Anthelmintic and Anti-Insect Activities of Memecylon umbellatum Burm Root Extracts

SG Killedar* and HN More

Department of Pharmacognosy, Bharati Vidyapeeth College of Pharmacy, Kolhapur, M. S., India.

ABSTRACT:

Different extracts of Memecylon umbellatum Burm root were screened for Anthelmintic and anti-insect activities using Indian earthworms (Pheretima posthuma) and red flour beetle, Tribolium castaneum (Herbst) respectively. The acetone and methanolic extracts exhibited significant (p<0.01) anthelmintic and anti-insect activities compared with the control and other extracts. Albendazole and Piperazine citrate were used as standards for anthelmintic activity while Neem Extract, Celphos (Aluminium phosphide) and citronellal for anti-insect activity. Chloroform, ethyl acetate and aqueous extracts showed weak activities. Acetone root extract showed most promising Anthelmintic activity at 20mg/mi compare to albendazole and insecticidal activity at 10mg concentration comparable to standard celphos (5%). Further investigations are however necessary to explore mechanism(s) of action involved in these pharmacological activities and constituents responsible for both the activities.

KEY WORDS: Memecylon umbellatum, Anthelmintic, anti-insect, Pheretima posthuma, Tribolium castaneum.

INTRODUCTION:

Traditional medicines are still very commonly used in India for anthelmintic and anti insect purposes. Chemicals such as pesticides in crop protection and insecticides for control of insects in stored grains have been utilized since long time. The indiscriminate use of chemical pesticides has given rise to many serious problems, including toxic residue, increasing cost of application, environment pollution, and hazards from handling. The development of cross and multi-resistant strains in many important species, resulting from the continuous use of chemical insecticides has been reported from all over the world.^{1, 2} Therefore, the development of bioinsecticides has been focused as a viable pest control strategy in recent years which offer an ecological, safety and economical viable alternative to synthetic one in managing pests below its injury level.^3,4,5 Moreover they are readily biodegradable and have more limited use than that of the potent and persistent synthetic pesticides.⁶ Hence the demand for search of more acceptable, effective natural plant products has been increased. Taking into account that the most important anthelmintic and insect repellant prototypes (e.g. Male fern, Pyrethrins, Neem) were originally derived from the plant sources, the study of plant species traditionally used as insecticides still be seen as a fruitful research strategy in the search of new drugs.⁷ In our way to investigate the local medicinal plants for their potential therapeutic uses, present study was aimed to screen the anthelmintic and anti-insect activity of different extracts of Memecylon umbellatum root. The plant (Family: Melastomataceae) is small evergreen shrub or tree having young terete branches and bears numerous umbellate cymes. The plant is known as “Anjani” in Sanskrit and “Ironwood tree” in English. Plants are distributed mostly in coastal regions of the Deccan peninsula, the eastern and southern part of India all along Western Ghats and in the Andaman islands.^{8, 9} The leaves have been reported to possess astringent properties and are given to treat leucorrhoea and gonorrhoea. Lotion prepared from leaves is used to treat eye troubles. The decoction of the root is used in the treatment of excessive menstrual discharge.¹⁰ Leaves are also reported to possess antiviral activity.¹¹ Bark is used in the treatment of bruises externally as lepa along with coconut kernels (Dymock).

The literature surveys reveal that leaves and roots of Memecylon umbellatum have been investigated for its hypoglycemic activity using alloxan induced hyperglycemic wistar albino rats.^12,13 Wound healing activity of ethanolic extract of leaves has also been reported.¹⁴ Plant contains wide variety of phytoconstituents such as umbellactone, β amyrine, oleanolic acid, ursolic acid, sitosterol and organic acids. ^{15, 16}

MATERIALS AND METHODS:

Plant Material:

The roots of Memecylon umbellatum were collected in the month of March-April from Gaganbavada hilly region of Kolhapur district, Maharashtra, India. The plant material was taxonomically indentified by Dr. S. R. Yadav, Department of Botany, Shivaji University, Kolhapur, India. The voucher herbarium (SGK-3) has been deposited in the department of Pharmacognosy, Bharati Vidyapeeth College of Pharmacy, Kolhapur.

Drugs and Chemicals:

Albendazole (Cipla), Piperazine citrate (GSK), Neem extract (0.15% azadirachtin- NICO ORGO Manures, Dakor), Citronella oil and Celphos (56% Aluminium phosphide-Excel Crop Care Ltd, Mumbai) were purchased. Solvents and chemicals used for experimental work were of AR grade (Loba and Merck).

