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Short Note

Investigation of in-vitro anthelmintic activity ofethanolic leaf extract of Boerhavia diffusa(Nyctaginaceae) including pharmacognostical andphytochemical screening

Ramasubramania Raja Rajagopal*

Assistant Professor, Department of Pharmacognosy, Jagan’s College of Pharmacy, Nellore, Andhra Pradesh, India

a r t i c l e i n f o

Article history:

Received 25 July 2013

Accepted 8 August 2013

Available online 3 September 2013

Keywords:

Boerhavia diffusa

Pheretima posthuma

Paralyse

Death

Anthelmintic

* Tel.: þ91 (0) 9494516207 (mobile).E-mail address: rsmr_raj@yahoo.co.in.

0974-6943/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.jopr.2013.08.009

a b s t r a c t

The present study clearly indicated that the crude ethanol extract of Boerhavia diffusa did

produce anthelmintic activity against Indian earthworm Pheretima posthuma. The plant

possesses significant anthelmintic activity at 100 mg/ml concentration measured by time

taken for paralyze/death of the earth worms. The current investigation leads to conclusion

that the leaves of B. diffusa have potent anthelmintic activity of conventionally used drug.

The results did not, however, exclude the possibility that doses of the extract with lower

anthelmintic activity in this study might be efficacious against other species of helminths.

Further studies using in vivo models and to isolate active constituents from extract are

required to carry out and established the effectiveness and pharmacological rational for

the use of B. diffusa as an anthelmintic drug.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction chronic, debilitating nature; they probably cause more

Helminthes infections, repeatedly entitled helminthiasis are

among the most pervasive infection and a foremost degen-

erative disease distressing a large proportion of world’s

population. In developing countries, they pose a large threat

to public health and contribute to the prevalence of malnu-

trition, anemia, eosinophilia and pneumonia. The helminths

parasites mainly subsist in human body in intestinal tract,

but they are also found in tissue, as their larvae migrate to-

wards them. Most diseases caused by helminthes1 are of a

2013, JPR Solutions; Publi

morbidity and greater economic and social deprivation

among humans and animals than any single group of para-

sites. Chemical control of helminthes coupled with improved

management has been the important worm control strategy

throughout the world. However, development of resistance in

helminthes against conventional anthelmintics is a foremost

problem in treatment of helminthes diseases. Henceforth it is

important to look for alternative strategies against gastroin-

testinal nematodes, which have led to the proposal of

screening medicinal plants for their anthelmintic activity. In

shed by Reed Elsevier India Pvt. Ltd. All rights reserved.

Table 2 e Fluorescence analysis study of Boerhavia diffusaleaf powder.

S. no Treatment for leafpowder

Visible light Long UV365 nm

1. Powder Greenish yellow Dark green

2. Powder þ 1 N HCl Dark green Dark brown

3. Powder þ 50% of H2SO4 Dark brownish

green

Greenish brown

4. Powder þ 10% of NaOH

(aqueous)

Brown Dark brown

5. Powder þ 10% of NaOH

(alcoholic)

Dark green Pale reddish

6. Pet ether Yellowish

brown

Red

7. Benzene extract Black Black

8. Acetone extract Pale green Dark green

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the present study, an attempt has been made to enrich the

knowledge of Anthelmintic activity of ethanolic leaf extract

of Boerhavia diffusa.

2. Materials and methods

2.1. Plant material

The plant of B. diffusa2 was collected from Thirumalaisamu-

dram7 kmaway fromThanjavur (Tamil Nadu) in themonth of

January 2013. The plant was identified by local people of that

village and authenticated by Dr. N. Ravichandran, Asst. Pro-

fessor, Drug Testing Laboratory, CARISM, SASTRA University

Thanjavur, and the Voucher specimen is preserved in labo-

ratory for future reference.

9. Alcoholic extract Dark green Pale green

10. Aqueous extract Dark green Pale olive green

2.2. Chemicals

All the reagents used were of analytical grade obtained from

S.D Fine Chemicals, Ltd, and Hi Media, Mumbai.

2.3. Pharmacognostical screening of plant

Macroscopic characters, microscopic characters and physi-

ochemical parameters of B. diffusa and leaf powder3:

The macroscopic evaluation was carried out for shape,

size, color, odor, taste and fracture of the drug. The micro-

scopic evaluation was performed the transverse section of

midrib and lamina region of the leaf.

The physiochemical parameters of (Table 1) different

physioechemical values such as ash value, extractive values,

loss on drying, foreign organic matter, crude fiber content,

were determined.

