Leaves and seeds are used in the treatment of bronchitis, asthma, thirst, biliousness, dysentery, ulcers. Syzygium cumini is a medicinal plant, whose parts were pharmacologically proved to possess hypoglycemic, antibacterial, anti-HIV activity and anti-diarrhea effects⁶. The fruit pulp and seeds are used in treatment of diabetics and also in blood purifier; several studies which were done using modern techniques have authenticated its use in complications like nephropathy, cataract, insulin resistant. According to the Indian system the extracts of the bark, seeds and leaves are used for the treatment of diabetes. A decoction of the bark and powdered seeds is used in asthma, bronchitis, diarrhea, dysentery, dyspepsia, mouth washes for the astringent effect on mouth ulcers, spongy gums and stomatitis. The leaves have antibacterial properties and used for strengthening teeth and gums. The fruit is used as stomachic, carminative, antiscorbutic and diuretic. For ringworm treatment water diluted juice is used as lotion^7,8. The main chemical constituents present in the leaves are determined by analyses which results in crude protein, 9.1%; fat, 4.3%; crude fiber, 17.0%; ash, 6.0%; calcium, 1.3%; phosphorus, 0.19%. They are rich in tannin and contain the enzymes esterase and galloyl carboxylase which are presumed to be active in the biosynthesis of the tannins⁹. Apart from this it consists of 1-galloyl glucose, 3,6-hexahydroxy-diphenoyl glucose, 3-galloyl glucose, 4,6-hexahydroxy-diphenoyl glucose, 6-galloyl glucose, acetophenone, alanine, alpha-pinene, alpha-humulene, alpha-terpineol, astragaline, beta-caryophelene^10,11.

Achyranthes aspera:

Achyranthes aspera is commonly known as prickly chaff flower, chirchidi and belonging to the family Amaranthaceae¹². These mostly found in dry places and waste lands as a weed. It is found throughout Tropical Asia, Africa, Australia and America. It can adapt to a wide range of environments. It frequently occurs in waste areas, along road sides, foot paths, rail road and sand dunes. In Ayurveda two varieties of plants, red and white are mentioned. Different parts of the plant form ingredients in many native prescriptions in combination with more native remedies. It is described in ‘Nighantas’ as purgative, pungent, digestive, a remedy for inflammation of the internal organs, piles, itch, abdominal enlargement and enlarged cervical glands¹³. It is also used as astringent, diuretic, alterative and antiperiodic properties. Plant is used in skin eruptions, opthalmia, dysentery, eye and liver complaints, rheumatism, scabies, bronchial infections and in leprosy. Leaves are useful in gonorrhea. Roots are used in cancer, stomach troubles and bladder stones. Seeds are useful in renal dropsy, bronchial infections and in leprosy. Branches and roots are useful as tooth brushes. It is also used as laxative, increases appetite, discomfort, pain, asthma, cough and an anti-periodic in preventing the return of such diseases which recur, like malaria^14-16. The plant contains triterpenoid saponins possessing oleanolic acid as aglycone, saponins A, B, C and D as major chemical constituents. Other constituents of the plant are ecdysterone, long chain alcohol, saponin 17-penta triacontanol, 27-cyclohexyl heptaeosan-7-ol, 16-hydroxyl 26-methyl heptacosan-2one and 36, 47-dihydroxy hen-pentacontan-4one. It also contains a water soluble base, betaine. Chemical constituents of Achyranthes aspera are Saponins from alcoholic extract of defatted seeds, Oleanic acid from seeds, Saponins A and B, Saponins C and D from unripe fruits, Amino acids, Aldehydes, protein, Iron, Calcium, phosphorous, Achyranthine, N-methyl pyrrolidine –3 carboxylic acid, Water soluble base, betaine, Vitamin-C, Ecdysterone and Inokosterone ecdysterone in callus and tissue culture^14,17. It does not cause any toxic effect if taken in recommended doses. If it is taken in excess doses causes nausea, vomiting and convulsions, diarrhea, spasms in abdomen and dehydration¹⁸.

MODELS USED TO TEST ANTHELMINTIC ACTIVITY:

The mostly used models to test anthelmintic activity of crude drugs are

Pheretima posthuma (earth worm)

Haemonchus contortus (red stomach worm)

Ascaridia galli (parasitic round worm of birds)

Pheritima posthuma:

It is found in the wet soil which is rich in organic matter. They belong to class oligochaeta. They are found in lawns, fields and gardens. They are nocturnal and omnivorous. They are fossorial. The circular and longitudinal muscle helps in locomotion. They are hermaphrodite and protandrous. Their head is made up of prostomium and peristomium. They have great power of regeneration. They show cross fertilization in the ootheca. They have a clitellum which is found from 14-16 segments. Each segment has a curved and chitinous structure known as setae^19,20.

