In-Vitro Anthelmintic Activity of Thespesia populnea Linn Roots

 

SM Shaheedha*1, R Uma2, Ramya K, CH Phanindra and                 MD Dhanaraju1

 

1Research Lab, GIET School of Pharmacy, NH 5, Chaitanya Nagar, Rajahmundry, India-533294

2Dept. of Chemistry, Pachaiyappa’s College, Chennai -600 030, India.

 

ABSTRACT:

The aim of the proposed study was to evaluate anthelmintic potential of Thespesia populnea root which was extracted with petroleum ether (60°-80°C), chloroform, ethyl acetate and methanol. The extracts were prepared in the percentage of 0.1%, 0.2% and 0.5%. These fractions were tested in the bioassay, which involved the determination of time taken for paralysis and death on the Indian helminthes Pheritima posthuma and Ascaris lumbricoides.  Piperazine citrate was included as reference standard and 10% DMSO in normal saline as vehicle. Petroleum ether extract (0.5%) showed significant activity against the Indian varieties Pheritima posthuma, and Ascaris lumbricoides. The time taken for paralysis and death was found to be 9.25 min and 15.8min against Pheritima posthuma and 4.50 min and 9.25 min against Ascaris lumbricoides respectively. The results proved that the extracts were significant at higher concentration (0.5% pet ether) and had almost the same activity as that of the standard Piperazine citrate. The traditional use of the root of Thespesia populnea as an anthelmintic has been confirmed.

 

KEYWORDS: Anthelmintic activity, Thespesia populnea, Pheritima posthuma, Ascaris lumbricoides, Piperazine citrate. 

 

INTRODUCTION:

Thespesia populnea Linn belonging to the family Malvaceae 1 popularly known as Indian tulip tree. It is a compact quick growing evergreen tree distributed mainly along the coastal regions throughout India. It thrives in moist and warm situations, but can withstand temperature as low as -4° C. The presence of gossypol and beta sitosterol has been reported in the roots. Also lupeol, lupenone, thespesone, thespone, kaempferol, quercetin, rutin, rutinoside, herbacetin and populnin have been reported .2  The plant has been claimed to be used as astringent, depurative, anti-inflammatory, haemostatic, vulnerary, alterant, antidiarrhoeal and in skin diseases3.  The root has been reported to be toxic against micrococcus pyogenes and Escherichia coli and has been used in dysentery, cholera, hemorrhoids and in dermatitis like scabies and psoriasis4 .The present study was to evaluate the invitro anthelmintic activity of crude extracts of Thespesia populnea against Pheritima posthuma   and   Ascaris lumbricoides.

 

MATERIALS AND METHODS:

Plant material:

Fresh roots of Thespesia populnea were collected from the rural parts of Chennai and were authenticated by the taxonomist of Captain Srinivasa Murti Drug Research Institute for Ayurveda, Chennai. After authentication the roots were washed, shade dried until a constant weight was obtained and then milled to coarse powder by a mechanical grinder.

 

Worms collection:

Indian helminthes Pheritima posthuma and Ascaris lumbricoides were collected from the department of soil biology, New College, Chennai.

 

Table 1: Time taken for paralysis and death in mins Species: Pheritima posthuma

Treatment

0.1%

0.2%

0.5%

Paralysis

Death

Paralysis

Death

Paralysis

Death

Control

-

-

-

-

-

-

Standard

38.0±1.78

66.2±3.84

15.5±0.86

18.8±1.11

11.2±2.78

16.8±0.75

Pet ether ext*

35.5±2.10

59.0±3.69

12.5±0.28

14.5±0.28

9.25±3.27

15.8±0.25

Chloroform ext

60.0±2.43

80.5±2.32

37.5±3.62

58.5±1.55

39.5±2.50

50.2±2.13

Ethyl acetate ext

70.2±0.85

91.0±2.27

52.2±1.93

89.5±4.99

52.8±0.25

51.5±2.32

Methanol ext

70.2±3.01

87.0±1.29

58.0±0.00

77.5±1.85

38.0±0.00

61.0±1.47

* Significance P< 0.05;  Results are expressed as mean ± SEM 

 

Table 2: Species: Ascaris lumbricoides

Treatment

0.1%

0.2%

0.5%

Paralysis

Death

Paralysis

Death

Paralysis

Death

Control

-

-

-

-

-

-

Standard

14.2±1.65

57.8±1.93

12.5±0.64

37.8±2.55

7.0±1.22

11.8±1.18

Pet ether ext*

16.8±1.18

54.0±1.47

11.5±0.64

33.0±2.04

4.50±0.64

9.25±0.63

Chloroform ext

62.5±1.89

85.8±2.01

43.0±1.68

64.8±1.37

22.8±1.88

51.8±0.85

Ethyl acetate ext

62.2±1.11

83.0±1.53

42.5±1.50

73.8±1.78

44.8±0.63

70.5±0.64

Methanol ext

56.5±3.30

87.0±0.64

42.2±0.47

75.8±1.60

60±0.402

61.2±0.63

*Significance P<0.05; Results are expressed as mean± SEM

 

 

Preparation of extract:

The powdered plant material was extracted by successive solvent extraction using soxhlet apparatus with petroleum ether (60°-80°C), chloroform, ethyl acetate and methanol. The solvent was then removed under reduced pressure.