2.4. Florescence analysis study of B. diffusa leavespowder

Florescence analysis study of (Table 2) powdered drug mate-

rial with different reagents was carried out observe the color

reactions.

Table 1 e Physiochemical parameters of Boerhavia diffusaleaf powder.

S. no Parameters Boerhavia diffusa % W/W

1. Pet ether soluble extractive 2

2. Chloroform soluble extractive 10

3. Acetone soluble extractive 6

4. Methanol soluble extractive 10

5. Ethanol soluble extractive 17

6. Methanol soluble extractive 20

7. Water soluble extractive 22

8. Foreign organic matter 2.6

9. Loss on drying 3.0

10. Crude fiber content 23.5

11. Total ash 3.2

12. Acid insoluble ash 1.8

13. Sulfated ash 3.4

2.5. Plant cell inclusion of B. diffusa leaves powder

A plant cell inclusion study of (Table 3) powdered drug ma-

terial with different reagents was carried out to observe the

color reactions.

2.6. Preparation of extracts from B. diffusa leavespowder

B. diffusa leaveswere dried under shade, powdered and passed

through 40 meshes and stored in closed vessel for further use.

The dried powder material (20 g) was subjected to Soxhlet

extraction with ethanol for continuous hot extraction for 6 h.

The extracts were concentrated under reduced pressure to

obtain the extracts solid residues. The percentage value of the

extracts was 9.35%w/w.

2.7. Phytochemical evaluation of crude powder andethanolic leaf extract of B. diffusa

The crude powder and ethanolic leaf extract of B. diffusa (leaf)

was subjected to preliminary phytochemical test (Tables 4 and

Table 3 e Plant cell inclusions of Boerhavia diffusa leaf.

Reaction of cell walls

Cellulose Positive

Lignin Positive

Suberin Positive

Chitin Positive

Reaction of cell contents

Starch Positive

Mucilage Positive

Proteins Positive

Fixed oils Positive

Volatile oil Positive

Alkaloids Positive

Tannins Positive

Calcium oxalate Positive

Calcium carbonate Positive

Table 5 e Preliminary phytochemical screening of theethanolic extracts of Boerhavia diffusa.

Phytoconstituents Boerhavia diffusa

Alkaloids þProtein e

Alkaloids þGlycosides þFlavonoids e

j o u rn a l o f p h a rma c y r e s e a r c h 7 ( 2 0 1 3 ) 7 7 4e7 8 0776

5) followed by themethods of Harbome (1998), and Trease and

Evans (1983) and the phytoconstituents reported in table.

2.8. Screening of thin layer chromatography

The ethanolic leaf extract of B. diffusa (leaf) was subjected to

screening of thin layer chromatography (Table 6) with

different mobile phases.

TLC for alkaloids

Stationary phase Silica gel G

Mobile phase Butanol:acetic acid:water (4:5:1)

Chloroform: methanol: ammonia (8:4:1:5)

Chloroform:Di ethyl amine (9:1)

Detecting reagent Dragendroff’s reagent

TLC for terpenes

Stationary phase Silica gel G

Mobile phase Toluene:chloroform:ethyl alcohol

(4:5:4:5:1)

Detecting reagent Iodine chamber

TLC for saponins:

Stationary phase Silica gel G

Mobile phase Chloroform:methanol:water (7:4:1)

Chloroform:acetate acid:methanol:water

(6:4:3:2:1:0:8)

Ethylacetate:methanol (9.7:0.3)

Detecting reagent Iodine chamber

TLC for flavonoids:

Stationary phase Silica gel G

Mobile phase Chloroform:ethylacetate (6:4)

Toluene:ethylacetate:formic acid (5:4:1)

Toluene:ethyl acetate (9.5:0.5)

Detecting regent Iodine champer

TLC for phenolic compounds:

Stationary phase Silica gel G

Mobile phase Butane-2-ol:Acetic acid:water (14:1:5)

Detecting reagent Ammonia vapor

Table 4 e Preliminary phytochemical screening ofBoerhavia diffusa crude drug powder.

Phytoconstituents Boerhavia diffusa

Alkaloids þGlycosides þVolatile oils þAminoacids þCarbohydrates þProteins e

Catechins e

Flavonoids e

Phenolic groups e

Fixed oils þSaponins þSteroids e

Tannins þTriterpenes þ

þ Present, e absent.

Saponins þFixed oil þVolatile oil e

Tannins þAmino acids þPhenolic compounds þSteroids e

þ Present e absent.

