INTRODUCTION:

Plants which are rich in phytochemical constituents are diversed to pharmacological properties. Many human harbor helminths (worms) of one species or another in some cases causes infections, result mainly in discomfort and do not cause substantial ill health, an example being, thread worms in children, other worm infections such as schistosomiasis and hook worm disease can produce very serious morbidity¹. In many countries, particularly those in tropical and sub-tropical regions, all the indigenous population is infected with hook worm or other helmintis and the problem of the treatment of helminthiasis is therefore one of very great practical importance. In addition worm infections are also a major cause for concern in vetinary medicine, affecting both domestic pets and farm animals. Primarily as the result of stepped-up advocacy by the World Health Organization (WHO), the World Bank there is increasing appreciation for the impact of helminth infections on the health and education of school age children².

Anthelmintics are drugs that act either locally to expel worms from the gastrointestinal tract or systemically to irradiate adult helmintis or developmental forms that invade organs and tissue. Worms’s pamogenic for human are metazoan, classified in to round worms and two types of flatworms, fluker and tapeworms.

Due to discover and development of anthelmintic, particularly for veterinary application, physician now have effective and in some cases broad-spectrum agents that will were or control most human infections caused by either fluker or intestinal helminths. Because metazoan parasites are long- lived and have complex life cycle acquired resistance to anthelmintic in human has yet to become a major factor limiting clinical efficacy³.

The development of anthelmintic resistance and the high cost of conventional anthelmintic drug led to the evaluation of medicinal plants as an alternation source of anthelmintic, In the current study, in vitro experiment were conducted to determine the possible anthelmintic effect of ethyl alcohol and aqueous extract of the whole part of Mussaenda frondosa (Family: Rubiaceae)

Mussaenda frondosa syn Mussaenda glabrata is a rambling shrub seen in western guts, andamans, konkan, malabar and tirunelvelly hills. Leaves are broadly elliptic, shortly acuminate and more or less pubescent. Flowers are terminal open cymes berries 10-13cm, subglobose or obovoid, glabrous. Half a tola of the root is given with cow’s urine in leprosy .In jaundice 2 tolas of the whole leaves are given in milk. In Indochina, the flowers considered pectoral and diuretic, they are given in asthma, intermittent fever and dropsy. Externally they are given as a detergent to ulcers. It has been used in chinese folk medicine as a diuretic, antiphlogestic and antipyretic. It is also used to detoxify mushroom poisons and terminate early pregnancy^{4, 5}

Iridoids, flavanoids and trieterpenes as the common chemical ingredients distributed in mussaenda species.astragalin isoquercetine, kaempferol-3-o-beta drutinoside were isolated from leaves⁶. A new compounds sanzhil actone along with mussaenside, barlerine lupeol and beta-d-glucose has been obtained from the stem⁷. Quercetin, rutin, hyperin, ferulic acid, sinapic acid, beta sitosterol, saponins has also been isolated⁸. Mussaenda frondosa distributed in central Nepal, India and Sreelanka. The juice of the routine is used in blemishes on tongue and the sepals are diuretics. Mussaenda frondosa has been found to be process anti bacterial effect⁹.

MATERIALS AND METHODS:

Plant Material: The leaves of Mussaenda frondosa were collected from Kannur district in Kerala, India in January 2009 and get authenticated by Dr. Radhika. C .P Dept of Dravya Guna, Govt Ayurveda College, Kannur ,Kerala. A voucher specimen PC-05/2009 was submitted at Academy Of Pharmaceutical Science, Pariyaram Medical College, Kannur for future reference. Dried whole plant were ground to course powder, passed through sieve no 24 and stored in air tight container and used for further extraction.

Table 1 Phytochemical constituents of Mussaenda frondosa-

Phytochemical constituents	Ethyl Alcohol Extract	Aqueous Extract
Carbohydrates	+	+
Steroids	+	-
Alkaloids	+	+
Saponins	-	-
Terpenoides	+	-
Flavonoids	+	+
Tannins	+	+
Polyphenols	+	+

(+): Present (-): Absent

Preparation of Extract:

Ethyl alcohol extract: The powdered material was exhaustively extracted with 95% ethyl alcohol using a soxhlet apparatus. The extract was concentrated in Vaccuo to a syrupy consistency. The percentage yield of extract was found to be 4.26 %.

Aqueous extract:

The dried powders (200 g) were kept for maceration with 1000 ml of distilled water for 24 hrs. The extract was double filtered by using muslin cloth and what mann no.1filter paper and concentrated by evaporation on water bath. The extract was dried and used as a powder. The percentage yield of extract in 6.31 %

Animals:

Indian adult earthworms (Pheretima posthuma), round worms (Ascaridia galli) and tape worms (Raillietina spiralis) were used to evaluate in vitro anthelmintic activity. Earth worms were collected from moist soil and washed with normal saline to remove all fecal matter were used for the anthelmintic study. The earth worms of 3-5 cm in length and 0.1-0.2 cm in width were used for all the experimental protocol. Round worms and tape worms were from intestine of freshly slaughtered fowls. The intestines were then dissected and worms were collected and kept in normal saline solution. The average size of earth worn was 3-5 cm, average size of round worn was 5-7 cm and average size of tape worn was 6-8 cm^10-14.

