- Email: [email protected]
Investigation of in-vitro anthelmintic activity of ethanolic leaf extract of Boerhavia diffusa (Nyctaginaceae) including pharmacognostical and phytochemical screening
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 7 7 4 e7 8 0
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Short Note
Investigation of in-vitro anthelmintic activity of ethanolic leaf extract of Boerhavia diffusa (Nyctaginaceae) including pharmacognostical and phytochemical screening Ramasubramania Raja Rajagopal* Assistant Professor, Department of Pharmacognosy, Jagan’s College of Pharmacy, Nellore, Andhra Pradesh, India
article info
abstract
Article history:
The present study clearly indicated that the crude ethanol extract of Boerhavia diffusa did
Received 25 July 2013
produce anthelmintic activity against Indian earthworm Pheretima posthuma. The plant
Accepted 8 August 2013
possesses significant anthelmintic activity at 100 mg/ml concentration measured by time
Available online 3 September 2013
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.
Keywords:
The results did not, however, exclude the possibility that doses of the extract with lower
Boerhavia diffusa
anthelmintic activity in this study might be efficacious against other species of helminths.
Pheretima posthuma
Further studies using in vivo models and to isolate active constituents from extract are
Paralyse
required to carry out and established the effectiveness and pharmacological rational for
Death
the use of B. diffusa as an anthelmintic drug.
Anthelmintic
Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Helminthes infections, repeatedly entitled helminthiasis are among the most pervasive infection and a foremost degenerative 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 malnutrition, 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 towards them. Most diseases caused by helminthes1 are of a
chronic, debilitating nature; they probably cause more morbidity and greater economic and social deprivation among humans and animals than any single group of parasites. 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 gastrointestinal nematodes, which have led to the proposal of screening medicinal plants for their anthelmintic activity. In
* Tel.: þ91 (0) 9494516207 (mobile). E-mail address: [email protected]. 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.08.009
775
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 7 7 4 e7 8 0
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
S. no 1. 2. 3.
The plant of B. diffusa2 was collected from Thirumalaisamudram 7 km away from Thanjavur (Tamil Nadu) in the month of January 2013. The plant was identified by local people of that village and authenticated by Dr. N. Ravichandran, Asst. Professor, Drug Testing Laboratory, CARISM, SASTRA University Thanjavur, and the Voucher specimen is preserved in laboratory for future reference.
2.2.
Table 2 e Fluorescence analysis study of Boerhavia diffusa leaf powder.
4. 5. 6. 7. 8. 9. 10.
Treatment for leaf powder
Visible light
Powder Greenish yellow Powder þ 1 N HCl Dark green Powder þ 50% of H2SO4 Dark brownish green Powder þ 10% of NaOH Brown (aqueous) Powder þ 10% of NaOH Dark green (alcoholic) Pet ether Yellowish brown Benzene extract Black Acetone extract Pale green Alcoholic extract Dark green Aqueous extract Dark green
Long UV 365 nm Dark green Dark brown Greenish brown Dark brown Pale reddish Red Black Dark green Pale green Pale olive green
Chemicals
All the reagents used were of analytical grade obtained from S.D Fine Chemicals, Ltd, and Hi Media, Mumbai.
2.5.
2.3.
A plant cell inclusion study of (Table 3) powdered drug material with different reagents was carried out to observe the color reactions.
Pharmacognostical screening of plant
Macroscopic characters, microscopic characters and physiochemical 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 microscopic 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 leaves powder Florescence analysis study of (Table 2) powdered drug material with different reagents was carried out observe the color reactions.
Table 1 e Physiochemical parameters of Boerhavia diffusa leaf powder. S. no 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Parameters
Boerhavia diffusa % W/W
Pet ether soluble extractive Chloroform soluble extractive Acetone soluble extractive Methanol soluble extractive Ethanol soluble extractive Methanol soluble extractive Water soluble extractive Foreign organic matter Loss on drying Crude fiber content Total ash Acid insoluble ash Sulfated ash
2 10 6 10 17 20 22 2.6 3.0 23.5 3.2 1.8 3.4
Plant cell inclusion of B. diffusa leaves powder
2.6. Preparation of extracts from B. diffusa leaves powder B. diffusa leaves were 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 and ethanolic 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 Lignin Suberin Chitin Reaction of cell contents Starch Mucilage Proteins Fixed oils Volatile oil Alkaloids Tannins Calcium oxalate Calcium carbonate
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive
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5) followed by the methods 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 Mobile phase
Detecting reagent TLC for terpenes Stationary phase Mobile phase Detecting reagent TLC for saponins: Stationary phase Mobile phase
Detecting reagent TLC for flavonoids: Stationary phase Mobile phase
Silica gel G Butanol:acetic acid:water (4:5:1) Chloroform: methanol: ammonia (8:4:1:5) Chloroform:Di ethyl amine (9:1) Dragendroff’s reagent Silica gel G Toluene:chloroform:ethyl alcohol (4:5:4:5:1) Iodine chamber Silica gel G Chloroform:methanol:water (7:4:1) Chloroform:acetate acid:methanol:water (6:4:3:2:1:0:8) Ethylacetate:methanol (9.7:0.3) Iodine chamber
Silica gel G 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 5 e Preliminary phytochemical screening of the ethanolic extracts of Boerhavia diffusa. Phytoconstituents
Boerhavia diffusa
Alkaloids Protein Alkaloids Glycosides Flavonoids Saponins Fixed oil Volatile oil Tannins Amino acids Phenolic compounds Steroids
þ e þ þ e þ þ e þ þ þ 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 resemblance 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.
