Studies on Antibacterial, Anthelmintic and Larvicidal Efficacy of Pothos scandens L

KS Vinayaka^1*, TR Prashith Kekuda², N Rajkumar¹, Chandrashekar MB¹, Shivakumar Banakar³ and Shruti V Hegde¹

¹Dept. of Studies and Research in Applied Botany, Jnanasahyadri, Shankaraghatta-577451, Karnataka

²S.R.N.M.N College of Applied Sciences, NES Campus, Balraj Urs Road, Shivamogga-577201, Karnataka

³Dept. of Studies and Research in Microbiology, Jnanasahyadri, Shankaraghatta-577451, Karnataka

*Corresponding Author E-mail: ks.vinayaka@rediffmail.com

ABSTRACT

The present study describes the phytochemical, antibacterial, anthelmintic and larvicidal activity of extracts of a traditionally used medicinal plant of Western Ghats Pothos scandens L. The powdered plant material was subjected to soxhlet extraction using methanol solvent. The extracts were tested for the presence of various phytoconstituents. Antibacterial activity by Agar well diffusion method. Wormicidal activity was tested using adult Indian earthworm model by determining paralysis and death of worms in the presence of extracts. The larvicidal activity in terms of Percentage larval mortality was determined using second instar larvae of Aedes aegypti. The preliminary phytochemical analysis showed the presence of terpenoids, alkaloids, steroids, and saponins. The methanol extract showed antibacterial activity against gram positive and gram negative bacteria. The minimum inhibitory concentration of the extract was determined for each of the test bacteria. Gram positive bacteria were inhibited at low concentrations than Gram negative bacteria. The activity may be attributed to the presence of various phytochemicals present in them. Further, the extracts could be used against infections by bacteria, mosquito vectors and the parasitic worms. Further experiments in animal models could possibly reveal the in vivo efficacy of the extract.

KEYWORDS: Pothos scandens, Agar well diffusion, Larvicidal activity, Anthelmintic activity

INTRODUCTION:

Plants produce a diverse range of bioactive molecules, making them rich source of different types of medicines. The medicinal value of plants lies in some chemical substances that produce a definite physiological action on the human body. The most important of these bioactive constituents of plants are alkaloids, tannins, flavonoids, and phenolic compounds. Phytomedicines derived from plants have shown great promise in the treatment of various diseases including viral infections. Single and poly herbal preparations have been used throughout history for the treatment of various types of illness ¹. Mosquitoes are the most important single group of insects acting as vector for many tropical and subtropical diseases such as dengue fever, yellow fever, malaria, filariasis, Japanese encephalitis and others ². The approach to combat these diseases largely relied on interruption of the disease transmission cycle by either targeting the mosquito larvae through spraying of stagnant water breeding sites or by killing the adult mosquitoes using insecticides ³.

Parasitic helminthes affect human being and animals by causing considerable hardship and stunted growth. Most diseases caused by helminthes are of a chronic and debilitating in nature ⁴. Pothos scandens belongs to the family Araceae. It is locally known as Akkigida and is a climbing shrub with aerial roots, leaves obliquely linear to ovate, flower hermaphrodite, spadix yellow, fruits are berries. The leaves are traditionally used as fodder, increases milk in cows, leaf powder is used for small pox, and root is bruised and fried in oil for application on abscesses ⁵. Literatures on biological activity of the plant are quite lacking. Knowing the significance of the plant in traditional medicine, the present study has been carried out to find out the efficacy of methanol extract of Pothos scandens L, against bacteria, worms and larvae.

Pothos scandens L.

MATERIALS AND METHODS:

Collection and identification of plant materials:

Pothos scandns L was collected in the evergreen forests of Western Ghats of Karnataka. The plant was authenticated in Dept. of Studies and Research in Applied Botany, Jnanasahyadri, Shankaraghatta and voucher specimen (KU/AB/KSV/315) was deposited in the department for future reference.

Table-1: Phytochemical constituents detected in methanol extract of P. scandens

Phytochemical group	methanol extract
Tannins	-
Terpenoid	+
Alkaloid	+
Steroid	+
Saponins	+
Flavonoids	-

The result of antibacterial activity of solvent extract is shown in Table-2. Results were recorded as presence or absence of zones of inhibition around the well. The inhibitory zone around the well indicated the absence of bacterial growth and it as reported as positive and absence of zone as negative ¹⁴. It was found that the methanol extract of P. scandens and the standard antibiotic Chloramphenicol have shown inhibition of all tested bacteria. The control (10% DMSO) did not reveal inhibition of any of the tested bacteria.