3. In-vitro anthelmintic activity4

3.1. Experimental worms

All the experiments were carried out in Indian adult earth

worms (Pheretima posthuma) due to its anatomical resem-

blance with the intestinal roundworm parasites of human

beings. They were collected from moist soil and washed with

water to remove all fecal matters.

3.2. Administration of Metronidazole

Metronidazole (10 mg/ml) was prepared by using 0.5% w/v of

CMC as a suspending agent as administered as per method of

extract.

3.3. Experimental design

The anthelmintic activity was performed according to the

method. On adult Indian earth worm P. posthuma as it has

anatomical and physiological resemblance with the intestinal

roundworm parasites of human beings. P. posthuma was

placed in petri dish containing two different concentrations

(25, 50 & 100 mg/ml) of ethanolic extract of leaves of B. diffusa.

Each petri dish was placed with one worms and observed for

paralysis or death.Mean time for paralysiswas notedwhenno

movement of any sort could be observed, except when the

worm was shaken vigorously; the time death of worm (min)

Table 6 e Screening of thin layer chromatography ofethanolic extract of Boerhavia diffusa.

Name ofphytoconstituents

Ethanolic extract of Boerhavia diffusaRf values

Alkaloids 0.67

0.51

0.26

Phenolic groups 0.35

Saponins 0.23

0.74

Terpenes 0.68

Table 7eAnthelmintic potency of ethanolic leaf extract ofBoerhavia diffusa.

Drug/extract Concentration(mg/ml)

Time takenfor paralysis

(min)

Time takenfor death(min)

Metronidazole 10 5 � 2 6 � 3

Boerhavia diffusa

ethanolic extract

100 5 � 2 8 � 3

50 10 � 2 13 � 3

25 14 � 4 18 � 4

Control e e e

Fig. 2 e Transverse section of Boerhavia diffusa leaf (midrip

region). Uep e upper epidermis, Trc e trichome, Lep e

lower epidermis, Vb e vascular bundle, Pa e parenchyma.

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was recorded after ascertaining that worms neither moved

when shaken norwhen given external stimuli. The test results

(Table 7) were compared with Reference compound Metroni-

dazole (10 mg/ml) treated samples.

4. Results

4.1. Macroscopical characters of B. diffusa leaf

The B. diffusa Fig. 1 leaves-opposite in unequal pairs, larger

ones 25e37 mm long and smaller ones 12e18 mm long ovate-

oblong or suborbicular, apex rounded or slightly pointed, base

subcordate or rounded, green and glabrous above, whitish

below, margin entire or subundulate, dorsal side pinkish in

certain cases, thick in texture, petioles nearly as long as the

blade, slender.

Stem-greenish purple, stiff, slender, cylindrical, swollen at

nodes, minutely pubescent or early glabrous, prostrate

divericately branched, branches from common stalk, often

more than a meter long.

Fig. 3 e Transverse section of Boerhavia diffusa leaf blade.

Uep e upper epidermis, Lep e lower epidermis, Pbs e

peripheral bundle sheath.

4.2. Microscopical characters of B. diffusa leaf

Transverse section of leaf shows Figs. 2e7. The Transverse

section of Leaf shows anomocytic stomata on both sides,

numerous, a few short hairs, 3e4 celled, present on the

margin and on veins, palisade one layered, spongy paren-

chyma 2e4 layered with small air spaces, idioblasts

Fig. 1 e Plant of Boerhavia diffusa.

Fig. 4 e Transverse section of Boerhavia diffusa midrib

shows raphids. r e raphids, Pa e parenchyma, Ph e

phloem, Xy e xylem, Pbs e peripheral bundle sheath.

Fig. 5 e Transverse section of Boerhavia diffusa leaf blade

shows peripheral bundle sheath. Ph e phloem, Xy e

xylem, Pbs e peripheral bundle sheath.

Fig. 6 e Transverse section of Boerhavia diffusa leaf shows

trichomes. Trc e trichomes.

Fig. 7 e Transverse section of Boerhavia diffusa shows

vascular bundles and acicular calcium crystals. Ac e

acicular calcium crystals, Xy e xylem Ph e phloem, Pa e

parenchyma.

Fig. 8 e Anthelmintic potency of ethanolic leaf extract of

Boerhavia diffusa.

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containing raphides, occasionally cluster crystal of calcium

oxalate and orange-red resinousmatter present in mesophyll.

