ANTHELMINTIC POTENTIAL OF PTEROSPERMUM.pdf

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Volume 1, Issue 2, March April 2010; Article 020 ISSN 0976 044X

International Journal of Pharmaceutical Sciences Review and Research Page 107Available online at www.globalresearchonline.net

ANTHELMINTIC POTENTIAL OF CRUDE EXTRACTS AND ITS VARIOUS FRACTIONS

OF DIFFERENT PARTS OFPTEROSPERMUM ACERIFOLIUMLINN.

Sambit Parida1*, V. Jagannath Patro1, Uma Shankar Mishra2, Lucy Mohapatra3, Santanu Sannigrahi41College of Pharmaceutical Sciences, Mohuda, Berhempur, Gm(Orissa)-India-760002.2Royal college of Pharmacy & Health Sciences, Berhempur, Gm(Orissa)-India-760001.

3Roland Institute of Pharmaceutical Sciences, Berhempur, Gm(Orissa)-India-760001.4St. Peters Institute of Pharm. Sciences, Hanamkonda, Warangal(AP)-India-506001.

*E-mail: [email protected]

ABSTRACT

The aim of the present study was to determine the anthelmintic activity of crude extracts and different fractions from leaves, barks andflowers ofPterospermum Acerifolium Linn. Anthelmintic activity of crude extracts and fractions were investigated against earthworms(Pheretima posthuma), roundworms (Ascardia galli) and tapeworms (Raillietinaspiralis) using Albendazole and Piperazine citrate asreference standards. The results ofanthelmintic activity revealed that the ethyl acetate fraction of all the parts were most potent whichwere well comparable with both standard drugs followed by n-butanol fractions of those parts, but at higher doses. All other frations,

petroleum extracts and remaining crude extract after fractionations of those three parts of the plant were endowed with minuteanthelmintic property, which were not up to standards. Thepresent study prooves the potential usefulness of Pterospermum Acerifoliumas good anthelmintic agent.

Keywords: Pterospermum Acerifolium Linn., Anthelmintic activity, Pheretima posthuma, Raillietinaspiralis, Ascardia galli.

INTRODUCTION

Resistance of the parasites to existing drugs1 and their highcost warrants the search for newer anthelmintic molecules.The origin of many effective drugs is found in thetraditional medicine practices and in view of this severalresearchers have undertaken studies to evaluate folklore

medicinal plants for their proclaimed anthelminticefficacy2. Pterospermum acerifolium (L) Willd (Family:Sterculiaceae) commonly known as Dinner plate tree(English) and Muchukunda (Hindi), is widelydistributed in North Canada and in many parts India i.e.Himalayan tracts, Dehradun, Bengal, Assam andManipur3,4. The flowers are sharply bitter, laxative,disinfectant, anthelminthic, removes cough (In Ayurveda),useful in leucorrhoea, ulcer, inflammation and leprosy.Leaves are used as haemostatic agent5. Barks are used asanthelmintic in treating animals6. Flavonoids likekeampferol, keampferide, luteolin, steroids andtriterpenoids like sitosterol, taraxerol, friedelin, sugars,

fatty acids are present in the plant7,8. As the peopleconsume this plant to cure helminthic infections as per theliterature, we attempted to investigate this medicinal plantfor its claimed anthelmintic activity.

MATERIALS AND METHODS

Plant material

The plant was identified and authenticated by taxonomistDr. S. K. Dash- H.O.D of Bioscience; College of Pharm.Science-Mohuda. The voucher herbarium specimen (no.-CPS/HS-008) was diposited in the herbarium of P.G.Dept.

of Phytochemistry- College of pharm. Sciences-Mohudafor future reference. After authentication, fresh leaves,barks and flowers were collected separately (during itsflowering time in Mar-April-2008) in bulk from young

matured plants from the rural hill area of Mohuda,Berhempur - Orissa.