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.
Table 4 e Preliminary phytochemical screening of Boerhavia diffusa crude drug powder. Phytoconstituents Alkaloids Glycosides Volatile oils Aminoacids Carbohydrates Proteins Catechins Flavonoids Phenolic groups Fixed oils Saponins Steroids Tannins Triterpenes þ Present, e absent.
Boerhavia diffusa þ þ þ þ þ e e e e þ þ e þ þ
Administration of Metronidazole
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 paralysis was noted when no 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 of ethanolic extract of Boerhavia diffusa. Name of phytoconstituents Alkaloids
Phenolic groups Saponins Terpenes
Ethanolic extract of Boerhavia diffusa Rf values 0.67 0.51 0.26 0.35 0.23 0.74 0.68
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 7 7 4 e7 8 0
777
Table 7 e Anthelmintic potency of ethanolic leaf extract of Boerhavia diffusa. Drug/extract
Metronidazole Boerhavia diffusa ethanolic extract Control
Concentration Time taken Time taken (mg/ml) for paralysis for death (min) (min) 10 100 50 25 e
5 5 10 14
e
2 2 2 4
6 8 13 18
e
3 3 3 4
was recorded after ascertaining that worms neither moved when shaken nor when given external stimuli. The test results (Table 7) were compared with Reference compound Metronidazole (10 mg/ml) treated samples.
4.
Results
4.1.
Macroscopical characters of B. diffusa leaf
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.
The B. diffusa Fig. 1 leaves-opposite in unequal pairs, larger ones 25e37 mm long and smaller ones 12e18 mm long ovateoblong 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.
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 parenchyma 2e4 layered with small air spaces, idioblasts
Fig. 1 e Plant of Boerhavia diffusa.
Fig. 3 e Transverse section of Boerhavia diffusa leaf blade. Uep e upper epidermis, Lep e lower epidermis, Pbs e peripheral bundle sheath.
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.
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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. 8 e Anthelmintic potency of ethanolic leaf extract of Boerhavia diffusa.
containing raphides, occasionally cluster crystal of calcium oxalate and orange-red resinous matter present in mesophyll.
5.
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.
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 percentage 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 activity 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 Metronidazole 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.
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 7 7 4 e7 8 0
Fig. 10 e Anthelmintic activity of control (vehicle).
779
Fig. 13 e Anthelmintic activity of ethanolic leaf extract of Boerhavia diffusa (25 mg).
Evaluation of activities of medicinal plants claimed for possessing 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 concentration dependent of the extracts. The activity of the extracts was found to be inversely proportional to the time taken for paralyse/death of the earth worms. Fig. 11 e Anthelmintic activity of ethanolic leaf extract of Boerhavia diffusa (100 mg).
from the developing countries. However tremendous advances 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 effective medicine 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.
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 concentration 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 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.
Conflicts of interest The author has none to declare.
references
Fig. 12 e Anthelmintic activity of ethanolic leaf extract of Boerhavia diffusa (50 mg).
1. Bundy DA. Immunoepidemiology of intestinal helminthic infection: the global burden of intestinal nematode disease. Trans R Soc Trop Med Hyg. 1994;8:259e261.
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j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 7 7 4 e7 8 0
2. Singh A. Boerhaavia diffusa: an over-exploited plant of medicinal importance in eastern Uttar Pradesh. Curr Sci. 2007;93(4):446. 3. Kumarmayan K, Irchhaiya R, Mahendra Singh. Morphology, phytochemistry & pharmacological profile of Boerhaavia diffusa; an overview. Intern J Curr Pharm Res. 2012;4(3):4e8.
