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Evaluation of Anthelmintic Activity (In -vitro) of Boerhaavia diffusa Linn (Punarnava) Root Extract Including Phytochemical Screening

Shelu R. S. Jangde C. R. Nanotkar R. Y. Limsay R. P. Awasthy V. B. Singh R. P.
Vol 7(5), 209-219
DOI- http://dx.doi.org/10.5455/ijlr.20170330053039

The plant Boerhaavia diffusa is a herb found throughout the India. The roots of the plant have been described in various herbal natural medicine literatures to possess many medicinal properties like anti-inflammatory, antibacterial, diuretic, antiurethritis, hepatoprotective, anti-convulscent, antinematodal and antifibrinolytic etc. In view of various medicinal uses, the root of the plant was selected for screening of anthelmintic property. The helminths including Monezia expansa, Haemonchous contortus and Amphistomes were used for study. The B. diffusa alcoholic extract at 10, 20, 30 mg/ml concentration can produced paralysis and death of worm within half to five hrs of exposure. Plant root extract at higher dose (30mg/ml) reported significant anthelmintic activity by causing paralysis and death, when compared to standard drug against three of heminths. The extractability percentage with absolute alcohol was worked out to be 8%. The phytochemical screening reveals the presence of sterol, alkaloid, amino acid, protein, reducing sugar, glycoside, saponins, tannins, and flavonoides. This study provides scientific evidence that the root of B diffusa has anthelmintic property.


Keywords : Anthelmitic Boerhaavia diffusa Linn Helminths Paralysis

Introduction

Helminths due to their adverse effects are recognized as a major constraint to livestock production throughout the tropics and elsewhere (Githiori et al., 2004). These adverse effects include retarded growth (Kochapakdee et al., 1995), lowered productivity (Perry and Randolph, 1999), mortality (Sykes, 1994) and high economic losses (Iqbal et al., 1993). Haemonchosis caused by Haemonchus contortus is a predominant, highly pathogenic and economically important disease of sheep and goats (Mortensen et al., 2003). These parasites are common blood feeders that cause anaemia and reduced productivity and can lead to death in heavily infected animals (Githigia et al., 2001). Amphistomes (stomach flukes) are parasite of ruminants which particularly affect cattle and sheep. The flukes are often found in small numbers but only seriously affect livestock under certain conditions, in heavy infections, and in certain growth stages. The disease is characterized by severe enteritis, diarrhoea, dehydration, oedema, polydipsia, anaemia, listlessness and weight loss. Young cattle are the usual subjects of the disease (Swarnakar et al., 2014). Heavy infection with immature flukes in the upper intestine can cause serious ill-health and death (Urquhart et al., 2000). Moniezia expansa is commonly known as sheep tapeworm or double-pored ruminant tapeworm. It is a large tapeworm inhabiting the small intestines of ruminants such as sheepgoats and cattle. M. expansa infections are generally asymptomatic, even when the tapeworms are present in large numbers in young lambs. However heavy infection may cause intestinal obstruction, diarrhea and weight loss (Elliott et al., 1986). Chemotherapeutic control practices have evolved a number of problems including resistance of helminths to various groups of anthelmintic (Chartier et al., 2001), chemical residues, toxicity problems, increased cost of treatment, non-adaptability of drugs and non-availability of the medicine in remote areas. Such problems diverted the researchers’ attentions towards the development of alternate and safe methods for the treatment of helminthiasis. Due to the good efficacy and cost effectiveness herbal medicine have gained much importance in recent years (Bharali et al., 2003). Medicinal plants are the backbone of herbal drugs. It is estimated that about 25% of all modern medicine are directly or indirectly derived from plant. WHO has recently define traditional medicine (including herbal drugs ) as comprising therapeutic practices that have been in existence, often for hundreds of years, before the development and spread of modern medicine and are still in use today (Kamboj, 2000).