Table-2: Antibacterial activity of methanol extract of P. scandens

Test bacteria	Methanol extract	Standard	Control
E. coli	+	+	-
S. aureus	+	+	-
K. pneumoniae	+	+	-
P. aeruginosa	+	+	-
C. perfringens	+	+	-
S. typhimurium	+	+	-

‘+’ Inhibition; ‘-’ No inhibition

The results of MIC of extracts are shown in Table-3. From the table it is clear that the methanol extract was effective in inhibiting Gram positive bacteria in low concentration than Gram negative bacteria. The methanol extract at concentrations 250μg/ml and 200μg/ml was effective in inhibiting S. aureus and C. perfringens respectively. Gram negative bacteria were inhibited at concentration higher than that of Gram positive bacteria. Among Gram negative bacteria tested, K. pneumoniae revealed MIC of 400μg/ml followed by E. coli (450μg/ml), S. typhimurium (450μg/ml) and P. aeruginosa (500μg/ml). Among Gram negative bacteria, P. aeruginosa has shown susceptibility at higher concentration than others.

Table-3: MIC of methanol extract of P. scandens

Test bacteria	MIC (μg/ml)
E. coli	450
S. aureus	250
K. pneumoniae	400
P. aeruginosa	500
C. perfringens	200
S. typhimurium	450

The methanolic extract exhibited dose dependent activity against larvae of Aedes aegypti (Table-4). At concentration of 1, 2.5 and 5mg/ml, the larval mortality percentage was found to be 60%, 85% and 100% respectively. In control, no mortality of larvae was observed. Earlier studies observed that phytochemicals have major role in mosquito control programme ^{15, 16}. It is observed that the carbohydrates, saponins, phytosterols, phenols, flavonoids and tannins in the plant extract are having mosquito larvicidal activity ¹¹. It is reported the use of commercial saponin from Quillaja saponaria bark as a natural larvicidal against Aedes aegypti and Culex pipens ¹⁷. Cardiac glycoside was found to have an acaricidal effect against larva and adult stages of the camel tick ¹⁸.

Table-4: Larvicidal efficacy of methanol extract of P. scandens

Treatment	Concentration (in mg/ml)	Number of larvae dead	% larval mortality
*Methanol extract*	1	12/20	60
	2.5	17/20	85
	5	20/20	100

The anthelmintic activity of different concentrations of standard drug and P. scandens is shown in table- 5. The results revealed dose dependent activity. The mean paralysis and death time in 1%, 3% and 5% Piperazine citrate was found to be 78, 23 and 16 minutes and 104, 43 and 28 minutes respectively. The average paralysis and death time in 1%, 3% and 5% of P. scandens was found to be 61, 46 and 37 minutes and 78, 59 and 47 minutes respectively. From the results, it is clear that P. scandens was found to be more effective in killing worms at 1% concentration when compared to standard drug Piperazine citrate. The origin of many effective drugs is found in the traditional medicine practices and in view of this several workers have undertaken studies pertaining to testing of folklore medicinal plants for their proclaimed anthelmantic activity ¹³.

Extraction of plant materials using solvents:

The plant materials were washed thoroughly 2-3 times with running tap water and once with sterile water, shade dried, powdered and used for extraction. The powdered plant material was extracted with methanol. A known amount of powdered material (500gm) was subjected to soxhlet extraction and exhaustively extracted with solvent for about 48 hours. The extracts were filtered and concentrated in vacuum under reduced pressure using rotary flash evaporator and dried in the desiccator ⁶.Methanolic extract of the plant was subjected to preliminary phytochemical screening to screen the presence of various secondary metabolites in the solvent extracts ⁷.

Preliminary screening of solvent extracts for antibacterial activity:

Six species of bacteria belonging to Gram positive (Staphylococcus aureus MTCC-902 and Clostridium perfringens MTCC-450) and Gram negative (Escherichia coli MTCC-405, Klebsiella pneumoniae MTCC-432, Salmonella typhimurium MTCC-1252 and Pseudomonas aeruginosa MTCC-1934) were used in the antibacterial study. The bacteria were procured from Institute of Microbial Technology, Chandigarh. The bacteria were screened for their sensitivity towards the plant extracts by Agar well diffusion method ⁸. In this method, 24 hours old Muller-Hinton broth cultures of test bacteria were swabbed uniformly on solidified sterile Muller-Hinton agar plates using sterile cotton swab. Then, aseptically wells of 6mm diameter were bored in the inoculated plates with the help of gel puncher and the extract (10mg/ml of DMSO), Standard (Chloramphenicol, 1mg/ml) and Control (10% DMSO) were added into the respectively labeled wells. The plates were incubated at 37^oC for 24 hours in upright position and the zone of inhibition was recorded. The experiment was carried in triplicates to get average reading.

