Anthelmintic and in vitro antioxidant evaluation of fractions of methanol extract of Leea asiatica leaves.

Leea asiatica, a folk medicinal plant of India, is used in the treatment of worm infection and other oxidative stress-related disorders, traditionally. In the present study, the in vitro anthelmintic and in vitro antioxidant activity of different fractions of the methanol extract from the Leea asiatica leaves were evaluated. The fraction displayed significant anthelmintic activity against Indian adult earthworms (Pheretima posthuma). The ethyl acetate fraction showed a better paralysis activity (13.99 ± 0.59), while the methanol fraction showed a better death time (63.76 ± 0.73 minutes), when compared with other fractions, at a dose of 50 mg/ml concentration. The anthelmintic activity of methanol and the ethyl acetate fraction were almost similar and comparable to the standard drug, piperazine citrate. The petroleum ether fraction did not produce a potent anthelmintic effect compared to the standard. The in vitro antioxidant activity was evaluated by using the diphenyl-picrylhydrazyl (DPPH) radical scavenging assay, nitric oxide radical scavenging assay, lipid peroxidation assay, and the ferric thiocyanate method. The ethyl acetate fraction showed better antioxidant activity in all tested methods. The IC(50) value of the ethyl acetate fraction in the DPPH radical, nitric oxide radical scavenging assay, and lipid peroxidation assay were 9.5, 13.0, and 57.0 μg/ml, respectively. The fractions significantly (P < 0.05) inhibited the peroxidation of linoleic acid. The results confirmed the folk use of Leea asiatica in warm infection and the plant could be viewed as a potential source of natural anthelmintic and antioxidant compound.

INTRODUCTION

Helminth infections are among the most prevalent infections in human beings, affecting a huge proportion of the world's population, particularly in tropical countries.[12] According to the World Health Organization a staggering two billion people harbor parasitic worm infections.[3] Poorer personal and environmental hygienic conditions, inadequate sanitary facilities, and lack of supply of pure water, coupled with poverty and illiteracy, are the major fundamental factors responsible for the spreading of helminthiasis in developing countries.[1] Helminth infections also contribute to the prevalence of undernourishment, anemia, eosinophilia, and pneumonia. Although several drugs are available for the treatment of parasitic diseases, yet helminthiasis is principally responsible for the ruthless morbidity affecting the population in endemic areas. Gastrointestinal helminth infections have become resistant to the currently available synthetic anthelmintic drugs, which cause the foremost problem in the treatment of helminths diseases.[145] Hence, the demand for natural anthelmintics is increasing.

The recent abundant evidence confirmed the involvement of oxidative stress in the pathogenesis of several acute, chronic disorders and diseases. Exogenous antioxidants help the endogenous enzymatic and non-enzymatic antioxidant defense systems to control the production of reactive oxygen or nitrogen species.[6] Antioxidant phytochemicals provide enormous scope in correcting the redox imbalance and replace synthetic antioxidants that are being limited due to several side effects like carcinogenicity, inflammations, and atherosclerosis.[6-8] Of late, attention toward the finding of natural antioxidants for the maintenance of human health, prevention, and treatment of diseases is increasing.[9]

Leea asiatica (L.) Ridsdale a folk medicinal plant of India, is used by the ethnic communities in the healthcare system. The plant is used by the tribes of Karnataka to treat bone fractures.[10] The roots of the plant are pounded and applied on boils and blisters in North Andaman Islands, while the leaves are used to treat worm infections and liver disorders by the tribes of Tripura, India.[1112] However, there is no scientific evidence with regard to the anthelmintic and antioxidant activities of the plant. Therefore, this study has been undertaken to examine the in vitro anthelmintic and in vitro antioxidant potency of the fractions of the methanol extract of Leea asiatica leaves.

MATERIALS AND METHODS

Plant materials

Leaves Leea asiatica (L.) Ridsdale were collected at Agartala, Tripura, India, and authenticated by Dr. B K Datta, Department of Botany, Tripura University, Tripura, India. A voucher specimen (TU/BOT/HEB/SS23072011c) has been kept in the herbarium of Plant Taxonomy and Biodiversity Laboratory, Tripura University.

