± SE, * = p<0.01, SCE = Sitopaladi     churna extract

 

The disruption of mast cells is an important feature of anaphylaxis20. An attempt was made to find out whether sitopaladi churna extract has any effect on the rate of disruption of mast cells following compound 48/80. Compound 48/80 a condensation product of N-methyl-p-methoxy-phenylamine with formaldehyde21 widely used as a selective histamine release agent or degranulator from mast cells in rats and mice. Histamine release from mast cells is closely related to an activation of phosphatidylinositol turnover and Ca2+ mobilization through guanine nucleotide binding regulatory protein22, 23. In this study, sitopladi churna extract offered significant protection against compound 48/80-induced mast cell degranulation. All   these findings clearly reveal

 

that antihistaminic activity of sitopaladi churna extract may be due to stabilization of the mast cell membrane or inhibition of antigen-induced histamine release by some of the components of the churna extract.

 

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Received on 23.04.2008                               Modified on 06.05.2008

Accepted on 10.05.2008                            © RJPT All right reserved

Research J. Pharm. and Tech. 1(2): April-June. 2008;Page 89-92