Test organism:

Indian adult earthworms Pheretima posthuma were obtained from marshy area near by Shivaji University and insect red floor beetle Tribolium castaneum from local grain market and identified by Entamology Dept. Agriculture College, Kolhapur.

Preparation of extracts:^{17, 18}

The roots were thoroughly washed and dried under shade. Coarsely powdered root (1kg) was subjected for extraction in soxhlet apparatus using different solvents with increasing polarity for 18 h just below the boiling point of individual solvents. Each time the powder was dried overnight in oven at 45⁰C. The aqueous extract (Chloroform water) was prepared by maceration using orbital shaker (Remi India) with 60 rpm at room temperature for 48h. All the extracts were characterised and concentrated using rotary vacuum evaporator (Buchi) and dried by vacuum drier (Desiccator), preserved in refrigerator, and were used for testing the activities. The extracts were short named such as Petroleum ether (40-60⁰C) [PEMuR], Chloroform [CHMuR], Ethyl acetate [EAMuR], Acetone [AMuR], Methanol [MMuR] and Chloroform water [CWMuR].

Phytochemical screening:¹⁹

All the extracts were screened for Phytochemical screening using standard reagents and results are given in table-1

Anthelmintic activity:²⁰

The anthelmintic activity was evaluated on adult Indian earthworms, Pheretima posthuma due to its anatomical and physiological resemblance with the intestinal roundworm parasites of human beings^{21, 22}. Twenty seven groups of approximately equal sized (7±1cm) Indian earthworms consisting six earthworms in each group were selected. All selected worms were washed thoroughly first with running water and then with normal saline and released six worms in to 10ml of desired formulation in each Petri dish at room temperature. The groups were tested as follows,

I : Control- 5% DMF solution in normal saline.

II : Piperazine Citrate (15mg/ml in 5% DMF in normal saline).

III : Albendazole (20 mg/ml in 5% DMF in normal saline).

IV-VII : PEMuR ( 5-20 mg/ml in 5% DMF in normal saline)

VIII-XI : CHMuR (5-20 mg/ml in 5% DMF in normal saline).

XII-XV : EAMuR (5-20 mg/ml in 5% DMF in normal saline).

XVI-IXX : AMuR (5-20 mg/ml in 5% DMF in normal saline).

XX-XIII : MMuR (5-20 mg/ml in 5% DMF in normal saline).

XXIV-XXVII : CWMuR (5-20 mg/ml in 5% DMF in normal saline).

The time taken to complete paralysis (stoppage of movement by pin test) and death (fading of color or no movement in hot water at 50⁰C) were recorded. The mean paralysis and mean lethal time for each group were reported in table-2.

Anti-insect activity:

A) Insect repellent activity:

Insect repellent activity was evaluated on adult red flour beetle Tribolium castaneum. The repellency test was carried out as per reported method with some modifications.²³ Adults of the insects were collected from the local grain market and authenticated. Insects were released in glass jar (10 x 15 cm) containing wheat flour admixed with 5% yeast powder. Before culturing, the flour was kept at 60±1⁰C in oven for two hours to eliminate contamination of other organisms. The culture jars were placed in incubator maintained at 30 ± 1⁰C and 70 ± 1% relative humidity after seven days of oviposition period, the adults were removed and the eggs were allowed to develop to the pupae stage. The pupae were sifted from the flour with a 40 mesh sieve and put into small glass jar (5x10 cm) containing wheat flour and yeast. From these jars, the adults of F₁ generation (2-3 weeks) were obtained for experiment. The filter paper disc (Whatman no.40, diameter 8cm) were prepared and cut into two equal halves. On one of the half 1ml of 5, 10, 15 and 20% solution of each extracts were added and on another half 1ml solvent was added as a control. These halves were air –dried for 10-30min. and each treated half disc was then attached to control half disc lengthwise, edge to edge with adhesive tape and placed in a Petri dish (dia.8 cm). The inner edge of the Petri dish was smeared with glue stick in order to prevent escaping of insects. The orientation of the seam was changed in replicates to avoid the effect of any external directional stimulus affecting the distribution of the test insects. Thirty adult insects were released in the middle of each filter paper circle and a plastic cover with some holes was placed on the Petri dish. The distribution of insect in the two halves is observed after 1h and then hourly intervals for 5h. Three replicates were maintained for each treatment. The average of the count was converted to percentage repellency (PR) using Talukder and howse formula²⁴. The results are given in table-3.

Table 1: Phytochemical screening of Memecylon umbellatum root extracts

Extract

Sugars

Alk.

