PHYTOCHEMICAL AND IN-VITRO ASSESSMENT OF ANTIHISTAMINIC AND ANTICHOLINERGIC ACTIVITY OF LEAVES OF HIBISCUS SABDARIFFA LINN.

PHYTOCHEMICAL AND IN-VITRO ASSESSMENT OF ANTIHISTAMINIC AND ANTICHOLINERGIC ACTIVITY OF LEAVES OF HIBISCUS SABDARIFFA LINN.

Md. Rageeb Md. Usman ^* and Mangalsing K. Kachhava

Department of Pharmacognosy, Smt. Sharadchandrika Suresh Patil College of Pharmacy, Chopda - 425107, Maharashtra, India.

ABSTRACT: Objective: The aim of the present study was to find out the herbal drug which has potential antihistaminic properties. Studying the in-vitro antagonistic effect of crude extracts on isolated goat tracheal chain preparation for assessment of direct antihistaminic activity using two agonists like histamine and acetylcholine. Methods: The ethanolic extract of leaves of Hibiscus sabdariffa was found quite useful in showing antihistaminic activities when tested on an experimental isolated goat tracheal. They were divided into five groups. Results: PTEE exerted an antagonistic effect on histamine and acetylcholine-induced contraction (P < 0.05). Significance is seen at a dose of 2, 4, 10 mg/ml for histamine and acetylcholine Figure 6.2 and 6.4 in a dose-dependent manner. Histamine antagonistic effect seen as (70.12 ± 1.727, 56.09 ± 1.2, 48.17 ± 1.321) similarly the acetylcholine antagonistic effect seen as (85.60 ± 2.489, 60.20 ± 2.456, 44.00 ± 1.141). Conclusion: The present study a notable contraction produced by histamine at a dose 1.6µg/ml, as 82 mm taken as 100% while notable contraction was produced by acetylcholine at a dose 1.6 µg/ml, as 92 mm taken as 100% were observed.

Hibiscus sabdariffa Linn., Histamine, Acetylcholine, Leaves extract

INTRODUCTION: Hibiscus sabdariffa is a medicinal plant that is consumed for its health benefits, juice/concoction prepared from the plant is taken as a preventive/curative measure against diabetes and hypertension. The antihypertensive and other pharmacological properties such as antibacterial, anti-oxidant, nephro- and hepato-protective, renal/diuretic effect, anti-cholesterol, and anti-diabetic effects of Hibiscus sabdariffa have been demonstrated in several studies.

Constituents of different plant parts of Hibiscus sabdariffa include phenolic acids, organic acid, flavonoids, and anthocyanins which may contribute to the pharmacological effects of the plant. There is a growing market for nutraceutical and functional foods, while a study on natural sources of antioxidants and their potential as nutraceutical and functional foods is on the increase ¹.

One plant that has attracted much attention over the years for its health benefits is roselle (Hibiscus sabdariffa); many studies on the plant, its numerous preparation, and constituents focused on its antioxidant properties. Hibiscus sabdariffa L. (roselle) belongs to the family Malvaceae. It exists as herbs or shrubs, often with fibrous stems ². The leaves are deeply three- to five-lobed, 8–15 cm long, arranged alternately on the stems. Vernacular names, in addition to roselle, in English-speaking regions are rozelle, sorrel, red sorrel, and Florida cranberry. In North Africa and the Near East, Hibiscus sabdariffa is called karkadé or carcadé ³. Hibiscus sabdariffa is believed to have originated from India and Malaysia. In India, Africa, and Mexico, all above-ground parts of the Hibiscus sabdariffa plant are valued in native medicine. Infusions of the leaves or calyces are regarded as diuretic, cholerectic, febrifugal, and hypotensive, decreasing the viscosity of the blood and stimulating intestinal peristalsis. The fresh calyx of Hibiscus sabdariffa is eaten raw in salads, is cooked, and used as a flavouring in cakes; presently, it is consumed worldwide as a cold beverage and as a hot drink (sour tea) ^{4, 5, 6}. The red anthocyanin pigments in the calyces are used as food colouring agents ⁷. Seeds of Hibiscus sabdariffa are used in oily soups, sauces, and coffee substitute ^{8, 9}. Root of Hibiscus sabdariffa is edible but very fibrousy, mucilaginous, without very much flavor ¹⁰. The aim of the present study was to find out the herbal drug which has potential antihistaminic properties Fig. 1.

FIG. 1: HIBISCUS SABDARIFFA PLANT

MATERIALS AND METHODS: The plant specimens for the proposed study were collected from the deep forest of Satpuda hills with the help of forest officers of Chopda Tahsil, Dist. Jalgaon, Maharashtra (India) in the month of Dec. 2018, care was taken to select healthy plants and for normal organs. The plant Hibiscus sabdariffa was authenticated (Ref. No. BSI/WRC/IDEN.CER/ 2018/H3/115) by Prof. (Dr.) Priyanka A Ingle, scientist, BSI (Botanical Survey of India), Pune (M.S.).