Extraction and fractionation

All the plant materials were washed, shade dried and thenmilled in to coarse powder by a mechanical grinder. All

the powders of those different parts were passed throughsieve number 40 and used for further studies. Powder ofLeaves (2 kg), barks (2 kg) and flowers (2 kg) wereseparately extracted successively with petroleum ether(60-80C) and methanol using Soxhlet apparatus. Thesolvents were then removed under reduced pressure toobtained sticky residues. The different crude methanolicextracts (leaves-105 g, barks-78g and flowers-89g), afterremoval of the solvents, were dissolved in 10% sulfuricacid solution and partitioned with chloroform, ethylacetate and n-buatnol successively to give chloroformfraction (leaf-2.5 g, bark-1.9g,flower-2.2g), Ethyl acetatefraction (leaf-5.5 g, bark-3.9g,flower-4.2g), n-BuOH

fraction (leaf-7.8 g, bark-5.9g, flower-6.2g) andremaining crude water soluble fractions (scheme 1).

All extracts, fractions and remaining crude extracts afterfractionations were encoded as:

PEL -Pet. Ether Extract of leaf ; CFL-chloroform fractionof leaf ; EFL-ethyl acetate fraction of leaf ; NFL -n-butanol fraction of leaf ; CRL-remaining crude extractafter fractionation from leaf ; PEB-Pet. Ether Extract ofbark ; CFB-chloroform fraction of bark ; EFB-ethylacetate fraction of bark ; NFB -n-butanol fraction of bark ;CRB-remaining crude extract after fractionation frombark; PEF-Pet. Ether Extract of flower ; CFF-chloroform

fraction of flower ; EFF-ethyl acetate fraction of flower ;NFF-n-butanol fraction of flower; CRF-remaining crudeextract after fractionation from flower.


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Chemicals & Drugs used- Petrolium Ether (60-800c),chloroform, ethyl acetate, n-butanol and methanol-(allsolvents from Merck Ltd.); saline water (ClarisLifesciences Ltd., Ahmedabad), Albendazole (Alkem Ltd.)and Piperazine citrate (Glaxo Smithkline Ltd.) were usedas reference standards for anthelmintic study.

Preliminary phytochemical screening

Standard methods9, 10 were used for preliminaryphytochemical screening of the extracts to know the natureof phytoconstituents present in it (Table. 1).

Anthelmintic activity

Adult earthworms Pheretima posthuma L.Vaill(Annelida), Roundworm Ascaridia galli Schrank(Nematode) and Tapeworms (Raillietina spiralis) wereused to evaluate anthelmintic activity in vitro. Earthwormswere collected near the swampy water, roundworms andtapeworms were obtained from intestine of freshly

slaughtered fowls Gallus gallus Spadiceus (Phasianidae).Infested intestines of fowls were collected from the localslaughter house and washed with normal saline solution toremove all the faecal matter.

Scheme-1: Scheme of extraction of different parts of P. acerifolium

Table 1: Preliminary phytochemical screening of different parts of P. acerifolium

TEST FOR

P

E

L

C

F

L

E

F

L

N

F

L

C

R

L

P

E

B

C

F

B

E

F

B

N

F

B

C

R

B

P

E

F

C

F

F

E

F

F

N

F

F

C

R

F

Alkaloids + + - - - + - - - - + + - - -

Carbohydrates - - - - + - - - - + - - - - +

Glycosides - - + + - - - - + + - - - + +

Triterpenoids - - + - - - - + - - - - + - -

Tannins-phenolic compounds - - + + - - - + + - - + + - -

Protein & amino acids + + - - - - - - - - - - - - -

Gum & mucilage - - - - + - - - - + - - - - +

Flavones & flavonoids - + + - - - - + + - - + + - -

Saponins - - - + + - - - + - - - - + +

Steroids & sterols + + - - - + + - - - + + - - -

+ stands for present and stands for absent

PEL -Pet. Ether Extract of leaf ; CFL-chloroform fraction of leaf ; EFL-ethyl acetate fraction of leaf ; NFL -n-butanol fraction of leaf ;

CRL-remaining crude extract after fractionation from leaf ; PEB-Pet. Ether Extract of bark ; CFB-chloroform fraction of bark ; EFB-ethyl acetate fraction of bark ; NFB -n-butanol fraction of bark ; CRB-remaining crude extract after fractionation from bark ; PEF-Pet.Ether Extract of flower ; CFF-chloroform fraction of flower ; EFF-ethyl acetate fraction of flower ; NFF-n-butanol fraction of flower;CRF-remaining crude extract after fractionation from flower.


