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ANTI-HISTAMINIC AND ANTI-SPASMODIC POTENTIAL OF ETHANOLIC EXTRACT OF TEPHROSIA PURPUREA (Linn.) PERS. AERIAL PARTS

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ABOUT AUTHORS:
Paresh Lallubhai Gajera*, Dalal Mittal V.
Department of Pharmacology, ROFEL,
Shri G. M. Bilakhia College of Pharmacy,
Vapi (Gujarat)
*paresh.gajera@gmail.com

ABSTRACT
The aim of present study was to evaluate anti-histaminic and anti-spasmodic potential of ethanolic extract of aerial parts of Tephrosia purpurea (Linn.) against experimental animal models. In this study ethanolic extract of aerial parts of Tephrosia purpurea (L.) was prepared and anti-histaminic and anti-spasmodic activity evaluated on isolated goat tracheal chain and rat ileum preparation by dose response curve of histamine and acetylcholine in absence and in presence of ethanolic extract was plotted respectively. The results of present study revealed that EETP inhibits moderately significant (*p <0.05) percentage contraction at 50µg/ml while EETP inhibits significantly (**p<0.01) percentage contraction at 100µg/ml induced by histamine (10µg/ml) and acetylcholine (1µg/ml) in isolated goat tracheal chain and rat ileum preparation respectively. To, concluded, EETP possesses dose dependent antihistaminic and antispasmodic potential. However, future studies are required to focus on the molecular mechanism of responsible phytochemical constituents and establish exact mode of action involved in it.

REFERENCE ID: PHARMATUTOR-ART-1941

INTRODUCTION
Tephrosia purpurea
(Linn.) Pers. (Fabaceae) commonly known in Sanskrit as Sharapunkha is a highly branched, sub-erect, herbaceous perennial herb. It is one of the excellent plants for human being made and gifted by the nature having composition of all the essential constituents that are required for normal and good human health.[1,2] In Ayurvedic system of medicine various parts of this plant are used as remedy for impotency, asthma, diarrhoea, gonorrhoea, rheumatism, ulcer and urinary disorders. In the Ayurvedic system of medicine, the whole plant has been used to cure tumours, ulcers, leprosy, allergic and inflammatory conditions such as rheumatism, asthma and bronchitis.[3] The phytochemical investigations on Tephrosia purpurea have revealed the presence of glycosides, rotenoids, isoflavones, flavanones, chalcones, flavanols, and sterols.[4] Recent investigations have shown that the LD50 of the ethanolic extract of aerial parts of Tephrosia purpurea is 5.12g/kg and it inhibits type I hypersensitivity reactions.[5,6] The extract also inhibited the late inflammatory phase of an allergic reaction.[7] Ayurveda offers a unique insight into comprehensive approach to asthma management through proper care of the respiratory tract. More than 400 medicinal plant species have been used ethno-pharmacologically and traditionally to treat the symptoms of asthmatic and allergic disorders worldwide. The world health organization (WHO) has recognized herbal medicine as an essential building block for primary health care of vast countries like India and China.[8] Tephrosia purpurea (Linn) seems to be a promising plant for treatment of bronchial asthma because of its reported anti-oxidant, immunomodulatory and anti-inflammatory activity.[9-11] There are hardly any reports on anti-histaminic and anti-spasmodic activity of this plant. Hence, the present study was investigated based on traditional claims of showing anti-histaminic and anti-spasmodic activity; we have tested the plant extract for both anti-histaminic and anti-spasmodic activity.

MATERIAL AND METHODOLOGY

Procurement of Plant and its authentication
The plant required for the study i.e. Tephrosia purpurea (Linn.) Pers. was collected from Anand Agriculture University and the field around it in the month of January’ 2011, during flowering stage. The plant, Tephrosia purpurea (Linn.), was authenticated by Dr. Minoo H. Parabia, advisor of Shri R. M. Dhariwal Ayurved College and Research Centre, Waghaldhara, Valsad, Gujarat. And voucher specimen submitted to herbarium department of this college.

Preparation of ethanolic extract
Aerial parts of the plants were collected, cleaned and dried in shade. After 7-days of drying, the dried plant was powdered by grinding and sieved with a 40# sieve. The powder was extracted in soxhlet apparatus for 24 hours with 95% ethanol. The extract was filter and concentrated in vacuum under reduced pressure using a rotary flash evaporator (yield 4.8 % w/w). The concentrated mass was used.