Tannins

Glycosides

Steroids

Proteins/ Amino acids

Organic acid

R

NR

HT

CT

a

c

s

f

co

ST

TT

C

O

T

PEMuR

-

+

-

-

-

-

-

-

-

-

+

+

-

-

-

-

CHMuR

+

+

-

-

-

-

-

-

-

-

+

+

-

-

-

-

EAMuR

+

+

-

+

+

-

-

-

-

-

-

+

+

-

-

-

AMuR

+

+

-

+

-

-

+

+

+

+

+

+

+

+

+

-

MMuR

+

+

-

+

+

-

+

+

+

+

-

+

+

-

+

+

CHMuR

+

+

-

+

-

-

-

-

-

-

-

-

-

-

+

+

Alk.- Alkaloids, R-Reducing sugar, NR-Non Reducing sugar, HT-Hydrolysable Tannins, CT-Condensed Tannins, a-anthracene, c-cardiac, s-saponin, f-flavanoidal, co- coumarin, ST-Steroids, TT-Triterpenoids, C-Citric, O-Oxalic, T-Tartaric acid

Table 2: Anthelmintic activity of different extracts of Memecylon umbellatum root

Group	Treatment	Concentration used (mg/ml)	Time taken for paralysis* (min)	Time taken for death *(min)
I	Control 5% DMF	--	--	--
II	Piperazine	15.0	18.50 ± 0.35	54.25 ± 0.38
III	Albendazole	20.0	17.28 ± 0.70	34.42 ± 0.72
IV	PEMuR	5.0	45.38 ± 0.66	--
V		10.0	37.48 ± 0.34	--
VI		15.0	30.56 ± 0.76	105.43 ± 0.42
VII		20.0	24.32± 0.27	80.58 ± 0.18
VIII	CHMuR	5.0	47.28 ± 0.64	--
IX		10.0	39.58 ± 0.24	--
X		15.0	33.51 ± 0.63	110.73 ± 0.38
XI		20.0	28.17± 0.52	84.26 ± 0.18
XII	EAMuR	5.0	51.32 ± 0.68	--
XIII		10.0	47.45 ± 0.36	109.08 ± 0.32
XIV		15.0	38.36 ± 0.56	105.43 ± 0.42
XV		20.0	35.32 ± 0.22	80.58 ± 0.18
XVI	AMuR	5.0	30. 50 ± 0.39	65.45 ± 0.31
XVII		10.0	27.53 ±0.37	53.17 ± 0.42
XVIII		15.0	24.23 ±0.13	41.21 ± 0.18
XIX		20.0	20.28 ± 0.20**	35.22 ± 0.24**
XX	MMuR	5.0	37.25 ± 0.16	78.24 ± 0.32
XXI		10.0	33.48 ± 0.40	69.12 ± 0.16
XXII		15.0	31.46 ± 0.53	60.25 ± 0.40
XXIII		20.0	28.35 ± 0.36	51.33 ± 0.32
XXIV	CWMuR	5.0	--	--
XXV		10.0	78.23 ± 0.43	--
XXVI		15.0	69.45 ± 0.28	108.11 ± 0.50
XXVII		20.0	58.34 ± 0.17	93.47 ± 0.16

* Average of six determinations ± SEM, -- No results even after 2h, ** significant at p<0.01 compare to Albendazole.

Table No. 3: Insect repellent activity of different extracts of Memecylon umbellatum root

Extract	Concentration in mg (%)	Avg. repellency* after					% Repellency over 5h duration (mean ±SD)
		1h	2h	3h	4h	5h
PEMuR	5.0	3	4	5	5	6	20.00
	10.0	4	4	5	6	7	23.33
	15.0	6	6	7	8	8	26.66
	20.0	7	7	8	9	9	30.00
CHMuR	5.0	1	1	2	2	2	06.66
	10.0	1	2	2	2	2	06.66
	15.0	2	3	3	3	4	13.33
	20.0	3	3	4	4	5	16.66
EAMuR	5.0	2	2	3	3	4	13.33
	10.0	3	3	4	5	5	16.66
	15.0	5	5	6	7	7	23.33
	20.0	7	8	8	9	9	30.00
AMuR	5.0	11	13	14	14	17	56.66
	10.0	16	18	20	21	24	80.00
	15.0	20	24	25	27	28	93.33**
	20.0	25	28	30	30	30	100.00**
MMuR	5.0	7	7	8	8	9	30.00
	10.0	10	12	13	15	16	53.33
	15.0	14	16	18	19	20	66.66
	20.0	15	17	20	23	24	80.00
CWMuR	5.0	2	2	3	3	3	10.00
	10.0	3	3	4	4	5	16.33
	15.0	4	5	6	6	6	20.00
	20.0	5	7	9	9	9	30.00
Citronella oil	5.0% v/v	13	14	14	15	15	50.00
	7.0% v/v	17	18	18	19	19	63.33
	10.0% v/v	22	26	27	28	28	93.33