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Table 2: Anthelmintic Activity of leaves of Pterospermum acerifoliumTime taken for paralysis (P) and death (D) of worms in min

Pheretima posthuma Ascardia galli RaillietinaspiralisConcn.

Mg/ml P D P D P D

Control - - - - - - -

10 590.17 920.13 580.02 -- 630.54 --

20 440.68 830.02 450.33 930.31 490.12 930.92

PEL

40 350.92 770.81 370.61 820.02 380.45 790.61

10 350.71 800.75 410.13 910.81 600.17 --

20 270.44 660.08 320.67 790.32 450.09 870.33

CFL

40 210.29 590.55 240.15 690.12 330.87 740.28

10 230.17 620.73 130.23 650.98 260.32 670.12

20 170.31 550.12 080.36 530.27 180.66 550.51

EFL

40 130.88 450.77 040.76 420.54 130.43 510.72

10 290.12 680.54 230.23 710.13 310.64 740.79

20 220.07 580.11 170.61 580.11 220.93 590.09

NFL

40 170.28 470.76 130.32 480.65 160.65 470.02

10 910.11 -- 890.87 -- 780.19 970.26

20 830.53 -- 760.47 850.88 630.56 930.61

CRL

40 650.67 980.86 530.12 580.23 410.33 780.38

P=paralysis; D= death, Each value represents mean SEM (N=6).

These intestines were then dissected and worms werecollected and kept in normal saline solution. The averagesize of earthworm was 8-9 cm, average size of roundworm was 5-7 cm and average size of tapeworm was 6-8cm. Earthworm and helminths were identified in Dept. ofZoology, Khallikote Aut. College- Berhempur, Orissa andservices of veterinary practioners were utilized to confirmthe identity of worms.

The anthelmintic assay was carried out as per the methodof Ajaiyeoba et al11. The assay was performed in vitrousing adult earthworm (Pheretima posthuma) owing to itsanatomical and physiological resemblance with theintestinal roundworm parasites Ascaris lumbricoids ofhuman beings for preliminary evaluation anthelminticactivity12,13.Use ofAscaridia galli andRaillietina speciesas a suitable model for screening of anthelmintic drug wasadvocated earlier14,15. Test samples of each extract and

fraction was prepared at the concentrations, 10, 20 and 40mg/ml in distilled water and six worms i.e. Pheretimaposthuma, Ascaridia galli and Raillietina spiralis ofapproximately equal size (same type) were placed in eachnine cm Petri dish containing 25 ml of above test solutionof extracts. Albendazole (10 mg/ml) and Piperazine citrate(10 mg/ml) was used as reference standard and salinewater as control16,17,18. This procedure was adopted for allthree different types of worms. All the test solution andstandard drug solution were prepared freshly beforestarting the experiments. Observations were made for thetime taken for paralysis was noted when no movement ofany sort could be observed except when the worms wereshaken vigorously. Time for death of worms wererecorded after ascertaining that worms neither movedwhen shaken vigorously nor when dipped in warm water(500C). All the results were shown in (Table. 2, 3, 4) andexpressed as a mean SEM of six worms in each group.

Table 3: Anthelmintic Activity of barks of Pterospermum acerifoliumTime taken for paralysis (P) and death (D) of worms in min

Pheretima posthuma Ascardia galli RaillietinaspiralisConcn.