Preparation of extract and fractionation

The fresh leaves of Leea asiatica were collected and cleaned, to remove the unwanted material. The leaves were air dried under shade, pulverized with a mechanical grinder, and extracted with methanol using the Soxhlet apparatus. The extract was concentrated to dryness under reduced pressure, to obtain the methanol extract. The methanol extract was further fractionated with the following solvents in the order of increasing polarity, namely, petroleum ether, ethyl acetate, and methanol. The yields afforded petroleum ether (PFLA, 6.8% w/w), ethyl acetate (EFLA, 37.5% w/w), and methanol (MFLA, 40.3% w/w) fractions.

Drugs and chemicals

2, 2-diphenyl-picrylhydrazyl (DPPH) and sodium nitroprusside (SNP) were purchased from Sigma Aldrich (Bangalore, India). Trichloro acetic acid (TCA), thio barbituric acid (TBA), and quercetin were procured from SD Fine Ltd., Mumbai. Linoleic acid, ammonium thiocyanate, ascorbic acid, and α-tocopherol were obtained from Sisco Research Laboratories Pvt. Ltd. (SRL), Mumbai. All other chemicals used in the study were obtained commercially and were of analytical grade.

Experimental animals

Indian adult earthworms (Pheretima posthuma) were used for anthelmintic activity. The earthworms were collected from moist soil and washed with normal saline to remove all fecal matter. Earthworms 3-5 cm in length and 0.1-0.2 cm in width were used for in vitro anthelmintic activity.

A healthy Wistar rat (150-200 g) was used for evaluation of the lipid peroxidation inhibition activity of fractions. The rat was maintained under controlled environmental conditions, with ad libitum access to standard rodent chow and water. The study was approved by the Institutional Animal Ethical Committee (Reg. No. 1305/ac/09/CPCSEA).

Anthelmintic evaluation

The anatomical and physiological features of an Indian earthworm resembles the intestinal round worm parasite in human beings, therefore, Pheretima posthuma was taken in this study to evaluate anthelmintic activity of fractions. Indian earthworms are divided into twelve groups each containing six earthworms approximately of equal size, in the following manner,

Group I: Control (3% Tween 80 in normal saline)

Group II-IV: MFLA (10, 20, and 50 mg/ml)

Group V-VII: EFLA (10, 20, and 50 mg/ml)

Group VIII-X: PFLA (10, 20, and 50 mg/ml)

Group XI-XII: Reference standard piperazine citrate (10 and 20 mg/ml)

The earthworms of each group were released into 50 ml of solution of the respective standard and test drugs. Observations were made for the time taken to paralyze and death of the individual worms. Time taken to paralysis was recorded when no movement could be noticed except when the worms were shaken vigorously. Time taken for death of the worms was noted after ascertaining that the worms lost their motility completely and did not respond even when shaken vigorously or dipped in warm water at 50°C, followed by fading of their body color.[13]

Antioxidant activities of fractions

2, 2-diphenyl-1-picryl-hydrazyl radical scavenging assay

The free-radical scavenging activities of fractions and ascorbic acid were investigated by the ability of the fraction to bleach the stable radical DPPH. Briefly, 1.0 ml of 0.1 mmol DPPH solution in methanol was mixed with 3.0 ml of the sample or standard drug solution at different concentrations. The mixture was incubated at room temperature, in darkness, for 30 minutes, and the absorbance was recorded at 517 nm.[14] The DPPH solution (1.0 ml) with methanol (3.0 ml) served as the control. All the experiments were performed in triplicate.

Nitric oxide radical scavenging assay

The nitric oxide scavenging activity of the fractions was estimated using the method followed by Yen et al. (2011).[15] Briefly, 4.0 ml of the fraction solution was added to 1.0 ml of a 25 mmol sodium nitroprusside solution, and the mixture was incubated at 37°C for two hours. At the end of the incubation period, 2.0 ml reaction mixture was mixed with 1.2 ml Griess reagent (1% sulfanilamide in 5% H3PO4 and 0.1% naphthyl ethylenediamine dihydrochloride in equal amounts). The chromophore produced during diazotization of the nitrite with sulfanilamide, its subsequent coupling with naphthyl ethylenediamine dihydrochloride, and the absorbance of the reaction solution was instantly read at 570 nm. Ascorbic acid was used as standard.