 

Preparation of test sample:

0.1%, 0.2% and 0.5% dilutions of petroleum ether, chloroform, ethyl acetate and methanolic extracts were prepared with 10 % DMSO in normal saline. Piperazine citrate was diluted with normal saline to 0.1%, 0.2% and 0.5% which served as standard and 10 % DMSO as control. All the dilutions were poured into Petri dishes for the assessment of in vitro anthelmintic activity.

 

Anthelmintic assay:

The anthelmintic assay was carried as per the method of Ajaiyeoba E.O.etal.5 the assay was performed on adult Indian helminthes Pheritima posthuma and Ascaris lumbricoides due to its physiological resemblance with the intestinal round worm parasite of human beings6-9. Because of easy availability, earth worms have been used widely for initial evaluation of anthelmintic compounds in vitro10-13. Sixteen groups each consisting of six helminthes of approximately equal size were placed into 25ml of desired formulation for each species.

 

Each group was treated with one of the following:

Vehicle (10% DMSO in normal saline), Piperazine citrate 0.1%, 0.2% and 0.5% in normal saline and extracts  0.1%, 0.2% and 0.5% in normal saline containing 10 % DMSO. Observations were made for the time taken for the complete paralysis and death of individual worms up to four hours of the test period. External stimuli were applied to ascertain the paralysis time when the worms did not show any motility. The time taken by the worms to lose its motility was considered as paralysis time and the lethal time was ascertained by the death of the non motile worms followed with fading away of their body colour14-16

 

Statistical Analysis:

ANOVA test was used to study the statistical significance between the treated groups and the control, followed by Dunnets’t’ test. The results are expressed as Mean ± SEM.

 

 

RESULTS AND DISCUSSION:

The petroleum ether (60°-80° C) extract of Thespesia populnea and its different fractions exhibited anthelmintic activity in dose-dependent manner giving shortest time of paralysis and death with 0.5%. The time taken to paralysis and death for 0.5% petroleum ether extract was 9.25min and 15.8 min against Pheritima posthuma Whereas, 4.50min and 9.25 min against Ascaris lumbricoides respectively. Results showed that, amongst four extracts petroleum ether extract showed significant anthelmintic activity followed by chloroform, methanol and ethyl acetate extracts against Pheritima posthuma and Ascaris lumbricoides. Piperazine citrate exhibited similar effects at 11.2min and 16.8 min for Pheritima posthuma and 7.0min and 11.8 min for Ascaris lumbricoides .The predominant effect of piperazine citrate on helminthes is to cause a flaccid paralysis .The values are tabulated in tables 1 and 2.

 

The above studies demonstrated that 0.5% petroleum ether extract posses potent anthelmintic activity which is almost equal in effectiveness to standard Piperazine citrate.

 

Piperazine citrate, by increasing chloride ion conductance of worm muscle membrane produces hyper polarization and reduced excitability that leads to muscle relaxation and flaccid paralysis17.The root extract of Thespesia populnea not only demonstrated paralysis, but also caused death of worms especially at higher concentration of 0.5%, in shorter time as compared to reference drug Piperazine citrate. The perusal of the data reveals that the petroleum ether extract exhibited quick onset of paralysis and death rate when compared to that of the standard piperazine citrate. The other test concentrations of chloroform, ethyl acetate and methanolic extracts showed marked degree of anthelmintic activity but showed delayed paralysis and death rate. Phytochemical analysis of the crude extract revealed the presence of tannins among other chemical constituents contained within them. Chemically tannins are polyphenolic compounds18which produces anthelmintic activities19 by binding to free proteins in the gastrointestinal tract of host animal20 or glycoprotein on the cuticle of the parasite21 and may cause death.

 

The present study reveals that the petroleum ether extract was more potent than the other extracts, even though all the extracts were endowed with anthelmintic property. Potency of the extracts was found to be inversely proportional to the time taken for the paralysis and death. Further studies are in process to identify the possible phytoconstituents responsible for anthelmintic activity and to establish the mechanism of action required.

 

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17.   Martin RJ. Y-Aminobutyric acid and Piperazine activated single channel currents from Ascaris suum body muscle. Br. J. Pharmacol. 1985; 84(2): 445-61.

18.   Bate-Smith EC.   The phenolic constituents of plants and their taxonomic significance, Dicotyledond. J.Linn.Soc.Bot. 1962; 58: 95-173.

19.   Niezen JH, Waghorn GC, and Charleston WA. Growth and gastrointestinal nematode parasitism in lambs grazing either Lucerne (Medicago sativa) or (Hedysarum coronarium), which contains condensed tannins. J. Agri. Sci. 125: 281.

20.   Athnasiadou S, Kyriazakis F, Jackson RL, and Coop. Direct anthelmintic effects of              condensed tannins towards different gastrointestinal nematodes of sheep: In vivo studies. Vet. Parasitol.2001; 99:19.

21.   Thompson DP, and Geary TG. The structure and function of helminth surfaces. In: J. J. Marr, eds. Biochemistry and Molecular Biology of Parasites.1995; 1: pp 203-232.

 

 

Received on 24.11.2009

Accepted on 10.01.2010

© A&V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 2(1): Jan.-Feb. 2010, 64-66