4. Tripathi KP. Essentials of Medicinal Pharmacology. 5th ed. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd.; 2003:759. 5. Coles GC. Nematode control practices and anthelmintic resistance on British sheep farms. Vet Rec. 1997;141:91e93. 6. Mehtha Preeti, Phutane Shweta, Sutar Shreyas. In-vitro anthelmintic activity of whole plant of Pheretima posthuma. Asian J Pharm Clin Res. 2012;5:200e201.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Short Note
Investigation of in-vitro anthelmintic activity of ethanolic leaf extract of Boerhavia diffusa (Nyctaginaceae) including pharmacognostical and phytochemical screening Ramasubramania Raja Rajagopal* Assistant Professor, Department of Pharmacognosy, Jagan’s College of Pharmacy, Nellore, Andhra Pradesh, India
article info
abstract
Article history:
The present study clearly indicated that the crude ethanol extract of Boerhavia diffusa did
Received 25 July 2013
produce anthelmintic activity against Indian earthworm Pheretima posthuma. The plant
Accepted 8 August 2013
possesses significant anthelmintic activity at 100 mg/ml concentration measured by time
Available online 3 September 2013
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.
Keywords:
The results did not, however, exclude the possibility that doses of the extract with lower
Boerhavia diffusa
anthelmintic activity in this study might be efficacious against other species of helminths.
Pheretima posthuma
Further studies using in vivo models and to isolate active constituents from extract are
Paralyse
required to carry out and established the effectiveness and pharmacological rational for
Death
the use of B. diffusa as an anthelmintic drug.
Anthelmintic
Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Helminthes infections, repeatedly entitled helminthiasis are among the most pervasive infection and a foremost degenerative 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 malnutrition, 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 towards them. Most diseases caused by helminthes1 are of a
chronic, debilitating nature; they probably cause more morbidity and greater economic and social deprivation among humans and animals than any single group of parasites. 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 gastrointestinal nematodes, which have led to the proposal of screening medicinal plants for their anthelmintic activity. In
* Tel.: þ91 (0) 9494516207 (mobile). E-mail address: [email protected]. 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.08.009
775
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 7 7 4 e7 8 0
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
S. no 1. 2. 3.
The plant of B. diffusa2 was collected from Thirumalaisamudram 7 km away from Thanjavur (Tamil Nadu) in the month of January 2013. The plant was identified by local people of that village and authenticated by Dr. N. Ravichandran, Asst. Professor, Drug Testing Laboratory, CARISM, SASTRA University Thanjavur, and the Voucher specimen is preserved in laboratory for future reference.
2.2.
Table 2 e Fluorescence analysis study of Boerhavia diffusa leaf powder.
4. 5. 6. 7. 8. 9. 10.
Treatment for leaf powder
Visible light
Powder Greenish yellow Powder þ 1 N HCl Dark green Powder þ 50% of H2SO4 Dark brownish green Powder þ 10% of NaOH Brown (aqueous) Powder þ 10% of NaOH Dark green (alcoholic) Pet ether Yellowish brown Benzene extract Black Acetone extract Pale green Alcoholic extract Dark green Aqueous extract Dark green
Long UV 365 nm Dark green Dark brown Greenish brown Dark brown Pale reddish Red Black Dark green Pale green Pale olive green
Chemicals
All the reagents used were of analytical grade obtained from S.D Fine Chemicals, Ltd, and Hi Media, Mumbai.
2.5.
2.3.
A plant cell inclusion study of (Table 3) powdered drug material with different reagents was carried out to observe the color reactions.
Pharmacognostical screening of plant
Macroscopic characters, microscopic characters and physiochemical 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 microscopic 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 leaves powder Florescence analysis study of (Table 2) powdered drug material with different reagents was carried out observe the color reactions.