Determination of Minimum inhibitory concentration (MIC):

MIC was determined by micro-dilution method using serially diluted (2 folds) extracts (0 to 2000µg/ml in nutrient broth tubes. Specifically 0.1 ml of standardized inoculums (10⁷cfu/ml) was added in each tube. The tubes were incubated aerobically at 37^oC for 18-24 h. Two control tubes were maintained for each test batch. The lowest concentration (highest dilution) of the extract that produced no visible growth (no turbidity) when compared with the control tubes were regarded as MIC ^9,
10.

Screening of methanolic extracts for Larvicidal activity:

Larvae of Aedes aegypti mosquito were collected from water stagnate area and identified in the Dept. of Entomology, UAS, Shivamogga, Karnataka, India. The larvae were maintained under suitable temperature and humidity. Different concentrations of methanolic extracts (1, 2.5 and 5mg/ml) were prepared in 10% DMSO and added to sterile labeled beakers containing about 100ml of water. Twenty larvae were placed in each of the beakers containing extracts. A control was kept containing DMSO. After adding the larvae, the beakers were kept in the growth room maintained at room temperature. The larvicidal effect of extracts was determined by counting the number of dead larvae after 24 hours. Dead larvae were identified when they failed to move after probing with a needle in siphon or cervical region. Each test was repeated thrice; the percentage of larval mortality was determined ¹¹.

Screening of methanolic extracts for wormicidal activity:

The assay was performed on adult Indian earthworm due to its anatomical and physiological resemblance with the intestinal roundworm parasite of human beings. Adult Indian earthworms (Pheretima pasthuma) collected from the local earthworm breeder in the outskirts of Shivamogga city were used for the Anthelmintic study. Equal sized (8±1 cm) worms were selected for the study. The worms were washed with normal saline to remove all the extraneous matter. Different concentrations of standard drug (Piperazine citrate) and methanolic extracts namely 1.0, 3.0 and 5.0mg/ml were prepared in normal saline (0.85%) were prepared in normal saline and poured into respective labeled Petri plates (50 ml in each plate). Six worms of equal size (or nearly equal) were introduced into each of the plates. Observations were made for the time taken to paralysis and death of individual worm. Paralysis was said to occur when the worms were not able to move even in normal saline. Death was concluded when the worms lost their motility followed with fading away of their body colors¹².Death was also confirmed by dipping the worms in slightly warm water. The mortality of parasite was assumed to have occurred when all signs of movement had ceased ¹³.

RESULTS AND DISCUSSION:

The presence of various secondary metabolites in the methanol extract of P. scandens is given in Table-1. The phytoconstituents namely terpenoids, alkaloids, steroids and saponins were found to be present in methanol extract. The tannins and flavonoids were not detected in the extract.

CONCLUSION:

The plant selected for this study has shown a good activity against the tested bacteria. The extracts could be used to treat infections caused by these bacteria. The presence of various phytoconstituents in the extracts highlights the antibacterial efficacy of the plants. The extracts could be used in the treatment of several types of infections such as nosocomial infections, enteric infections, urinary tract infections, food poisoning, wound infections etc. Plant extracts have been used in the control of mosquito borne diseases as the chemical agents have caused some ill effects and also the mosquitoes developed resistance against them. The results of the present study are in justification of this and the extracts of plants selected in this study could be used in control of arboviral infections transmitted by Aedes aegypti. The results of the study have justified the traditional use of the plants in curing diseases. Further studies in animal models have to be carried out.

ACKNOWLEDGEMENT:

The authors express their sincere thanks to Principal, S.R.N.M.N College of Applied Sciences, Shivamogga and Chairman, Department of Applied Botany, Kuvempu University, Shankaraghatta for providing all facilities and moral support to conduct the work.

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Received on 30.08.2009 Modified on 15.10.2009

Research J. Pharm. and Tech.2 (4): Oct.-Dec. 2009; Page 850-853

Compound	Concentration	No. of worms	Mean paralysis time in min	Mean death time in min
Piperazine citrate	1	6	78	104
	3	6	23	43
	5	6	16	28
*P. scandens*	1	6	61	78
	3	6	46	59
	5	6	37	48