Haemonchus contortus:

Haemonchus contortus, also known as red stomach worm, wire worm or Barber's pole worm, is very common parasite and one the most pathogenic nematode of ruminants. Adult worms are attached to abomasal mucosa and feed on the blood. H. contortus is a member of the superfamily trichostrongyloidea (Strongylida) which contains most of the economically important parasitic nematodes of grazing livestock. This parasite is responsible for anemia, bottle jaw, and death of infected sheep and goats, mainly during summer months in warm, humid climates. The infection, called Haemonchosis, causes large economic losses for farmers around the world, especially for those living in the warmer climates. Anthelmintic are used to combat these, and other, worm infections for a long time, but resistance of the parasites against these chemicals is growing^21,22.

Ascaridia galli:

Ascaridia galli is a parasitic roundworm belonging to the phylum Nematoda. Nematodes of the genus Ascaridia are essentially intestinal parasites of birds. A. galli is the most prevalent and pathogenic species, especially in domestic fowl, Gallus domesticus Linnaeus. It causes ascaridiasis, a disease of poultry due to heavy worm infection, particularly in chicken and turkeys. It inhabits the small intestine, and can be occasionally seen in commercial eggs²³.

In the present study we used Pheretima posthuma –Indian adult earth worm as model to test anthelmintic activity.

MATERIALS AND METHODS:

PLANT MATERIAL:

The plant material (leaves) of Syzygium cumini and Achyranthes aspera were collected and authenticated. These leaves were dried in shade for 15 days and then homogenized to get a coarse powder and passed through 80mesh to give uniform particles.

CHEMICALS AND SOLVENTS:

n-Hexane, Benzene, Chloroform, Methanol, Water, Gum acacia and Sodium chloride. All solvents, chemicals used were of analytical grade procured from local suppliers. Albendazole is obtained from medical store.

PREPARATION OF CRUDE EXTRACTS:

Syzygium cumini extracts:

The leaf powder of Syzygium cumini 150gm is subjected to maceration using different solvents like n-Hexane, Benzene, Chloroform, Methanol. In this process the powder and the solvent are taken in the conical flask and under constant shaking and controlled conditions for 6 days. Then it is filtered by applying vacuum condition and stored. Then the marc is again treated with solvent and shaken for 3 days and again filtered. Now both the combined filtrates were mixed and then concentrated and dried. The dried extracts were stored in refrigerator until used.

Achyranthes aspera extracts:

The powder of the leaves of Achyranthes aspera 60gm which is collected after grinding is extracted with water and methanol solvents using Soxhlet apparatus. It is subjected to reflex heating for 8 hours and it is filtered under vacuum condition at a temperature of 40 C. Then the extract is dried under vacuum conditions to get the dried powder product in a china dish. This is stored in refrigerator until used.

Preparation of test samples:

Samples of the anthelmintic study were prepared by dissolving 2.5gm of crude extract in 25ml of 1% gum acacia prepared in normal saline to obtain a stock solution of 100mg/ml, from this stock solution different working dilutions were prepared.

Preparation of standard samples:

A standard drug, Albendazole of 400mg drug is taken and weighed and then powdered. Now weighing the required quantity of the powder a solution of 50mg/ml solution is prepared in 1% gum acacia prepared in normal saline solution.

Animals:

Indian adult earthworms (Pheretima posthuma) were used to study anthelmintic activity. The earthworms were collected from moist soil and washed with normal saline to remove all fecal matter. The earthworms of 3-5 cm in length and 0.1-0.2 cm in width were used for all experimental protocols. The earthworm resembles both anatomically and physiologically to the intestinal roundworm parasites of human beings, hence can be used to study the anthelmintic activity.

Screening for anthelmintic activity:

Fourteen groups of approximately equal size Indian earth worms consisting of six earth worms in each group were used for the study. Each group was treated with the following solution:

DRUG-I Syzygium cumini leaf extracts:

Group 1(G₁): n-hexane extract (50mg/ml)

Group 2(G₂): n-hexane extract (100mg/ml)

Group 3(G₃): benzene extract (50mg/ml)

Group 4(G₄): benzene extract (100mg/ml)

Group 5(G₅): chloroform extract (50mg/ml)

Group 6(G₆): chloroform extract (100mg/ml)

Group 7(G₇): methanol extract (50mg/ml)

Group 8(G₈): methanol extract (100mg/ml)

DRUG-II: Achyranthes aspera leaves extracts:

Group 9(G₉): alcohol extract (50mg/ml)

Group 10(G₁₀): alcohol extract (100mg/ml)

Group 11(G₁₁): water extract (50mg/ml)

Group 12(G₁₂): water extract (100mg/m)

STANDARD: ALBENDAZOLE:

Group 13(G₁₃): Albendazole solution (50mg/ml)

CONTROL:

Group 14(G₁₄): 1% gum acacia solution prepared in normal saline solution

PROCEDURE:

For the anthelmintic activity of leaf extracts of Syzygium cumini and Achyranthes aspera, Indian adult earthworms (Pheretima posthuma) were used. The animals were divided into six groups containing six earthworms in each group. All the extracts of Syzygium cumini and Achyranthes aspera were dissolved in minimum quantity of solvents respectively and then the volume was adjusted to 25 ml with normal saline solution. All the extracts and the standard drug solution were freshly prepared before starting the experiments. Different extracts and the standard drug solution were poured in different Petri dishes. All the earthworms were washed in normal saline solution before they were introduced into Petri dishes.