Table 1 e Physiochemical parameters of Boerhavia diffusa leaf powder. S. no 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Parameters
Boerhavia diffusa % W/W
Pet ether soluble extractive Chloroform soluble extractive Acetone soluble extractive Methanol soluble extractive Ethanol soluble extractive Methanol soluble extractive Water soluble extractive Foreign organic matter Loss on drying Crude fiber content Total ash Acid insoluble ash Sulfated ash
2 10 6 10 17 20 22 2.6 3.0 23.5 3.2 1.8 3.4
Plant cell inclusion of B. diffusa leaves powder
2.6. Preparation of extracts from B. diffusa leaves powder B. diffusa leaves were 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 and ethanolic 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 Lignin Suberin Chitin Reaction of cell contents Starch Mucilage Proteins Fixed oils Volatile oil Alkaloids Tannins Calcium oxalate Calcium carbonate
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive
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5) followed by the methods 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 Mobile phase
Detecting reagent TLC for terpenes Stationary phase Mobile phase Detecting reagent TLC for saponins: Stationary phase Mobile phase
Detecting reagent TLC for flavonoids: Stationary phase Mobile phase
Silica gel G Butanol:acetic acid:water (4:5:1) Chloroform: methanol: ammonia (8:4:1:5) Chloroform:Di ethyl amine (9:1) Dragendroff’s reagent Silica gel G Toluene:chloroform:ethyl alcohol (4:5:4:5:1) Iodine chamber Silica gel G Chloroform:methanol:water (7:4:1) Chloroform:acetate acid:methanol:water (6:4:3:2:1:0:8) Ethylacetate:methanol (9.7:0.3) Iodine chamber
Silica gel G 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 5 e Preliminary phytochemical screening of the ethanolic extracts of Boerhavia diffusa. Phytoconstituents
Boerhavia diffusa
Alkaloids Protein Alkaloids Glycosides Flavonoids Saponins Fixed oil Volatile oil Tannins Amino acids Phenolic compounds Steroids
þ e þ þ e þ þ e þ þ þ 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 resemblance 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.
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.
Table 4 e Preliminary phytochemical screening of Boerhavia diffusa crude drug powder. Phytoconstituents Alkaloids Glycosides Volatile oils Aminoacids Carbohydrates Proteins Catechins Flavonoids Phenolic groups Fixed oils Saponins Steroids Tannins Triterpenes þ Present, e absent.
Boerhavia diffusa þ þ þ þ þ e e e e þ þ e þ þ
Administration of Metronidazole
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 paralysis was noted when no 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 of ethanolic extract of Boerhavia diffusa. Name of phytoconstituents Alkaloids
Phenolic groups Saponins Terpenes
Ethanolic extract of Boerhavia diffusa Rf values 0.67 0.51 0.26 0.35 0.23 0.74 0.68
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 7 7 4 e7 8 0
777
Table 7 e Anthelmintic potency of ethanolic leaf extract of Boerhavia diffusa. Drug/extract
Metronidazole Boerhavia diffusa ethanolic extract Control
Concentration Time taken Time taken (mg/ml) for paralysis for death (min) (min) 10 100 50 25 e
5 5 10 14
e
2 2 2 4
6 8 13 18
e
3 3 3 4
was recorded after ascertaining that worms neither moved when shaken nor when given external stimuli. The test results (Table 7) were compared with Reference compound Metronidazole (10 mg/ml) treated samples.
4.
Results
4.1.
Macroscopical characters of B. diffusa leaf
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.
The B. diffusa Fig. 1 leaves-opposite in unequal pairs, larger ones 25e37 mm long and smaller ones 12e18 mm long ovateoblong 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.
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 parenchyma 2e4 layered with small air spaces, idioblasts
Fig. 1 e Plant of Boerhavia diffusa.
Fig. 3 e Transverse section of Boerhavia diffusa leaf blade. Uep e upper epidermis, Lep e lower epidermis, Pbs e peripheral bundle sheath.
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.
778
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 7 7 4 e7 8 0
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. 8 e Anthelmintic potency of ethanolic leaf extract of Boerhavia diffusa.
containing raphides, occasionally cluster crystal of calcium oxalate and orange-red resinous matter present in mesophyll.
5.
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.
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 percentage 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 activity 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 Metronidazole 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.
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 7 7 4 e7 8 0
Fig. 10 e Anthelmintic activity of control (vehicle).
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Fig. 13 e Anthelmintic activity of ethanolic leaf extract of Boerhavia diffusa (25 mg).
Evaluation of activities of medicinal plants claimed for possessing 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 concentration dependent of the extracts. The activity of the extracts was found to be inversely proportional to the time taken for paralyse/death of the earth worms. Fig. 11 e Anthelmintic activity of ethanolic leaf extract of Boerhavia diffusa (100 mg).
from the developing countries. However tremendous advances 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 effective medicine 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.
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 concentration 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 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.
Conflicts of interest The author has none to declare.
references
Fig. 12 e Anthelmintic activity of ethanolic leaf extract of Boerhavia diffusa (50 mg).
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2. Singh A. Boerhaavia diffusa: an over-exploited plant of medicinal importance in eastern Uttar Pradesh. Curr Sci. 2007;93(4):446. 3. Kumarmayan K, Irchhaiya R, Mahendra Singh. Morphology, phytochemistry & pharmacological profile of Boerhaavia diffusa; an overview. Intern J Curr Pharm Res. 2012;4(3):4e8.
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