Investigation of in-vitro anthelmintic activity of Lindernia madayiparense

 

Umakrithika Chiranjeevi*, Kannan Kamarajan, Prabal Kumar Manna

Department of Pharmacy, Faculty of Engineering and Technology, Annamalai University,

Annamalainagar - 608002, Tamil Nadu, India

 

ABSTRACT:

Helminthiasis causes a large threatening to the public health and lead to the prevalence of malnutrition, anemia, eosinophilia and pneumonia in developing countries. Due to the high cost of currently available anthelmintic drugs and also the gastro-intestinal helminthes becomes resistant to the drugs; the research on herbal remedies as alternative anthelmintics has been increased. The present study was aimed to assess the anthelmintic activity of the plant, Lindernia madayiparense against Indian earthworms, Pheretima posthuma. Different extracts were prepared using decoction and continuous hot extraction methods. Preliminary qualitative analysis was carried out on all the extracts of L. madayiparanse to identify the presence of phytochemical constituents. Among the extracts tested, ethanol extract showed excellent anthelmintic activity as well as higher than other extracts at all the concentrations against the earth worms and well comparable with standard drug, Albendazole. All the extracts tested exhibited a dose dependent paralytic effect and the time of death. From these findings it was suggested that the observed anthelmintic activity might be due to the presence of these secondary metabolites such as alkaloids, flavanoids, terpenoids, tannins and saponins.

 

 

 

 

INTRODUCTION:

Helminths infections, generally known as helminthiasis are among the most invasive infection and a foremost degenerative disease suffering a large proportion of the population globally. Helminthiasis causes a large threatening to the public health and lead to the prevalence of malnutrition, anemia, eosinophilia and pneumonia in developing countries¹. Anthelmintics are drugs that expel parasitic worms (helminths) from the body, by either striking or killing the worms. Due to the high cost of currently available anthelmintic drugs and also the gastro-intestinal helminthes becomes resistant to the drugs; the treatment of helminthes diseases is one of the foremost problems ².

 

Therefore the research on herbal remedies as alternative anthelmintics has been increased. Medicinal plants have served through the era as a constant source of medicaments for the revelation of a various human ailments. As medicinal plants play a rich source of anthelmintic agents, they are used medicinally in different countries for the treatment of helminths infections³.

 

India is one of the countries having rich biodiversity and huge number of herbal plants. Lindernia madayiparense (Family: Linderniaceae) is a plant discovered first from the laterite hills, Madayipara, Kannur District, Northern Kerala, India. The Plant is used by the tribal inhabitants and local villagers for therapeutic purposes since long time^4,5. But there is no scientific evidence for the pharmacological effects of this plant. Therefore, a detailed phytopharmacological study is warranted to explore the major active compounds and related bioactivities of this plant. Hence, the present study was undertaken to assess the anthelmintic activity of the plant, L. madayiparense against Indian earthworms.

 

MATERIALS AND METHODS:

Plant materials:

Wild crafted plant, Linderina madayiparanse was collected during its flowering season in the month of October to December, 2013 in Kannur District, Kerala, India. The plant material was identified and authenticated by botanist Mr. P. Biju, Assistant Professor, Government College, Kasaragod, Kerala, India.

 

Preparation of plant extracts:

Decoction and continuous hot extraction methods were employed to obtain different extracts of L. madayiparanse. In the decoction method, the fresh plant, L. madayiparanse (500 gm) were washed with distilled water and chopped into small pieces as size of 0.5 cm to 1.0 cm and allowed to boil in one liter of distilled water for 20 min from the time the water started to boil or until the original volume was reduced to half. After 20 min, the mixture was allowed to cool and filtered using cheesecloth⁶. The collected decoction was concentrated using rotary vacuum evaporator under reduced pressure to remove the excessive solvent and stored in an air tight container and preserved in 8⁰C until further use. Lyophilizer was used to get an absolute dry decocted extracts⁷ and the percentage yield of the decocted extract was calculated.  In continuous hot extraction method, the whole plant, L. madayiparense was washed thoroughly with distilled water and dried under air-shade. The dried plant materials were pulverized mechanically into coarse powder. The dried coarse plant material (1000gm) has been successively extracted with four different solvents by changing the solvent polarity from non polar to polar solvents such as petroleum ether, ethyl acetate, ethanol and distilled water respectively using Soxhlet apparatus. At the end of each successive extraction, the mixture was collected and concentrated using rotary vacuum evaporator under reduced pressure to remove the excessive solvents⁸. To get an absolute extracts, Lyophiliser was used for water extract and others were freeze dried. All the extracts were stored properly and preserved in 8⁰C until further use⁷ and the percentage yield of all the extracts was calculated. 

 

Preliminary phytochemical studies:

Preliminary qualitative analysis was carried out on all the extracts of L. madayiparanse to identify the presence of phytochemical constituents such as alkaloids, carbohydrates, glycosides, flavonoids, steriods, triterpenoids, phenols, proteins, tannins etc. as per the standard methods⁹. The presence of the secondary metabolites was noted in table 1.