Anthelmintic activity:

The anthelmintic activity was carried out as per the method of Ajaiyeoba et al ¹⁵.The assay was performed in vitro using adult earth worms (pheretima posthuma) owing to its anatomical and physiological resemblance with the intestinal round worm parasites^{16, 17,
18}. Use of Ascaridia galli and Raillietina species on a suitable model for screening of anthelmintic activity^{19- 21}.

Test sample of the extract was prepared at concentration 10, 25,50 mg/ml in distilled water and six worms ( same type) were placed in each 9cm petridish containing 25 ml of above test solution of extracts. Piperazine citrate (10 mg/ml) was used as reference standard and distilled water as control^22,-24. This procedure in adopted for all three paralysis was noted when no movement of any sort cold be observed except when worms were shaken vigorously. Time for death of worms were recorded after ascertaining that worms neither moved when shaken vigorously nor when dipped in warm water (50⁰ c)

RESULT AND DICUSSION:

The photochemical screening subjected to detect the presence of some secondary plant metabolites following standard procedure shown in Table-1^25,26. Ethyl alcohol extract revealed the presence of carbohydrates, steroids, alkaloids, terpenoids, flavanoids, tannins and poly phenols, while aqueous extract showed presence of carbohydrates, alkaloids, flavanoids, tannins and poly phenols.

At higher concentration, the ethyl alcohol and aqueous extract of whole parts of Mussaenda frondosa displayed significant anthelmintic properties (Table 2). Both extract showed anthelmintic activities in dose dependent manner giving shortest time of paralysis (P) and death (D) with 50 mg /ml concentration, for all three types of worms. Evaluation of anthelmintic activity was compared with reference standard piperazine citrate (Table 2) .Piperazine citrate by increasing chloride conductance of worm muscle membrane produces hyperpolarization and reduced excitability that leads to muscle relaxation and flaccid paralysis.

Ethyl alcohol extract of Musaenda frondosa shows the paralysis as well as death of worm in a less time as compared to piperazine citrate especially at higher concentration (50 mg / ml). While water extract also shows significant activities. The ethyl alcohol and aqueous extract reveals the presence of flavanoids and polyphenolic compounds as one of the phytochemical constituents shows anthelmintic activity²⁷. Some of the synthetic polyanthelmintics, eg: niclosamide, oxclosanide are shown to interfere with energy generation in helmint parasites by on coupling oxidative phosphorelation²⁸. It is possible that phenolic compound in the extract of Musaenda frondosa produce similar effect.

Table 2 Anthelmintic activity of extracts of Mussaenda frondosa

Extract of Groups	Concentration mg/ml	Pheretima Posthuma		Ascardia Galli		Raillietina Spiralis
Extract of Groups	Concentration mg/ml	P	D	P	D	P	D
EA	10	26±0.42	70±1.21	21±1.61	57±1.46	28±1.22	61±1.09
	25	20±1.06	54±0.48	16±0.49	45±0.96	17±0.69	45±0.54
	50	15±0.06	34±0.49	9±1.22	31±0.46	10±0.33	29±0.48
AE	10	28±0.48	17±1.06	23±1.06	58±1.46	31±0.48	63±1.22
	25	23±1.76	57±0.48	18±0.46	48±0.98	20±0.68	48±0.56
	50	14±0.06	38±0.46	9±0.48	33±0.42	11±0.36	38±0.49
PC	10	21±1.06	15±0.76	10±1.23	37±1.06	22±0.76	53±0.88
Control	-	-	-	-	-	-	-

Where EA: Ethyl Alcohol extract, AE: Aqueous extract, PC: Piperazine citrate, P: Time taken for Paralysis (min), D: Time taken for Death of worms (min).

CONCLUSIONS:

This study has revealed the presence of many secondary metabolites (phytocostituents) in the plant of Mussaenda frondosa .It has further confirmed that the plant extract could be used against anthelmints. This result lend credence to the folkloric use of this plant in treating against worms and show that Mussaenda frondosa could be exploited for new potent anthelmintic .

ACKNOLEDGEMENTS:

The author’s thankful to Dr. Radhika C. P, Dept. of Dravyaguna, Govt. Ayurveda Medical College, Pariyaram, Kannur, for authentication of the plant specimen.

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Received on 15.08.2009 Modified on 23.09.2009

Research J. Pharm. and Tech. 3(1): Jan. - Mar. 2010; Page 151-153