Inhibition of lipid peroxidation

The liver of a healthy adult Wistar rat (200-250 g) was dissected out and homogenized in phosphate buffer saline. One millilitre of liver homogenate (5% w/v) was mixed with 0.1 ml of fraction solution, and incubated for two hours at 37°C. One milliliter of 15% TCA and 1.0 ml of 0.67% TBA were added to the mixture after the incubation period, and again incubated in a boiling water bath for 15 minutes and then cooled in room temperature. The volume of the solution was made up to 5.0 ml by adding deionized water and then centrifuged at 2800 rpm for 10 minutes. The absorbance of the supernatant solution was determined at 532 nm.[16] The control was prepared without fractions and Rutin was used as the standard. All the experiments were performed in triplicate.

Ferric thiocyanate method

The Ferric thiocyanate (FTC) method was carried out to find the amount of peroxide at the initial stage of lipid peroxidation. The reaction mixture contained 200 μg fractions in 4.0 ml of ethanol, 2.5% linoleic acid in 4.0 ml of ethanol, 8.0 ml of 0.05 mol phosphate buffer (pH 7.0), and 4.0 ml of distilled water. The mixture was kept in a screw cap vial and placed in an incubator at 40°C, in darkness. To evaluate the extent of antioxidant activity, 0.1 ml of the reaction mixture was mixed with 9.7 ml ethanol (75%) and 0.1 ml ammonium thiocyanate (30%). Three minutes later, 0.1 ml of 20 mmol ferrous chloride was added to the reaction solution, and the absorbance of the solution was determined at 500 nm, every 24 hours, until the absorbance of the control attained its maximum[17]. Linoleic acid solution, without the addition of fraction/standard, was used as the control, a-tocopherol was used as a positive control.

Statistical analysis

The results are expressed as mean ± SEM. The statistical difference was tested by using one-way analysis of variance (ANOVA) followed by Turkey tests. A level of P < 0.05 was used as the criterion for statistical significance.

RESULTS

Anthelmintic activity of fractions

The fractions exhibited more potent activity at a higher concentration (50 mg/ml) against Pheretima posthuma (earthworm). The results showed that at a 10 and 20 mg/ml concentration, piperazine citrate showed the potent activity for death time (68.33 ± 0.78 and 51.45 ± 0.54 minutes, respectively). The ethyl acetate fraction of L. asiatica showed better paralysis activity, while the methanol fraction showed better death time compared to other fractions. EFLA caused paralysis after 19.21 ± 0.84 minutes and 13.99 ± 0.59 minutes at 20 and 50 mg/ml concentrations, respectively, while death time reported for MFLA was equal to 74.54 ± 0.82 minutes and 63.76 ± 0.73 minutes at the same concentration. Both EFLA and MFLA demonstrated almost similar activity and paralysis in a lesser time as compared to piperazine citrate at a higher concentration of 50 mg/ml, although the standard drug caused the death of the worm in quicker time. The paralysis and death times of the fractions and standard drug are given in Table 1.

Table 1

In vitro anthelmintic effect of fractions of the methanol extract from Leea asiatica leaves against Pheritima posthuma

Antioxidant activity of fractions

The fraction showed a concentration-dependent DPPH· scavenging activity. The IC50 v value of ascorbic acid, MFLA, EFLA, and PFLA were 4.0, 12.5, 9.5, and 29.5 μg/ml, respectively. At a concentration of 32 μg/ml, the concentrations of MFLA, EFLA, and PFLA possessed 70.22, 76.22, and 51.99% scavenging activity [Figure 1].

Figure 1

DPPH radical scavenging effect of fractions of the metha-nol extract from Leea asiatica leaves. Each value is expressed as mean ± SEM (n = 3)

The fractions also possessed significant Nitric oxide (NO) scavenging activity. EFLA possessed the highest and a better nitric oxide radical scavenging activity with an IC50 value of 13.0 μg/ml, while IC50 v value of ascorbic acid, MFLA, EFLA, and PFLA were found to be 24.9, 17.1, 13.0, and 55.5 μg/ml. EFLA possessed 78.3% scavenging activity at 80 μg/ml concentration, while at the same concentration ascorbic acid possessed a 70.12% scavenging effect [Figure 2].

Figure 2

Nitric oxide radical scavenging effect of fractions of methanol extract from Leea asiatica leaves. Each value is expressed as mean ± SEM (n = 3)

Fractions significantly inhibit the formation of malondialdehyde, a cytotoxic product formed during lipid peroxidation. The IC50 v value of MFMS, EFMS, PFMS, and Rutin were 74.2, 57.0, 119.9, and 62.0 μg/ml. At a concentration of 120 μg/ml, EFLA and Rutin produced a 74.61 and 70.4% lipid peroxidation inhibition effect [Figure 3].