Boerhaavia diffusa Linn is belongs to family Nyctaginaceae. B diffusa is widely distributed in the tropics and subtropics (CSIR, 1988) It has a long history of uses by indigenous and tribal people and in Ayurvedic or natural herbal medicines (Dhar et al., 1968). The major active principle present in the root is punarnavine (Kirtikar and Basu, 1956). The some pharmacological studies have demonstrated that the root of B. diffusa exhibits a wide range of properties including anti-inflammatory (Bhalla et al., 1968), diuretic (Gaitonde et al., 1974), laxative (Chopra et al., 1956), antiurethritis (Nadkarni, 1976), anticonvulsant (Adesina, 1979), antinematodal (Vijayalakshmi et al., 1979), antifibrinolytic (Jain and Khanna, 1989),antibacterial (Olukoya et al., 1993), antihepatotoxic (Mishra, 1980; Chandan et al., 2012; Rawat et al., 2014).

B. diffusa is also indigenous to India, it is found throughout the warmer parts of the country up to an altitude of 2000 m in the Himalayan region. It grows well on wastelands and in fields after the rainy season (Chopra, 1969). The plant is also cultivated to some extent in West Bengal (CSIR, 1988). The roots and leaves with flowers have been found to be highly potent (CSIR, 1988). In ayurvedic medicine, different parts of this plant were reported to have various medicinal properties. It was used in renal ailments as diuretic (Anand, 1995); and to treat seminal weakness and blood pressure (Gaitonde et al., 1974). The present study was designed to explore the anthelmintic property of B. diffusa root along with phytochemical screening.The medicinal plant was selected in keeping mind that the growing interest in the development of ecofriendly, biodegradable and safer anthelmintic.

Materials and Methods

Plant Material Collection and Identification

The roots of B. diffusa were procured from the Nagpur region and were identified from University Department of Botany, Nagpur University, Nagpur (Voucher No-RT2301/NG).The plant material was dried at room temperature and powdered .The powder was stored in glass bottle in a cool and dry place away from direct sunlight and used for preparation of alcoholic extract.

Preparation of Extract

The methanolic extract was prepared by the method described by Rosenthaler (1930). Fifty grams of powered plant material was subjected to extraction in a “Soxhlet’s Extraction” apparatus, using methanol as solvent. This was heated on heating mantle till the colourless solvent startedreturning back to reservoir. Then the content was transferred to a dry, clean and already weighed petridish and kept at room temperature till the complete evaporation of the solvent. The petridish was again weighed to calculate the extractability percentage and finally stored in desiccator in a cool and dry place. A preliminary phytochemical screening of was carried out according to the method of Wagner et al. (1999). The Salkowski Test and Libermann Buchard Reaction were carried out for sterols. Dragendroff’s Reagent and Wangers Reagent Test were carried out for alkaloids. The Ninhydrin Test was carried out for amino acid detection. Xanthoprotein Test and Biuret Test were carried out for presence of proteins. . Benedict’s reagent and Fehling’s reagent tests were carried out for reducing sugars. Foam test was carried out for Saponins. Lead acetate and ferric chloride test carried out for detection of tannins. Bantrager test was carried out for antraquinones. Flavonoid test carried out for detection of flavonoids. Alcoholic extract test was carried out for resins.

Experimental Protocol for In vitro Anthelmintic Activity of Extract

The in vitro trials for anthelmintic activity of alcoholic extract of B. diffusa was conducted on mature live Haemonchus contortus, Moniezia expansa, and Amphistome as per method described by Agrawal et al. (1979). Briefly, the mature worms were collected from the abomasum of freshly slaughtered sheep in the local abattoir governed by Nagpur Municipal Corporation. The worms were collected by blunt forceps then washed and finally suspended in tyrode solution. The worms were identified from department of Parasitology, Nagpur Veterinary College, Nagpur. The experiment was started after two hours of collection of worms. These worms were used for screening the anthelmintic activity of extract by petridish method (Agrawal et al., 1979).