Mg/ml P D P D P DControl - - - - - - -

10 860.12 -- 670.28 980.13 770.31 980.18

20 510.37 860.71 530.23 810.08 580.22 830.04

PEB

40 430.23 710.09 390.56 770.57 430.36 670.1810 570.32 880.88 360.33 670.87 480.11 760.94

20 390.84 670.47 210.45 580.92 330.61 510.51

CFB

40 290.39 630.89 140.76 490.43 210.22 340.76

10 250.55 640.18 120.22 410.13 240.65 560.38

20 170.88 480.14 070.12 370.24 170.98 430.87

EFB

40 100.32 430.22 040.56 340.78 120.76 270.62

10 310.47 670.04 150.26 430.58 250.23 610.75

20 200.12 530.14 110.08 330.61 190.92 470.84

NFB

40 120.49 470.35 070.87 150.72 130.12 300.78

10 890.21 -- 880.61 -- 810.61 --20 710.53 -- 610.28 870.22 620.54 980.75

CRB

40 570.67 850.86 430.12 680.23 470.33 830.38P=paralysis; D= death, Each value represents mean SEM (N=6).


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Table 4: Anthelmintic Activity of flowers of Pterospermum acerifoliumTime taken for paralysis (P) and death (D) of worms in min

Pheretima posthuma Ascardia galli RaillietinaspiralisConcn

Mg/ml P D P D P D

Control - - - - - - -

10 760.91 -- 670.13 -- 710.83 --

20 630.02 950.81 480.25 780.09 590.22 910.19

PEF

40 490.09 830.17 330.17 680.28 400.37 740.0410 610.43 920.91 490.19 810.37 590.14 920.13

20 450.71 790.22 310.31 640.26 410.61 740.77

CFF

40 290.35 640.18 190.47 490.13 290.87 610.36

10 240.26 590.19 100.72 370.65 240.27 580.92

20 190.13 520.24 080.41 310.14 170.81 530.44

EFF

40 130.31 440.37 060.67 240.06 130.17 480.33

10 330.31 680.22 180.12 510.59 370.19 670.27

20 270.61 610.07 160.29 480.51 320.17 630.91

NFF

40 240.04 560.61 120.23 430.42 260.31 580.11

10 890.49 -- 750.22 -- 800.08 --20 710.53 990.17 510.66 840.54 590.88 930.76

CRF

40 570.67 880.86 430.12 780.23 410.33 780.38

Albendazole 10 22 0.28 59 0.78 100.14 380.84 220.64 540.26

Piperazine citrate 10 260.68 680.28 150.18 430.78 270.48 580.54P=paralysis; D= death, Each value represents mean SEM (N=6).

RESULTS AND DISCUSSION

In this present study it was observed that the ethyl acetatefractions of all the parts (i.e. EFL, EFB and EFF) weremore potent which is well comparable with both standarddrugs followed by n-butanol fractions (NFL, NFB andNFF), but at higher doses. Other fractions (CFL, CFB andCFF), petroleum extracts (PEL, PEB and PEF) andremaining crude extract after fractionations (CRL, CRBand CRF) of all the three parts of the plant were endowedwith minute anthelmintic property, which were not up tostandards.

The order of activity of all the extracts and fractionswere:-

EFL>NFL>CFL>PEL>CRL

EFB>NFB>CFB>PEB>CRB

EFF>NFF>CFF>PEF>CRF

The activity revealed concentration dependence nature ofthe different extracts. Potency of the extracts was found tobe inversely proportional to the time taken forparalysis/death of the worms (Table. 2,3,4). Ethyl acetate

extracts of all parts of the plant `(showing most potentanthelmintic activity) contains mainly flavones andflavonoids, triterpenoids, phenolic compounds andglycosides may be responsible for the anthelminticactivity19,20,21,22. Moderate anthelmintic activity19,20,21,22,23of the n-butanolic fractions of different parts may be dueto the presence of glycosides, phenolic compounds,saponins and flavonoids present in it. Our results from thepresent study indicate the potential usefulness ofPterospermum acerifoliumin the treatment ofhelminthiasis. Attempts for the isolation andcharacterisation of the active constituents responsible forsuch activities are currently under progress. Further

studies are necessary to understand the exact mechanismof action.

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