5. Discussions

The plant B. diffusa (Nyctaginaceae) was screened for its

macroscopical, microscopical, Physiochemical parameters,

and florescence analysis (day light, long UV), showed that they

all within limit. Made the ethanolic extracts of the plant leaves

by continuous hot extraction by Soxhlet apparatus, the per-

centage value of the extracts was 9.35%w/w. Preliminary

phytochemical analysis of ethanolic extracts showed the

presence of alkaloids, Amino acids, Carbohydrates, Saponins,

Tannins, and Triterpenes active phytoconstituents. Fig. 8 data

revealed that the ethanol extract showed anthelmintic activ-

ity at a concentration of 100 mg/ml, paralysis and death at

similar concentrations. The other test concentrations of the

extracts showed marked degree of anthelmintic activity. The

anthelmintic5 effect of extracts Figs. 10e13 is comparable with

that of the effect produced by the standard drug Metronida-

zole Fig. 9. Parasitic helminths affect animals and man,

causing considerable hardship and stunted growth. Hundreds

of millions if not billions of human infections by helminthes

exist worldwide and increased world travel and immigration

Fig. 9 e Anthelmintic activity of standard drug of

Metronidazole.

Fig. 10 e Anthelmintic activity of control (vehicle).

Fig. 11 e Anthelmintic activity of ethanolic leaf extract of

Boerhavia diffusa (100 mg).

Fig. 13 e Anthelmintic activity of ethanolic leaf extract of

Boerhavia diffusa (25 mg).

j o u r n a l o f p h a rm a c y r e s e a r c h 7 ( 2 0 1 3 ) 7 7 4e7 8 0 779

from the developing countries. However tremendous ad-

vances have been made during the previous decade and a

substantial number of synthetic precursors have been derived

to cope up the damage caused by parasite, but unfortunately

no effectivemedicine has been developed so far. Moreover the

problems associated with the use of such drugs like some

serious side effects and development of resistance drives the

severity of infection to the next level. These factors paved the

way for herbal remedies as alternative anthelmintics.

Fig. 12 e Anthelmintic activity of ethanolic leaf extract of

Boerhavia diffusa (50 mg).

Evaluation of activities of medicinal plants claimed for pos-

sessing the anthelmintic property is getting the attention

these days. Screening and proper evaluation of the claimed

medicinal plants could offer possible alternatives that may be

both sustainable and environmentally acceptable. The results

of this study have shown promising anthelmintic activity

suggesting the possible use of B. diffusa ethanolic leaf extracts

in intestinal nematode control. The anthelmintic activity of

ethanol extracts could be due to the constituents present. The

present study suggested that the ethanol extract was more

effective with anthelmintic property. The activity was con-

centration dependent of the extracts. The activity of the ex-

tracts was found to be inversely proportional to the time taken

for paralyse/death of the earth worms.

6. Conclusion

The results of the present study clearly indicated that the

crude ethanol extract of B. diffusa did produce anthelmintic

activity against Indian earthworm P. posthuma. The plant

possesses significant anthelmintic activity at 100 mg/ml con-

centration measured by time taken for paralyse/death of the

earth worms. The current investigation leads to conclusion

that the leaves of B. diffusa have potent anthelmintic activity

of conventionally used drug.6 In this study might be effica-

cious against other species of helminths. Further studies

using in vivo models and to isolate active constituents from

extract are required to carry out and established the effec-

tiveness and pharmacological rational for the use of B. diffusa

as an anthelmintic drug.

Conflicts of interest

The author has none to declare.

r e f e r e n c e s

1. Bundy DA. Immunoepidemiology of intestinal helminthicinfection: the global burden of intestinal nematode disease.Trans R Soc Trop Med Hyg. 1994;8:259e261.

j o u rn a l o f p h a rma c y r e s e a r c h 7 ( 2 0 1 3 ) 7 7 4e7 8 0780

2. Singh A. Boerhaavia diffusa: an over-exploited plant ofmedicinal importance in eastern Uttar Pradesh. Curr Sci.2007;93(4):446.

3. Kumarmayan K, Irchhaiya R, Mahendra Singh.Morphology, phytochemistry & pharmacological profile ofBoerhaavia diffusa; an overview. Intern J Curr Pharm Res.2012;4(3):4e8.

4. Tripathi KP. Essentials of Medicinal Pharmacology. 5th ed. NewDelhi: Jaypee Brothers Medical Publishers (P) Ltd.; 2003:759.

5. Coles GC. Nematode control practices and anthelminticresistance on British sheep farms. Vet Rec. 1997;141:91e93.

6. Mehtha Preeti, Phutane Shweta, Sutar Shreyas. In-vitroanthelmintic activity of whole plant of Pheretima posthuma.Asian J Pharm Clin Res. 2012;5:200e201.