Pharmacological Activity:

Experimental Tissue: Goat trachea was obtained from a slaughterhouse, brought in previously aerated Kreb’s solution (concentration in mM/lit: NaCl, 118; KCl, 4.7; CaCl₂, 2.5; MgSO₄, 1.2; NaHCO₃, 25.0; KH₂PO₄, 1.2; Glucose, 11.1) maintained at 37±10C.

Standard Drugs: All standard drugs required for the study were purchased, and solvents and chemicals used for physiological salt solution were laboratory grades.

• Histamine Sigma, USA
• Acetylcholine Research Lab. INDIA

Preparation of Standard Drug and Extract Solution: Histamine as well as acetylcholine respectively dissolved in physiological saline and stock solution of histamine & acetylcholine was prepared to have a concentration of 0.1 µg/ml and dosed as at 1 ml, 2 ml, 4 ml, 8 ml, 1.6 ml to study the effect of both agonist on tissue contraction at 0.1 µg, 0.2 µg, 0.4 µg, 0.8 µg, 1.6 µg respectively.

Solution of the extract was prepared in 5% Polyethylene glycol, and arbitrarily doses of all extracts were added to the reservoir as 2 mg/ ml, 4 mg/ml, and 10 mg/ml to study the effect of extracts on histamine & acetylcholine-induced contractions for in-vitro studies.

Storage of Drug Solution: Fresh drug solutions were prepared for each day’s work. The solutions were kept in air-tight amber coloured bottles and stored at room temperature till use.

Routes of Administration: All drugs were administered as per screening model procedures.

Statistical Analysis: The data were presented as mean ± SEM. The statistical significance between the groups has been tested by ANOVA followed by Dunnett’s test. A probability value less than 0.05 was considered significant.

In-vitro Assessment of Direct Antihistaminic and Anticholinergic Activity:

Isolated Goat Tracheal Chain Preparation: This method is used to assess the direct antihistaminic activity of plant extract. The pharmacological actions of histamine on trachea-bronchial muscle have been described in many species. In most animal species such as, rodents, dogs, and human trachea, histamine causes bronchoconstriction, whereas; in cats and sheep, it shows the relaxation of trachea-bronchial smooth muscles. The method is used for the study of action antispasmodic drugs on the tracheal musculature. The method is based upon the findings that the excised trachea will respond to many drugs with the characteristic actions for which the drugs are well known and that with proper magnification, the response can be recorded and measured for comparative purposes. Although the method is known for its suitability in the study of antispasmodic drugs in general, emphasis is given on its use in the testing of bronchodilators. This is because of the close anatomical and physiological association, which exists between tracheal and bronchial musculature. The bronchial muscle of Ox and Pig in-vitro has been often utilized for the study of contractile responses of agonists as well as antagonist evaluation. The rodents, dog, and man trachea respond with bronchoconstriction whereas, in cats, histamine has been shown to relax the tracheal smooth muscle ¹¹. The guinea pig tracheal chain is a classical preparation but requires some skill to prepare and is not very sensitive for many agonists. It is reported that isolated goat trachea contracts in response to acetylcholine (0.1-12.8 µg), histamine (0.1-102.4 µg), and barium chloride (0.1-51.2 µg) in a dose-dependent manner and to 5-HT in a narrow dose range. Pheniramine maleate (H1-receptor antagonist) blocks contractions to histamine, while cimetidine (H2-receptor antagonist) potentiates the contraction. These observations suggest the presence of both H1-excitatory and H2-inhibitory receptors for histamine on the isolated goat trachea. The Observed dose relative contractile responses of different agonists like acetylcholine, histamine, 5-hydroxytryptamine, and bradykinin on isolated goat trachea. With these agonists, the concentration necessary to produce contraction was less with goat tracheal chain than with guinea pig tracheal chain.

They also found that both goat tracheal chain and strip preparation were suitable for screening spasmogenic activity on respiratory smooth muscle, and goat tracheal chain is easier to handle and prepare and is also much more sensitive than a guinea-pig tracheal chain. They also found that both goat tracheal chain and strip preparation were suitable for screening spasmogenic activity on respiratory smooth muscle, and goat tracheal chain is easier to handle and prepare; it is also much more sensitive than guinea-pig tracheal chain ^{12, 13, 14, 15}.