The extract of B. diffusa Linn was used in the concentration of 10, 20 and 30 mg/ml. The dried extract was suspended in 1% w/v Carboxy Methyl Cellulose (CMC), prepared in normal saline water in three different conc. (10, 20, 30 mg/ml). Albendazole suspension of 5mg/ml conc. was taken as standard. The normal saline (0.90% concentration) water with 1% CMC was taken as a control. Worms were placed in petridish containing 15 ml of sample (drug) solution. Time for paralysis was noted either when any movement could not be observed except when the worms were shaken vigorously or when dipped in warm water (50 0C). Ten worms were exposed in triplicate and the time of transfer was noted. Observation were made with test drug at an interval of 30 minutes till the mortality of worms occurred. During observation the time required for paralysis of worm and complete cessation of motility was noted. The worms were gently pressed at either end with a blunt glass rod to confirm complete cessation of motility was noted. Death was included when the worms lost their motility followed by white secretions and fading away of their body colour. At the end of trial ,the worm were transferred to tyrode solution at 40 0C to check the motility if any and those worms showing no motility were considered as dead worms (Sharma and Sisodia,1976). The findings were confirmed by taking number of observation. All the experiments were conducted at room temperature varying between 97 0 F to 104 0F (Garg and Mehta, 1958).

Drugs and Chemicals

All the solvents, chemicals were used of analytical grade and the standard drug Albendazole (concentration 98%) obtained from local market (Alembic Animal Health Ltd., Mumbai).

Statistical Analysis

All the values in the test are presented as Mean ± SEM. Statistical differences between the means of the various groups were evaluated by F- test by using SPSS software at 5% level of significant(P<0.05).

Fig. 1: In vitro anthelmintic activity of roots of B. diffusa on Moniezia expansa. AEBd- alcoholic extract of B.diffusa

Fig. 2: In vitro anthelmintic activity of roots of B.diffusa on Haemonchus contortus. AEBd- alcoholic extract of B.diffusa

Fig. 3: In vitro anthelmintic activity of roots of B.diffusa on Amphistome at different doses. AEBd- alcoholic extract of B.diffusa

Result and Discussion

The extractability percentage of roots of B. diffusa calculated and was worked out to be 8 % with methanol. The colour of extract was dark brown and consistency was semisolid. Phytochemical analysis reveals the presence of sterol, alkaloid, amino acid, proteins, reducing sugar saponin, tannins and flavonoids are presented in Table 1.

Table 1: Showing result of phytochemical screening of methanolic root extract of B.diffusa

S. No. Active principle Test Applied Observation Result
1 Sterol Salkowski Test Development Of Red Colour in Chloroform Layer Positive
Libermann Buchard Reaction Development Of Red Colour in Chloroform Layer Positive
2 Alkaloid Dragendroff’s Reagent Development of orange colour Positive
Wangers Reagent Appearance of brown flocculent precipitate Positive
3 Amino acid Ninhydrin test Violet colour development Positive
4 Protein Xanthoprotein Test White precipitate was formed Positive
Biuret Test No violet pink colour development Negative
5 Reducing sugar Benedict’s Reagent Development of brownish red precipitate Positive
Fehling’s Reagent Red precipitate was formed Positive
6 Glycosides Benedict’s Reagent No development of brownish precipitate Negative
Fehling’s Reagent Further reduction was not observed Negative
7 Saponins Foam Test Formation of froth Positive
8 Tannins Lead Acetate Test Formation of precipitate Positive
Ferric Chloride Test Green colouration in filtrate Positive
9 Anthraquinones Bantrager test Ammonical layer did not acquire any pink colour Negative
10 Flavonoide Test For Flavonoides Development of pink or magnet colour Positive
11 Resin Alcholic Extract Test Absence of turbidity Negative

Siddiqui (2009) reported the presence of alkaloid in alcoholic extract. Chopra et al. (1923) studied that they are rich in proteins and fats. Awasthi et al. (1985) reported that roots are rich in basic proteins. Surwase (2000) reported the presence of sterol, alkaloid, amino acid, protein, reducing sugar, saponins, tannins, flavonoids. The observation in present study agrees the finding of mentioned researchers. Anthelmintic effects of plants are normally ascribed to secondary metabolites such as alkaloids, saponin and tannins or condensed tannins (CT). Condensed tannins are a diverse and widely-occurring group of compounds, and consist of polymers or hetero-polymers being common. They are found in plant material from both tropical and temperate areas, and have been widely investigated for their anthelmintic, antioxidant and anti-inflammatory properties (Martinez et al., 2012). It is also apparent that CT can have anthelmintic effects; reduced worm burdens have been reported in rats administered CT in the diet, or in livestock grazing forages containing CT (Butter et al., 2001). The anthelmintic activity of B.diffusa was screened against Monezia expansa, Heamonchous contortus, Amphistome at the different concentration of extract i.e. 10, 20, 30 mg/ml.

