PRELIMINARY PHYTOCHEMICAL AND IN-VITRO ANTHELMINTIC SCREENING OF HIBISCUS ROSA-SINENSIS LINN. FLOWER EXTRACTS (FAMILY: MALVACEAE)

About Author:
Krishn Kumar Agrawal*, Jeetendra Kumar Gupta, Anju Verma, Kishan Singh
Institute of Pharmaceutical Research,
GLA University 17-Km. stone, NH-2 Mathura-Delhi Highway,
P.O. Chaumuhan, Mathura-281406 (U.P.) India
*krishn.agrawal@rediffmail.com

Abstract
The objective of the present study to investigate the anthelmintic activity of aqueous and ethanolic extract of flower of Hibiscus rosa-sinensis L. (family: Malvaceae) against Indian earthworms (Pheretima posthuma). The preliminary phytochemical screening of Hibiscus rosa-sinensis showed the presence of sterols, glycosides, alkaloids, proteins, mucilage and flavonoids. Various standards such as Piperazine citrate, Albendazole and Pyrantel pamoate at concentration of 20mg/ml were used to compare anthelmintic activity with the extracts at the same concentration in terms of paralysis time and death time of worms. The result revealed the potential usefulness of aqueous and ethanolic extract of Hibiscus rosa-sinensis flower against helminthiasis infection.

Reference Id: PHARMATUTOR-ART-1400

1.      Introduction:
Hibiscus is a large genus that contains herbs, shrubs and trees widely distributed in the tropical and sub-tropical region of world. Hibiscus rosa-sinensis Linn. (Malvaceae) is commonly known as jasut in Hindi and China rose in English. It is a native of China and grown throughout in India as an ornamental plant. Plant can be propagated by cutting from mature wood of current growth. It is an evergreen woody, glabrous, showy shrub of 5-8 ft in height. Leaves are bright green, ovate, coarsely toothed above, flower are solitary, axillary, bell shaped, large 4-6 inch in diameter with pistil and stamens projecting from the centre ^[1].

Folklorically the flowers are used as demulcent, emollient, refrigerant, aphrodiasic emmenagogue and as anthelmintic ^[2]. A decoction of flower is used in bronchial catarrh. The dark red petals in the form of mucilaginous infusion are used in ardor-urinae, strangury, cystitis and other irritable conditions of the genito-urinary tract ^[3]. The flower of Hibiscus rosa-sinensis were reported to possess various activity such as analgesic^[4], anticonvulsant^[5], antidiabetic^[6], antipyretic^[7], wound healing^[8], antibacterial^[9], immunomodulatory^[10], antiestrous^[11], antioxidant^[12] and hair growth^[13].

Helminthiasis is the most prevalent infections globally but is more common in developing countries with under nourished people and environmental hygiene. They affect the host by secreting toxins, causing blood loss, injury to organs and intestinal obstruction. Anthelmintic drug have tendency to kill, expel or paralysis of worm and which are present in the intestinal canal. There is no systemic or scientific study on anthelmintic activity in spite of its traditional use. So the present study was focused to evaluate the anthelmintic activity of various extract of Hibiscus rosa-sinensis Linn flower.

2.      Material and Methods:
2.1 Plant material:
The flower of Hibiscus rosa-sinensis Linn were collected from the gardens of Mathura district, Uttar Pradesh and aunthentified by Birbal Sahni Institute of Palaeobotany, Lucknow, Uttar Pradesh, India and a herbarium specimen was deposited in the department of pharmacognosy with Ref. No. 13374.

2.2 Preparation of extracts:
The flower of Hibiscus rosa-sinensis were collected from gardens of Mathura (Uttar Pradesh) and dried in shade and coarsely powdered. It was than passed through the sieve no. 20. A weighted quantity (360g) of the powder drug was extracted with petroleum ether (60-80⁰C) using soxhlet extractor. Defatted drug was subjected to ethanolic extraction and extract was dried by distilling off the solvent and then dried in desiccator. The marc collected after ethanolic extraction was subjected to aqueous extraction by maceration process for seven days consecutively and then extract was dried by evaporating the water and stored for further activity.    

2.3 Animal:
Adult Indian earth worms (Pheretima posthuma) were collected from the moist soil as well as water logged area of Mathura district and washed with normal saline to remove all faecal matter were used for the anthelmintic activity. The earth worms of 3-6 cm in length and 0.1-0.3 cm in width were used for the experimental protocol due to their anatomical and physiological resemblance with the intestinal roundworms parasites of human beings.

2.4 Drugs and chemicals:
Piperazine citrate, albendazole and pyrantal pamoate were used as standard drugs. Petroleum ether (60-80), Ethanol, Di methyl sulfoxide (DMSO) and sodium chloride were used in various part of experiment.

2.5 Phytochemical screening:
The preliminary phytochemical screenings were conducted for the petroleum ether, ethanol and aqueous extracts of Hibiscus rosa-sinensis flower to find out the presence of various phytochemical constituents. The result of preliminary phytochemical screening was showed the presence of alkaloids, glycosides, tannins, flavonoids, mucilage and sterols. Data is shown in table.

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2.6 In-Vitro anthelmintic activity:
The In-vitro anthelmintic activity was evaluated on adult Indian earth worms (Pheritima posthuma). Worms were divided in the three groups of six worms in each group, to assess the anthelmintic activity of aqueous and ethanolic extract of Hibiscus rosa-sinensis Linn flower.