Procedure:

1. Isolated adult goat tracheal tissue was obtained immediately after slaughter of the animals.
2. Trachea was cut into individual rings and tied together in series to form a chain.
3. Trachea was suspended in a bath of Krebs solution of the following composition.
4. NaCl 6.9, KCl 0.35, CaCl₂ 0.28, MgSO₄ 0.28, NaHCO₃ 2.1, KH2PO4 0.16 and Glucose 2.0 gm/litre Which was continusoly aerated and maintained at 37 ± 0.5 ºC.
5. One end of the tracheal chain was attached to an S-shaped aerator tube and the other attached to an isotonic frontal writing lever to smoked drum (magnification 10-12 folds), a stream of 5% CO₂ in oxygen should be bubbled through the organ tube.
6. Tissue was allowed to equilibrate for 45 min. under a load of 400 mg.
7. A dose-response curve for histamine in variant molar concentrations, by maintaining 45 min time cycle was taken in following groups,

Group-I: Control

Group-II: Vehicle (PEG-400, 5% /ml)

Group-III: Test extract (2 mg/ ml)

Group-IV: Test extract (4 mg/ ml)

Group-V: Test extract (10 mg/ ml)

Graph of percentage of maximum contractile response on the ordinate and negative logarithm of the molar concentration of histamine & acetylcholine on abscissa was plotted to record dose-response curve of histamine & acetylcholine, in the absence and in presence test extract to study the possible antihistaminic & anticholinergic ability against histamine and acetylcholine precontracted goat tracheal preparation in-vitro (Table 3-6 and Fig. 2- 5)^16-20.

RESULTS AND DISCUSSION:

Preliminary Phytochemical Studies: Pet. Ether and Ethanolic extract of leaves of Hibiscus sabdariffa Linn. Showed the presence of various Phytoconstituents such as glycosides, saponins, triterpenoids, tannins and flavanoids Table 2 and 3.

TABLE 1: PERCENTAGE YIELD AND PHYSICAL CHARACTERISTICS OF HIBISCUS SABDARIFFA EXTRACTS

TABLE 2: PRELIMINARY PHYTOCHEMICAL TEST OF EXTRACTS OF TUBER OF HIBISCUS SABDARIFFA

Remark - (+) Present, (-) Absent, * - bioactive extract (PEE: Petroleum ether extract, EE: Ethanol extract

Pharmacological Activity:

TABLE 3: EFFECT OF VCPEE ON ISOLATED GOAT TRACHEAL CHAIN PREPARATION

n=4, values are in Mean ± SEM, Control = % Response of Histamine in the absence of (PTPEE) , Test I, II, III = D.R.C. of Histamine in the presence of (PTPEE) (2, 4, 10 mg/ml respectively) Statistically Non-significant data (90 mm as 100 %)

TABLE 4: EFFECT OF PTEE ON ISOLATED GOAT TRACHEAL CHAIN PREPARATION 

n=4, values are in Mean ± SEM. Control = % Response of Histamine in absence of (PTEE), Test I, II, III = D.R.C. of Histamine in presence of (PTEE) (2, 4, 10 mg/ml respectively) *p< 0.05 compared with histamine-induced contraction (82 mm as 100 %)

TABLE 5: EFFECT OF PTPEE ON ISOLATED GOAT TRACHEAL CHAIN PREPARATION

n=4, values are expressed in mean ± SEM, Statistically non-significant data (92 mm as 100 %)

TABLE 6: EFFECT OF PTEE ON ISOLATED GOAT TRACHEAL CHAIN PREPARATION

n=4, values are expressed in mean ± SEM, *p< 0.05 compared with acetylcholine induced contraction (92 mm as 100 %)

DISCUSSION AND CONCLUSION:

Effect of Crude extracts on Isolated Goat Tracheal Chain Preparation: Asthma is a heterogeneous disorder immunologically, physiologically, and biochemically and its etiology is multifactorial.

As different mediators are implicated in asthma, and the precise etiology is not known; also, multiple biochemical processes are triggered by different causative factors, it is difficult to have a single drug, which can effectively and simultaneously act upon different mediators involved in the pathogenesis of asthma. The present study was planned to evaluate the actions of test extracts of leaves of Hibiscus sabdariffa on some of the very primary aspects of asthma-like bronchoconstriction may be due to release of histamine and acetylcholine using in-vitro models.

Histamine and acetylcholine contract the tracheobronchial muscle of dog, horse, guinea pig and man. Our investigation showed the responses produce by histamine and acetylcholine in goat tracheal chain preparation in the presence of plant extract. PTEE exerted an antagonistic effect on histamine and acetylcholine-induced contraction (P < 0.05). Significance was seen at a dose of 2, 4, 10 mg/ml for histamine and acetylcholine Figure 6.2 and 6.4 in a dose-dependent manner.