* Average of three replicates and values for no. of insects repelled out of thirty. ** p<0.01 compare to standard citronella oil at (10%)

B) Insecticidal activity:²⁵

Different strengths of individual extracts (2.5, 5, 7.5, 10, 12.5, 15%) were prepared in 5ml of acetone by sonication. The film of different working concentrations (5ml) of acetone solution was prepared in Petri dishes (10cm diameter) and dried for 1h. Some wheat flour was placed on dish to provide food for insects. Thirty adult insects of red flour beetle were put in to each Petri dish covered by perforated plastic disc and maintained at 27⁰C. Mortality count was recorded after 24 h. Each set was run in triplicates. The experiment was repeated with promising extracts using suitable dilutions along with standards. Results are as given in table-4.

Table 4: Insecticidal activity of different extracts of Memecylon umbellatum root

Sample/ drug	Concentration (mg)	Average mortality count	% Mortality* Mean ± SD
PEMuR	2.5	12	40.00 ± 0.14
	5.0	13	43.33 ± 0.18
	7.5	15	50.00 ± 0.23
	10.0	16	53.33 ± 0.25
	12.5	17	56.66 ± 0.32
	15.0	17	56.66 ± 0.21
EAMuR	2.5	05	16.66 ± 0.26
	5.0	06	20.00 ± 0.28
	7.5	06	20.00 ± 0.31
	10.0	07	23.33 ± 0.22
	12.5	07	23.33 ± 0.32
	15.0	07	23.33 ± 0.43
AmuR	2.5	25	83.3 ± 0.81
	5.0	27	90.0 ± 0.81
	7.5	30	100 ± 0.49
	10.0	30	100 ± 0.49
	12.5	30	NT
	15.0	00	NT
MmuR	2.5	15	50.00 ± 0.23
	5.0	16	53.33 ± 0.25
	7.5	17	56.66 ± 0.32
	10.0	19	63.33 ± 0.21
	12.5	20	66.66 ± 0.22
	15.0	22	73.33 ± 0.14
Neem oil	0.15% v/v	22	73.33 ± 0.14
Celphos	5% w/v	29	96.66 ± 0.18
Control	Plain acetone	--	--

* Average of three replicates and values for no. of insects killed out of thirty, NT-not tested

Statistical Analysis:

Values are expressed as mean ± SEM from 6 earthworms. Statistical differences in mean were analyzed using one way ANOVA followed by Dunnett’s test. p<0.01 was considered significant.

RESULT AND DISCUSSION:

Insect control properties of plant essential oils, and their constituents and derivatives like Pongamia glabra, Cymbopogan citratus Allium sativa, Chrysanthemum cinnararifolium etc. have been described previously^26,27. Several polyphenolic compounds have been already reported to have insecticidal action^28-31. Numerous plant species have been reported to have pest control properties Azadirachta indica A. juss and bakain (Meliaceae) are the most entomological perspective being widely used for managing large number of pests³². The use of plant products as insecticides is gaining importance in recent years in view of the environmental and health hazards posed by synthetic insecticides. It is reported that the methanolic extract of Aedes oxyphylla possesses insecticidal action against larvae of D. melanogaster³³. The chloroform extract of Piper guanacastensis was also reported to have insecticidal action against A. atropalpus mosquito larvae³⁴. The results of the present study indicated that acetone and methanolic root extracts of Memecylon umbellatum exhibited strong Anthelmintic and anti-insect activity compare to control and other extracts. Acetone root extract showed significant Anthelmintic (p<0.01) and anti-insect activity (20mg/ml) compare to standards used. Chloroform, aqueous, petroleum ether and ethyl acetate extracts have showed very weak activities at all tested concentrations compare to standards. Study also reveals that both the activities are concentration dependent.

CONCLUSION:

As per the literature mostly polyphenolic compounds, triterpens and steroids are responsible for Anthelmintic and anti-insect activities. AMuR and MMuR extracts showed the presence of these constituents and responsible for their strong and significant activities. Further investigation is necessary for actual lead identification and mechanism of action involved.

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Received on 12.12.2009

Accepted on 20.01.2010

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Research J. Pharmacology and Pharmacodynamics 2(1): Jan. –Feb. 2010: 52-56