Figure 3

Inhibition of lipid peroxidation in liver tissues by the fractions of methanol extract from Leea asiatica leaves. Each value is expressed as mean ± SEM (n = 3)

The fractions significantly (P < 0.05) inhibited the peroxidation of linoleic acid. On the ninth day, the absorbance value of the control was decreased, which suggested that the formation of peroxides was stopped because of the non-availability of linoleic acid. The ethanol fraction showed a better activity than α-tocopherol, when observed during the incubation time [Figure 4].

Figure 4

Antioxidant activity of fractions of methanol extract from Leea asiatica leaves by the FTC method. Results are of triplicate measurements

DISCUSSION

This study has been undertaken to prove the traditional use (anthelmintic) of Leea asiatica, and find the antioxidant capacity of the fractions of the crude methanol extract from these plant leaves. Helminthic infections of the gastrointestinal tract of human beings and animals have been acknowledged to have adverse effects on the health standards, with a consequent lowering of resistance to other diseases. Nowadays, resistance to the available synthetic drugs is a major problem. Therefore, in recent years, a search for plant-derived drugs is the primary choice of researchers, as they are believed to have lesser side effects and are more compatible with the physiological flora.[418] In the search for a new drug with anthelmintic activity, different species of worms, like earthworms, Ascaris, Nippostrongylus, and Heterakis are used by the researchers. However, due to the availability and resemblances with intestinal ‘worms’ in their reaction to anthelmintics, earthworms have been used widely for the initial evaluation of anthelmintic compounds in vitro.[4] Piperazine citrate acts by enhancing the chloride ion conductance of the worm muscle membrane. It produces hyperpolarization and decreases the excitability, which leads to muscle relaxation and flaccid paralysis. This action may cause the death of the worm by damaging the mucopolysaccharide layer.[419] Different fractions of the Leea asiatica leaves methanol extract not only demonstrate paralysis, but also cause the death of worms, even at a low concentration of 10 mg/ml. The potent wormicidal activity of methanol and the ethyl acetate fraction of the methanol extract of Leea asiatica leaves against earthworms, suggests that it is effective against the parasitic infections of humans.

In the present study, the antioxidant activity of the fractions has also been investigated and the report shows that the fraction has a significant antioxidant activity. The DPPH· scavenging assay is based on the principle that the decolorization of DPPH will be caused by the antioxidant compounds. This DPPH· scavenging is also associated with the inhibition of lipid peroxidation.[20] The fractions show a significant DPPH radical scavenging activity; hence, the effect of fractions may be credited with a direct role in trapping the free radicals, by donating a hydrogen atom.

Nitric oxide is generated by the phagocytes and endothelial cells, to produce more reactive species like peroxynitrites, which can be involved in several toxic reactions, including, thiol group oxidation, protein tyrosine nitration, lipid peroxidation, and DNA modification. NO• is an imperative pleiotropic mediator of several physiological processes including neuronal signaling, smooth muscle relaxation, and regulation of cell-mediated toxicity, but overproduction may lead to tissue toxicity.[2122] The ethyl acetate fraction produces a better effect, which indicates that in EFLA, certain substances are potential antioxidants.

Lipid peroxidation can be explained as an oxidative deterioration process of polyunsaturated fatty acids, which can induce genomic and mtDNA damage, and ultimately lead to a disease condition. Inhibition of lipid peroxidation is thus a fundamental property of antioxidants, by virtue of which they can stop the initiation and/or propagation of a free radical reaction and can save health.[1623] Results suggest that fractions have a potential to inhibit lipid peroxidation significantly (P < 0.005) in liver tissue, entailing their defensive effect against lipid oxidation.

The FTC method was used to determine the total antioxidant capacity of the sample, which measured the amount of peroxides formed at the initial stage of lipid oxidation. In this assay, the ability of antioxidants to scavenge peroxyl radicals through hydrogen donation, during polyunsaturated fatty acids (PUFA) oxidation, was evaluated.[1724] The results clearly indicated the potent total antioxidant capacity of the fractions.

CONCLUSION

The folk use of Leea asiatica as an anthelmintic has been confirmed, as the fractions displayed activity against the worms used in the study. On the basis of the results obtained in the present study, it is concluded that this plant can be viewed as a potential source of natural anthelmintic and antioxidant compounds. Further studies are required to find the active phytoconstituents and establish the mechanism (s) of action.