 

Collection of earthworms:

The adult earthworms (Pheretima posthuma) were collected from moist soil and washed with normal saline solution. According to the experimental protocol of this method, the selected worms were 3-5cm in length and 0.1-0.2cm in diameter because of their anatomical and physiological similarities with human intestinal parasitic roundworms¹⁰.

 

Evaluation for anthelmintic activity:

The in vitro anthelmintic activity of L. madayiparense leaf extracts was evaluated using the method developed by Ajaiyeoba et. al., (2001) with minor modifications¹¹. Albendazole was used as the reference standard¹². Albendazole at a concentration of 15 mg/ml and the extracts of L. madayiparense at a concentration of 25 mg/ml, 50 mg/ml and 100 mg/ml were prepared by dissolving in 2% Tween 80 solution, and thus concentrations of extracts and albendazole were used in this study. The selected adult earthworms were divided into eighteen groups, consisting of six worms in each group and tested against 20mL of each solution (distilled water, 2% Tween 80, extracts and albendazole) in the Petridish. Group 1 was treated with distilled water and served as control. Groups 2 was treated with 2% Tween 80 and served as positive control. Group 3 was treated with Albendazole (15 mg/ml) and served as standard. Groups 4 - 18 were treated with all the extracts (25mg/mL, 50mg/mL and 100mg/mL) whereas groups. The spontaneous motility and evoked responses of earthworms in the petridishes were observed. The time taken for paralysis (min) was recorded when the earthworms did not move at all even after exposing to external stimuli such as normal saline, whereas the time taken for death (min) was noted once the worms lost their motility by neither vigorous shaking nor placing in the warm water of 50⁰C, and their body colour became faded. A maximum time period of 120 min was ascertained for the paralyzing as well as death time of earth worms¹³. All the results were tabulated as mean ± standard error of mean (SEM).

 

RESULTS AND DISCUSSION:

The preliminary phytochemical studies done on the extracts revealed that the presence of alkaloids, carbohydrates and glycosides, flavanoids, steroids, coumarins, terpenoids, tannins, phytosterols, saponins and poly phenols as the key phytoconstituents in the decocted, ethyl acetate and ethanol extracts but proteins and aminoacids, fats and oils/ resins are absent in these extracts (Table 1).

 

 

 

Table 1: Preliminary phytochemical analysis of the various extracts of the plant, L. madayiparense

Phytoconstituents	Decocted extract	Pet. ether extract	Ethyl acetate extract	Ethanol extract	Water extract
Alkaloids	+	-	+	+	-
Carbohydrates	+	-	+	+	+
Glycosides	+	-	+	+	-
Flavanoids	+	-	+	+	+
Proteins and Aminoacids	-	-	-	-	-
Steroids	+	-	+	+	+
Coumarins	+	+	+	+	+
Terpenoids	+	-	+	+	-
Tannins	+	-	+	+	+
Phytosterols	+	-	+	+	+
Saponins	+	+	+	+	-
Oils/ Resins	-	+	-	-	-
Poly phenols	+	-	+	+	+
Fats	-	+	-	-	-

 

All the extracts tested exhibited a dose dependent paralytic effect and the time of death (Table 2). Among the extracts tested, ethanol extract showed excellent anthelmintic activity as well as higher than decocted extract followed by ethyl acetate extract at all the concentrations against the earth worms and well comparable with standard drug.

 

Table 2: In vitro anthelmintic activy of extracts of Lindernia madayiparense

 

From these findings it was suggested that the observed anthelmintic activity might be due to the presence of these secondary metabolites in the ethanol, decocted, and ethyl acetate extracts for the treatment of helmintic infections by interrupting the worms’ life cycle and preventing their growth. The different spectrum of the anthelmintic activity of the different extract may be due to the variation in the concentration of these phytoconstituents. Among the secondary metabolites present in the medicinal plants, tannins contribute the most to produce anthelmintic activity against the worms. Few earlier studies supported that the anthelmintic activity of tannins^14,15 due to binding of tannins to free proteins in the gastrointestinal tract of host animal or glycoprotein on the cuticle of the parasite, leading to death^16,17. In addition that several earlier of studies are scientifically reported the responsible of alkaloids¹⁸, terpenoids¹⁹, saponins²⁰ and flavonoids²¹ for the anthelmintic activity against the worms.

CONCLUSION:

The present study could explore a rationale for the anthelmintic activity of the plant, Lindernia madayiparense against Indian earthworms, Pheretima posthuma. These findings evidently suggest that the extracts of L. madayiparense contain promising bioactive compounds which might be responsible anthelmintic efficacy of this plant. The present data would indeed to establish the potency of the plant, L. madayiparense as alternative for the available anthelmintic drugs. Therefore, further research is necessary to isolate and identify the bioactive components and to elucidate its mechanisms of action.

 

ACKNOWLEDGEMENTS:

The authors are grateful to the Management and Department of Pharmacy, Annamalai University for their continuous encouragement and providing necessary facilities. Authors are also thankful to Mr. P. Biju, Assistant Professor, Government College, Kasaragod, Kerala for his support during the collection and identification of the selected plant.

 

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Received on 04.08.2017         Modified on 20.11.2017

Accepted on 15.12.2017      © RJPT All right reserved