Observations were made for time taken to paralyze and time of death of individual worms. Paralysis was said to occur when the worm did not revive even in normal saline and Death was concluded when the worms lost their motility followed with color fading of their body^{(1) (4)}. All the results were expressed as a mean of time of paralysis and time of death of six animals in each group.

RESULTS:

The results were shown as tables as follows.

Syzygium cumini extracts yield:

EXTRACTS	Quantity of Powder (gm)	Weight of Extract (gm)	%YIELD
n-Hexane	100	4.50	3.00
Benzene	100	5.50	3.667
Chloroform	100	6.25	4.167
Methanol	100	5.93	3.95

Achyranthes aspera extracts yield:

EXTRACTS

Quantity of

Powder (gm)

Weight of

Extract (gm)

%YIELD

Alcohol

100

12.03

20.05

Water

100

10.15

16.91

PRELIMINARY PHYTOCHEMICAL INVESTIGATION:

Syzygium cumini extracts results:

	n-Hexane	Benzene	Chloroform	Methanol
Alkaloids	+	+	+	+
Amino acids	-	-	-	+
Anthraquniones	-	-	-	-
Flavanoids	-	-	-	+
Saponins	-	-	-	+
Glycosides	-	-	-	+
Steriodes	-	-	-	+
Tannins	+	+	+	+
Triterpinoids	-	-	-	+

Achyranthes aspera extract results:

	ALCOHOL	WATER
Alkaloids	+	+
Amino acids	-	-
Anthraquniones	-	-
Flavanoids	-	-
Saponins	-	+
Glycosides	+	+
Steriodes	-	-
Tannins	+	+
Triterpinoids	-	-

+ indicates positive results,- indicates negative results

CONCLUSION:

The anthelmintic activity of Syzygium cumini leaves extracts and Achyranthes aspera leaves extracts were carried out.

n-Hexane, Chloroform, Methanol extracts of Syzygium cumini leaves extracts exhibits better anthelmintic activity, when compared to standard Albendazole.

Benzene extract of Syzygium cumini leaves exhibit comparatively less anthelmintic activity to remaining extracts.

Alcohol and water extracts of Achyranthes aspera leaves also exhibit comparative anthelmintic activity to that of standard Albendazole.

Albendazole is benzimidazole derivative. The mechanism of action is blocking glucose uptake in susceptible helminths, thus depleting energy required for their survival²⁴. Tannins produced anthelmintic activity by binding to free protein in the gastrointestinal tract of the host animal or glycoprotein on the cuticle of the parasite. Phenolic compounds (tannins are polyphenolic compounds) by uncoupling oxidative phosphorylation hinder the energy production in helminth parasites^25-28.

Phytochemical analysis of leaves of Syzygium cumini and Achyranthes aspera revealed the presence of tannins as one of the constituent. Anthelmintic property of Syzygium cumini and Achyranthes aspera may due to either or both of above mechanism. The present study concludes that Syzygium cumini and Achyranthes aspera have potential anthelmintic activity. Further studies to determine the mechanism involved and individual constituent responsible for anthelmintic property is to be explored.

ASSESSMENT OF ANTHELMINTIC ACIVITY:

DRUG-I: Results of Syzygium cumini extracts:

Treatment vehicle	Concentration	Time of paralysis (min)	Time of death (min)
G₁	50mg/ml	20.17+0.15	22.16+0.17
G₃	50mg/ml	30.15+0.20	35.00+0.10
G₅	50mg/ml	19.25+0.10	21.30+0.05
G₇	50mg/ml	12.10+0.14	15.04+0.35

Treatment vehicle	Concentration	Time of paralysis (min)	Time of death (min)
G₂	100mg/ml	9.40+0.25	11.10+0.45
G₄	100mg/ml	11.05+0.35	13.16+0.33
G₆	100mg/ml	8.40+0.10	10.00+0.20
G₈	100mg/ml	5.34+0.4	7.50+0.50

DRUG-II: Results of Achyranthes aspera extracts:

Treatment vehicle	Concentration	Time of paralysis (min)	Time of death (min)
G₉	50mg/ml	30.15+0.39	35+0.10
G₁₁	50mg/ml	42+0.17	45+0.15
G₁₀	100mg/ml	15.15+0.20	18.05+0.10
G₁₂	100mg/ml	20+0.15	22.50+0.12

Results of standard and control:

Treatment vehicle	Concentration	Time of paralysis (min)	Time of death (min)
G₁₃	50mg/ml	25.05+0.45	30.00+0.20
G₁₄	Control	No paralysis	Alive

ACKNOWLEDGEMENTS:

The authors wish to thank the institution of KVSR Siddhartha College of Pharmaceutical Sciences, for providing the necessary facilities to carry out this work.

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Received on 07.03.2010

Accepted on 17.02.2010

Research Journal of Pharmacognosy and Phytochemistry. 2(6): Nov. - Dec. 2010, 441-445