The anthelmintic activity was compare with Albendazole @ 5 mg/ml as standard drug. The time taken for complete cessation of motility (paralysis) and mortality in hrs in different concentration are presented in Table 2, 3 and 4 (expressed as a mean± SEM of 10 worms in each group).

Table 2: Showing In vitro anthelmintic activity of alcoholic extracts of roots of B.diffusa on Moniezia expansa

Group Moniezia expansa
Paralysis Time(minutes) Death Time(minutes)
Control 216.66±4.40 485.33±2.02
Std (Albedazole 5mg/ml) 20.66±1.33 54±2.30
AEBd 10mg/ml 98±1.15 300±11.54
AEBd 20 mg/ml 40±2.88 120±4.61
AEBd 30 mg/ml 28±1.15* 68±1.73*

(P<0.05)* Significant at 5 % L.S. AEBd- alcoholic extract of B.diffusa, Std-standard drug

The table reveals that the activity observed was dose dependent. As the concentration was increased the death and paralysis occurred was earlier than the small amount of dose used. The extract at 10, 20, 30 mg/ml concentration produced paralysis and death of worm within half to five hrs of exposure. The paralysis time and death time of three helminths caused by different concentration of the extracts were compared with those caused by the standard drug albendazole (5 mg/ml).

Table 3: Showing in vitro anthelmintic activity of alcoholic extracts of roots of B.diffusa on Haemonchus contotortus

Group Haemonchus contortus
Paralysis Time(minutes) Death Time (minutes)
Control 180±17.3 360±17.32
Std (Albendadole 5mg/ml) 8±2.30* 25±2.88 *
AEBd 10mg/ml 34±2.30 52±1.15
AEBd 20 mg/ml 21±0.57 33±1.15
AEBd 30 mg/ml 10±2.30 * 28±1.73*

(P<0.05)* Significant at 5 % L.S. AEBd- alcoholic extract of B.diffusa, Std-standard drug

Table 4: Showing anthelmintic activity of alcoholic extracts of roots of B.diffusa on Amphistome

Group Amphistome
Paralysis Time(minutes) Death Time(minutes)
Control 166.66±6.66 360±20.8
Std (Albendadole 5mg/ml) 18.33±4.4* 33.33±8.8*
AEBd 10mg/ml 55±2.8 263.1±2.0
AEBd 20 mg/ml 27.66±1.4 101+2.0
AEBd 30 mg/ml 22.66±1.45* 46±3.0*

(P<0.05)* Significant at 5 % L.S. AEBd- alcoholic extract of B.diffusa, Std-standard drug

The activity of extract was observed for all the three types of worms used, but was more pronounced in case of higher doses (Fig.1, 2 and 3). The results were significantly observed. The In-vitro anthelmintic activity of leaves extract of B.diffusa reported by Ramasubramania R (2013) in dose dependent manner. The anthelmintic activity of methanolic extract of B. diffusa may be due to presence of more polar bioactive phytochemicals such as tannins, phenolics, alkaloids and saponins in polar solvent. Parasitic helminthes are worm like organisms that live and feed off living hosts, receiving nourishment and protection while disrupting their hosts’ nutrient absorption, causing weakness and disease in human and animals inflicting heavy production losses. Several phytochemicals have potential to alter metabolic pathways of worms and there by produce mortality in the worms and they have ability to bind with the free proteins present in the gastrointestinal tract of helminths and cause death (Tiwari et al., 2011; Rubini et al., 2012).

Conclusion

This study shows that the extract of B. diffusa offer a safe method or supplement treatment strategy to control helminth infestation. However, further detailed study is needed to isolate and purification of constituents from the plant root. Presence of various constituents e.g. phenolic, flavonoids, saponins, tannins etc. in the crude materials can be the reason of potent pharmacological activities. Further studies on isolation of phyto-constituents and their biological activities in vitro and in vivo, and developing of new anthelmintic medicines from B. diffusa are suggested to be carried out.

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