Procurement of Animals
Isolated adult goat tracheal tissue and albino rats (Wistar strain) of either sex weighing 200-250 gm were used for studies. Isolated adult goat tracheal tissue was obtained immediately after slaughter of the animal. Pieces of the trachea were collected in the ice cold oxygenated Krebs’ solution. The albino rats were obtained from animal house of Jai Research Foundation, Vapi, Gujarat. They were housed in polypropylene cages with standard pellet chow and water ad libitum. Permission was obtained, prior to the start of experiments, from Institutional Animal Ethics Committee (IAEC) of Rofel, Shri G. M. Bilakhia college of Pharmacy as per CPCSEA guidelines (Reg. no.: 403/01/a/CPCSEA; Rofel/2011/01).

Drugs and solvents
Kreb’s solution (NaCl 6.9, KCl 0.35, CaCl 0.28, MgSO4 0.28, NaHCO3 2.1, KH2PO4 0.16 and Glucose 2.0 g/ L), Ether, Ethanol (THOMAS BAKER Pvt. Ltd, Mumbai) Histamine, and Acetylcholine (Sigma).

Anti-histaminic activity

Histamine induced contraction of isolated goat trachea preparation[12]

Purpose and Rationale

The isolated tracheal chain of goat can be used to test for ß-blocking activity. In addition, this model can be used to test compounds which inhibit bronchospasm. It is used to detect ß-sympathomimetic, H1-receptor blocking and leukotrienes receptor blocking properties of test drugs. Histamine is an important mediator of immediate allergic (type 1) and inflammatory reactions. It causes bronchoconstriction by activating H1- receptors.

Procedure
Isolated adult Goat tracheal tissue was obtained immediately after slaughter of the animals.Trachea was cut into individual rings and tied together in series to form a chain.Trachea was suspended in bath of Kreb’s solution which was continuously aerated and maintained at 37±0.5
°C.One end of the tracheal chain was attached to an S-shaped aerator tube and other attached to isotonic frontal writing lever to smoked drum (magnification 10-12 folds). Tissue was allowed to equilibrate for 45 minute under a load of 1 gm.A dose response curve for histamine was taken in variant molar concentrations, by maintaining 15 min time cycle. After obtaining a dose response curve of histamine on trachea, the Tephrosia purpurea extracts was added to the respective reservoir and same doses of histamine were repeated.Graph of percentage of maximum contractile response on ordinate and negative logarithm of molar concentration of histamine was plotted to record dose response curve of histamine, in absence and in presence Tephrosia purpurea extracts. Molar conc. were calculated using following equations.

Calculation of the molar concentration of drug:


Where, Conc.- concentration; admin- administered; Qty.- quantity.

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Anti-spasmodic activity

Acetylcholine induced contraction of isolated rat ileum tissue preparation[13]

Aim: To evaluate anti-spasmodic activity of ethanolic extract of Tephrosia purpurea on rat ileum preparation.

Procedure: The assembly was set up and arrangements were made for experimental conditions. A guinea pig which was fasted over night was euthanized by cervical dislocation and sacrificed by cutting neck blood vessels. The abdominal cavity was quickly open and a piece of ileum was isolated. It was placed in a petridish containing Tyrode solution maintained at 37±0.5°C.  The mesentry of ileum was removed and the lumen of ileum was cleaned by passing warm Tyrode solution through it from a pipette held at an angle of about 20-30 degrees. The tissue was mounted in mammalian organ bath and connected to isotonic frontal writing lever. Then tissue was allowed to stabilize for 30 min. The responses of histamine were taken till the maximum effect was obtained. The normal Tyrode solution was changed with the Tyrode containing Tephrosia purpurea Linn. The responses of histamine was taken with same dose and continued till maximum effects were obtained. The graph was fixed and log-dose response curve was plotted to determine the nature of antagonism. Molar concentrations were calculated using above mentioned equations.

Statistical analysis
All values were expressed as Mean± SEM for both the models. Data was analyzed by Student’s t’-test and One way ANOVA followed by Dunnett’s t-test. The results were considered to be statistically significant when p<0.05

RESULTS

Effect of EETP on histamine induced contraction of isolated goat tracheal chain preparation: In the present study, it was observed that ethanolic extract of Tephrosia purpurea inhibits the contraction produced by histamine in this tissue preparation. Histamine (10µg/ml) was taken in different dose level and DRC was plotted in absence and in presence of EETP of 50 µg/ml and100 µg/ml concentration (figure 1). The result obtained from this study revealed that EETP inhibits moderately significant (*p <0.05) percentage contraction at 50 µg/ ml while EETP inhibits significantly (**p <0.01) percentage contraction at 100 µg/ ml induced by histamine in goat tracheal chain preparation (Table 1).