Histamine antagonistic effect seen as (70.12 ± 1.727*, 56.09 ± 1.2*, 48.17±1.321*) similarly the acetylcholine antagonistic effect seen as (85.60 ± 2.489*, 60.20 ± 2.456*, 44.00 ± 1.141*). A notable contraction produced by histamine at a dose 1.6 µg/ml, as 82 mm taken as 100% while notable contraction produced by acetylcholine at a dose 1.6 µg/ml, as 92 mm taken as 100% as shown in table 6.2 and 6.4.

This primarily justifies the presence of some of the component in ETHANOL extract might be responsible for the antihistaminic and anticholinergic effect. Further investigations are warranted for the necessary development of safer phytopharmaceuticals in the armament of asthma and related respiratory afflictions.

ACKNOWLEDGEMENT: The authors are extremely grateful to the Smt. S. S. Patil College of Pharmacy, Chopda, Dist. Jalgaon, Maharashtra, India, for providing financial support (faculty research) and research facilities to accomplish this study.

CONFLICTS OF INTEREST: There is no conflict of interest with this research.

REFERENCES:

1. Cevalls - casals BA and Cisneros-Zevallos L: Stoichimetric  and  kinetic  studies  of Antioxidants  from  Audean  Purple  Corn  & Red Fleshed. Chemistry 2003; 51: 3313-19.
2. Zobo so  fine  but;  Success  Digest  (Nigeria  No1  Life  changing  Magazine)  June, 2019 edition.
3. Roselle MJ: In: Fruits of warm climates. Julia F. Morton, Miami, FL 1987; 281-86.
4. Facciola S: Cornucopia - A Source Book of Edible Plants. Kampong Publications 1990 ISBN 0-9628087-0-9.
5. Ross, IA. Hibiscus sabdariffa. In Medicinal Plants of the World, Vol. 1, 2^nd Humana Press: New Jersey, 2003; 267-75.
6. Wang CJ, Wang JM and  Lin WL:  Protective  effect  of  Hibiscus  anthocyanins against  tert-butyl  hydroperoxide-induced hepatic  toxicity  in  Food Chem. Toxicol 2019; 38: 411-16.
7. Esselen WB and Sammy GM: Roselle: a natural red colorant for  foods? Food Prod. Develop. 2019; 7: 80–82.
8. Duke J: Handbook of Energy Crops. Published only on the Internet, excellent information on a wide range of plants 2018; 15-25.
9. Kunkel G: Plants for Human Consumption. Koeltz Scientific Books, 1984; ISBN 3874292169.
10. Cribb AB and Wild JW: Food in Australia. Fontana, 1976; ISBN 0-00-634436-4.
11. Mitra SK, Venkataranganna MV, Sundaram R and Gopumadhavan SJ: Antichlolestatic activity of HD-03, a herbal formulation in thioacetamide (TAA) induced experimental cholestatic. Indian Journal of Experimental Biology 1999; 37(4): 409-10.
12. Nayampalli S, Desai N and Ainapure S: Antiallergic activity of Tinospora cordifolia in animal models. Indian Journal of Pharmacology 2019; 18: 250-52.
13. Nayampalli S and Seth U: Evaluation of antiallergic activity of Tylophora indica using rat lung perfusion. Indian Journal of Pharmacology 2018; 11(3): 229- 232.
14. Nayampalli S: Antiallergic properties of Tinospora cordifolia in animal models. Indian Journal of Pharmacology 1996; 18: 250-52.
15. Kulshrestha S, Misra SS, Sharma AL, Sharma L and Singhal D: Response of the goat trachea to some autonomic drugs. Ind J Pharmac 1983; 15(2): 107-09.
16. Ghosh S, Shand A and Ferguson A: Ulcerative colitis. BMJ 2018; 320 (7242): 1119-23.
17. Gupta B and Saxena R: Tranquilizing, antihistaminic and purgative activity of Nyctanthes arbortristis leaf extract. Journal of Ethanopharmacol 2017; 81: 321-25.
18. Floreani N and Rennard S: The role of cigarette smoke in the pathogenesis of asthma and as a trigger for acute symptoms. Current Opinion in Pulmonary Medicine 1997; 3: 38-46.
19. Ahmadiani A and Shamsa F: Antihistaminic and anticholinergic activity of barberry fruit (Berberis vulgaris) in the guinea-pig ileum. J of Ethanopharmacol 1999; 64: 161-66.
20. Akah P and Ezike A: Evaluation of the anti-asthmatic property of Asystasia gangetica leaf extracts. Journal of Ethanopharmacol 2017; 89: 25-36.
    

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    How to cite this article:

    Rageeb M Usman M and Kachhava MK: Phytochemical and in-vitro assessment of antihistaminic and anticholinergic activity of leaves of Hibiscus sabdariffa linn. Int J Pharm Sci & Res 2021; 12(8): 4275-81. doi: 10.13040/IJPSR.0975-8232.12(8).4275-81.

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