Group 1 served as control group and worms in this group were placed in solution containing DMSO in distill water. Groups 2 was served as standard and further divided in to three sub groups of six animals in each sub groups and treated by piperazine citrate, albendazole and pyrantal pamoate at concentration of 20mg/ml in solution containing DMSO and distill water. Group 3 was act as test group and divided in to two sub groups and each sub groups was treated by aqueous and ethanolic extract at concentration of 20mg/ml. Observation were made for the time taken to paralyze and death of the individual worms. Mean time for paralysis in minute was noted when worms become motionless and to ascertain death, each worms was frequently applied with external stimuli, which stimulates and induce movement in the earthworms. Death was confirmed when no movement in worms even after application of external stimuli.  

2.7 Observation table:

Results are expressed as Mean± SEM, n=6 in each group
A=absent of activity,

3.      Result:
Results of the preliminary phytochemical screening of different extracts of Hibiscus rosa-sinensis are shown in table. The qualitative screening indicates that the extracts contain alkaloids, tannins, flavonoids, sterols, glycosides and mucilage.

In this anthelmintic study both the extracts (Aqueous and Ethanolic) of Hibiscus rosa-sinensis produce paralysis as well as death of the worms. As shown in table-2, aqueous and ethanolic extract exhibited anthelmintic activity as in standards (i.e. Piperazine citrate, Albendazole and Pyrantel pamoate). Although, Piperazine citrate caused only paralysis not death of the worms. Furthermore the anthelmintic effect of aqueous and ethanolic extract of Hibiscus rosa-sinensis was comparable with the standard drugs, although it caused paralysis as well as death of worms similar to albendazole and Pyrantel pamoate.

4.      Discussion:
The study was performed on Indian earthworm, Pheretima posthuma due to its anatomical and physiological resemblance with the intestinal round worms of human being and easily availability for in-vitro anthelmintic activity.

Piperazine citrate and Pyrantel pamoate causes spastic and flaccid paralysis of worms respectively, which may resulting in to expulsion of worms. On the other hand albendazole causes death of worms by inhibiting tubulin polymerization and blocking glucose uptake ^[14].

The mechanism action of Hibiscus rosa-sinensis is not yet fully understood, but the anthelmintic activity, as evident from the result of the present study was due to presence of flavonoids that justify its folkloric use in curing helminthiasis.

Therefore it can be concluded that the ethanolic and aqueous extract of Hibiscus rosa-sinensis have profound anthelmintic activity against tested worms but not better than standards. Further in future researches will also being carried out in our department to establish the prescribed mechanism of action and to identify phytochemical moiety which is responsible for this activity.

5.      Acknowledgement:
Authors are thankful to Director, Institute of Pharmaceutical Research, GLA University for providing the necessary facilities and support to carry out this work.

6.    References:
1.    Hibiscus rosa sinensis In. Sastri BN (chief editor) The Wealth of India.(vol.5) New Delhi National Institute of Science Communication CSIR, p.91
2.    stuartxchange.com/Gumamela.html assessed on 10-feb-2012
3.    Asolkar LV, Kakkar KK, Chakre OJ. Glossary of Indian medicinal plants with active principle part 1New Delhi National Institute of Science communication & Information Resource CSIR 2005, p.353
4.    Sawarkar A, Jangde CR, Thakre PD, Kadoo R and Shelu S, Analgesic Activity of Hibiscus rosa sinensis Linn in Rat, Veterinary World, 2009;2:353-354.
5.    Birari RB, Singh A, Giri IC, Saxena N, Shaikh MI and Singh A, Evaluation of anticonvulsant activity of Hibiscus rosa-sinensis flower extracts, Int. J. Pharm. Sci. Res. 2010; 1:83-88.
6.    Anti-diabetic activity of flowers of Hibiscus rosa-sinensis, Venkatesh S, Thilagavathi J, Shyam sundar D, Fitoterapia, 2008;79:79–81.
7.    Soni D, Gupta A, An evaluation of antipyretic and analgesic potentials of aqueous root extract of Hibiscus rosa sinesis Linn. (malvacae), Int. J. Res. Phytochem. Pharmacol., 2011; 1:184-186.
8.    Nayak BS, Raju S S, Orette FA, Chalapathi Rao AV, Effects of Hibiscus rosa sinensis L (Malvaceae) on wound healing activity: a preclinical study in a Sprague Dawley rat, Int J. Low Extrem. Wounds. 2007; 6:76-81.
9.    Hena JV, Antibacterial Potentiality of Hibiscus rosa-sinensis Solvent Extract and Aqueous Extracts Against Some Pathogenic Bacteria, Herbal Tech Industry , 2010, 21-23.
10.    Gaur K, Kori ML and Nema RK, Comparative Screening of Immunomodulatory Activity of Hydro-alcoholic Extract of Hibiscus rosa sinensis Linn. and Ethanolic Extract of Cleome gynandra Linn, Global J. Pharmacol., 2009; 3:85-89.
11.    Kholkute SD, Udupa KN, Antiestrogenic activity of Hibiscus rosa-sinensis Linn. flowers, Indian J. Exp. Biol. 1976;14:175-6.
12.    Bhaskar AA, Nithya V and Vidhya VG, Phytochemical screening and In Vitro antioxidant activities of the ethanolic extract of Hibiscus rosa sinensis L, Annals of Biol. Res., 2011; 2:653-661
13.    Adhirajan N, Kumar RT, Shanmugasundaram N, Babu M, In vivo and In Vitro evaluation of hair growth potential of Hibiscus rosa-sinensis Linn. J. Ethnopharmacol. 2003;88: 235–239.
14.    Tripathi KD, Essentials of medical pharmacology, 5th edition, jaypee brothers medical pulishers (P) ltd. New Delhi, 2003, 759-766

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