Effect of EETP on acetylcholine induced contraction of isolated rat ileum tissue preparation: In the present study, it was found that ethanolic extract of Tephrosia purpurea inhibits the contraction produced by acetylcholine in rat ileum tissue preparation. Acetylcholine (1 µg/ ml) was taken in different dose level and DRC was plotted in presence and in absence of EETP of 50 µg/ ml and 100 µg/ ml concentration (figure 2). Present study observed that EETP inhibits significantly (*p <0.05 and **p <0.01) percentage contraction at 100 µg/ ml but less inhibits percentage contraction at 50 µg/ ml induced by acetylcholine in rat ileum tissue preparation (Table 2).

DISCUSSION
The contraction of tracheal or bronchial smooth muscle in-vitro has often been utilized for the study of contractile/dilator responses of agonists as well as antagonists. Both goat tracheal chain and strip preparations are suitable for screening the activity of a drug on respiratory smooth muscles.[14] In this study two spasmogens – Histamine (goat trachea) and Acetylcholine (rat ileum) were used and the effects of EETP on this were studied.

Goat trachealchain is easier to handle and to prepare; it is also much more sensitive than guinea pig tracheal chain. Therefore goat tracheal chain was used to determine the antihistaminic effect of EETP. The result of this study revealed that EETP inhibits the contraction produced by histamine in isolated goat tracheal chain preparation.

Histamine produced dose dependent contraction of goat tracheal chain preparation, through H1 receptors.[12] EETP at a conc. of 50 μg/ml and 100 μg/ml in the perfused PSS significantly inhibited the histamine induced contraction of isolated goat trachea preparation.  This could be due to specific inhibition of H1 receptors or a non specific spasmolytic action.

Anticholinergic drugs have also been shown to be effective in relieving asthma symptoms. Hence, to validate the possible anticholinergic effects of Tephrosia purpurea (Linn.), the effect of EETP on Acetylcholine induced rat ileal contractions was checked.

Rat ileumwas used to determine the spasmolytic (anticholinergic) activity of EETP. It was found that EETP produced dose dependent inhibition of ileal contractions induced by acetylcholine. The parallel rightward shift in agonist concentration-response curves in the presence of increasing concentrations of EETP was indicating anti-spasmodic activity. The inhibition may be due to the antagonism of muscarinic receptors or nonspecific spasmolytic action.

CONCLUSION
It can be concluded from the results obtained from the result that EETP possesses significant spasmolytic activity that may be attributed to H1-antagonistic and anticholinergic activity. And also has significant mast cell stabilizing activity. Hence it may be used in prophylaxis and/or management of asthma.

ACKNOWLEDGEMENT
I am deeply indebted to my guide Prof. Mittal V. Dalal whose help, stimulation, suggestion and encouragement helped me in all the time. . I am greatly thankful to the Management and Principal of Rofel, Shri G. M. Bilakhia College of Pharmacy, Vapi, Gujarat, for providing necessary facilities. I am also acknowledging to Dr. Minoo H. Parabia, advisor of Shri R. M. Dhariwal Ayurved College and Research Centre, Waghaldhara, Valsad, Gujarat, for the authentication of the plant. I have been motivated by moral encouragement and support from my friends Prakash Gajera, Alpesh2, Mayur Gajera, Kishor, Vipul Gajera, Pravin Gajera, and Mukesh.

TABLES

Table 1: Effect of EETP (50 and 100 µg/ml) on histamine induced contraction of isolated goat trachea preparation:

Dose (histamine)

(10 µg/ ml)

-ve log molar conc. of histamine

Maximum contraction (%) (Mean ± SEM, n=6)

In absence of antagonist

In presence of EETP

(50 µg/ ml)

(100 µg/ ml)

0.1

6.87

25.80 ± 2.13

20.73 ± 1.79ns

18.29 ± 1.88*

0.2

6.57

39.40 ± 2.85

28.57 ± 2.19*

23.34 ± 1.75**

0.4

6.27

56.77 ± 1.58

37.60 ± 2.72**

28.87 ± 2.11**

0.8

5.96

71.66 ± 1.62

53.75 ± 2.00**

37.95 ± 3.18**

1.6

5.66

87.37 ± 0.89

68.48 ± 1.29**

50.71 ± 3.18**

3.2

5.36

97.05 ± 1.57

77.75 ± 1.86**

61.76 ± 3.51***

6.4

5.06

97.30 ± 1.30

86.43 ± 1.79*

72.54 ± 3.81**

N=6, Values are in Mean±SEM. Control=DRC of Histamine in absence of Tephrosia purpurea (Linn.) extract. EETP=DRC of Histamine in presence of ethanolic extract of Tephrosia purpurea Linn. (50 µg/ml and 100 µg/ml). Statistical analysis was done by one way ANOVA followed by Dunnett’s t- test. * p<0.05, ** p<0.01, *** p<0.001 compared to control.

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Dose

(ACh)

(1µg/ ml)

-ve log molar conc. of ACh

Maximum contraction produced by Acetylcholine

 (%) (Mean ± SEM, n=6)

In absence of antagonists

In presence of EETP

(50µg/ml)

(100µg/ml)

0.1

6.64

24.61 ± 1.96

21.11 ± 1.40ns

20.40 ± 1.07*

0.2

6.34

39.11 ± 2.56

31.56 ± 1.66*

26.73 ± 2.10**

0.4

6.04

55.36 ± 2.48

44.84 ± 1.18*

35.91 ± 1.86**

0.8

5.74

72.22 ± 2.80

59.30 ± 1.56**

41.84 ± 2.16**

1.6

5.44

86.72 ± 2.11

69.58 ± 2.13**

53.58 ± 3.50***

3.2

5.14

96.43 ± 1.00

82.03 ± 1.79**

61.26 ± 3.41***

6.4

4.83

99.43 ± 0.57

90.30 ± 1.87*

68.85 ± 3.58***

Table 2: Effect of EETP (50 and 100 µg/ml) on acetylcholine induced contraction of isolated rat ileum tissue preparation:

N=6, Values are in Mean±SEM. Control=DRC of Acetylcholine in absence of Tephrosia purpurea (Linn.) extract. EETP=DRC of Acetylcholine in presence of ethanolic extract of Tephrosia purpurea Linn. (50 µg/ml and 100 µg/ml). Statistical analysis was done by one way ANOVA followed by Dunnett’s t- test. *p<0.05, **p<0.01 and ***p<0.001 compared with control.

FIGURES


Figure 1: Effect of EETP (50 and 100 µg/ml) on histamine (10 µg/ml) induced contraction of isolated goat trachea preparation.


Figure 2: Effect of EETP (50 and 100 µg/ml) on acetylcholine induced contraction of isolated rat ileum tissue preparation.

REFERENCES
1.Chopra RN, Nayar SL, Chopra IC. Glossary of Indian medicinal plants. New Delhi, India: Council of Scientific and Industrial Research, 1956.
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3.Kirtikar KR, Basu BD. Indian medicinal plants.2nd edition. Allahabad, India: Lalit Mohan Basu, 1956.
4.Pelter A, Ward RS and Rao EV. Journal of Chemical Society. Perkin Trans, 1981; 1: 2491-2495.
5.Gokhale AB and Saraf MN. Tephrosia purpurea: a review of contemporary literature and medicinal properties. Indian Drugs. Indian J Pharm Sci, 2000; 37:228.
6.Damre AS, Kulkarni KR, Saraf MN, Gokhale AB and Dikshit VJ. Indian J Exp Biol 2000; 38:837.
7.Saraf MN and Gokhale AB. Indian Drugs, Tephrosia purpurea: a review of contemporary literature and medicinal properties. Indian J Pharm Sci 2000; 62:356.
8.Kumar V and Parmar NS. Herbs: A potential source for development of new phyto-medicinals. The Pharma Review. 2003; 1 (4): 59-63.
9.Shah R, Kathad H, Sheth R and Sheth N. Anti oxidant activity of Tephrosia purpurea. International J Pharmacy and Pharmaceutical Sci. 2010; 2 (3): 30-33.
10.Damre AS, Gokhale AB, Phadke AS, Kulkarni KR and Saraf MN. Tephrosia purpurea: immunomodulatory activity. Fitoterapia, 2003; 74: 257-261.
11.Shenoy S, Shwetha K, Prabhu K, Maradi R, Bairy KL and Shanbhag T. Tephrosia purpurea: anti-inflammatory activity in rats. Asian Pacific J of Tropical Medicine, 2010; 193-195.
12.Kulshrestha SS, Misra S, Sharma AL, Sharma P and Singhal D. Response of the goat trachea to some autonomic drugs. Indian J Pharmacol, 1983; 15: 107-109.
13.Goyal RK. Practicals in pharmacology, B S Shah prakashan, 2005, 33-34.
14.Nag Chaudhari AK and Lahiri SC. Use of Goat trachea isolated tracheal chain preparation. Indian J Pharmacol, 1974; 6:149-150.

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