Studies on the anthelmintic potentials of the roots of Asparagus racemosus willd. (Asparagaceae)

The Santhal tribe in Assam, India use the roots of Asparagus racemosus (Asparagaceae) as a deworming remedy. The study aimed to investigate the anthelmintic credentials of this plant, using two representative groups of helminth parasites. The in vitro testing was conducted against Hymenolepis diminuta (cestode) and Syphacia obvelata (nematode). Parasites were exposed to 10, 20 and 30 mg/ml concentrations of plant extract, and efficacy was adjudged on the basis of parasites paralysis and mortality. In vivo efficacy was examined using H. diminuta-rat and S. obvelata-mice models where animals were administered 125, 250 and 500 mg/kg doses of extract. In vitro assay, against H. diminuta revealed that at 30 mg/ml concentration the extract showed almost a comparable efficacy with that of reference drug praziquantel (PZQ) (1 mg/ml). The in vitro efficacy of extract against S. obvelata was however lower than H. diminuta. In vivo studies against H. diminuta at 500 mg/kg revealed 53.88 and 24 % reduction in eggs per gram (EPG) and worm counts respectively. Against S. obvelata the extract showed 26.61 and 30.93 % reduction for the same. The findings of this study present suggest that the roots of A. racemosus are effective against intestinal helminthic infections and justifies its use as an anthelmintic in the traditional medicine of the Santhals.

Experimental studies on this plant have shown that besides its efficacy as anti-dyspepsia and anti-ulcerogenic agent [6] this plant has been found to reverse memory loss [11] and also possesses immunoadjuvant, antioxidant, and anti-diarrheal properties [12][13][14]. The chemical studies on this plant have revealed presence of important steroidal saponins, called shatavarins, in its roots [15,16]. Besides, an isoflavone, 8-methoxy-5,6,4′trihydroxyisoflavone 7-o-β-d-glucopyranoside, has also been isolated from the roots of A. racemosus [17]. Owing to its multiple uses in traditional medicine, A. racemosus is highly demanded in Indian markets, but the supply is considered inadequate.
During a recent ethnomedicinal survey in Assam, India to collect information about anthelmintic plants, it emerged out that root decoction of A. racemosus is commonly used by the natives of the area to cure intestinal worm infections. In order to scientifically validate the aforesaid claims of the people, a systematic ethnomedicinal field survey was conducted to document some key facts about the usage pattern of this plant in the study area. In the present study, investigations on the in vitro and in vivo anthelmintic potentials of methanolic root extract of A. racemosus, involving a cestode, i.e. Hymenolepis diminuta-rat and a nematode, Syphacia obvelata-mice models was carried out, so as to comment about the usefulness of this plant as anthelmintic.

Plant material
Plant material was collected from natural habitats in Kokrajhar, Assam, India and identified by a taxonomist. Herbarium was made and submitted to the Department of Botany herbarium museum and assigned a voucher number (NEHU-12098). A copy of the herbarium has been retained in the Parasitology and Ethnopharmacology Lab. The taxonomic status of plant was also verified with the database of Kew working list of all known plant species (www.theplantlist.org). The roots were washed in tap water, air-dried and powdered in a blender. The powdered plant material was extracted in methanol using Soxhlet apparatus. The yield of extract was 7.98 % (w/w).

Experimental animals
Albino rats of either sex (Wistar strain), weighing about 180 to 220 g, and Swiss mice of both either sex, weighing about 25 to 30 g were used to perform in vivo experiments. Animals were maintained in acrylic cages and had ad libitum access to food and water. S. obvelata infection was maintained in Swiss mice as described by Gogoi and Yadav (2016) [18]. Infection of H. diminuta was maintained in Wistar rats as described by Tangpu et al. (2006) [19].
All the experiments on laboratory animals were done after the approval from the Institutional Ethics Committee (Animal models), North-Eastern Hill University, Shillong, vide, Member Secretary, IEC, NEHU, letter dated December 4, 2014. All experiments on animals adhere to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines and the EU Directive 2010/63/EU for animal experiments.

Ethnomedicinal survey
Firstly, data was collected from traditional healers, called as Ojhas, and local people, using a closed type semistructured questionnaire, about different medicinal plants used against intestinal-helminthic infections in different villages of Kokrajhar district, Assam, India. The study involved 300 respondents, interviewed from April, 2014 to April, 2015. The questionnaire included queries such as, the local names of various plants that are used by the natives against worm infections, the form of their preparations, their dosage, perceived efficacy, and opinion about adverse effects.

Anthelmintic assays
In vitro study H. diminuta and S. obvelata were collected from infections maintained in rats and mice respectively in the laboratory. The parasites were washed in phosphatebuffered saline (PBS) and exposed to 10, 20 and 30 mg/ ml concentrations of extract in separate petridishes (n = 5) inside an incubator at 37 ± 1°C. A group of worms (n = 5) were placed in PBS alone and served as negative control. In addition, another set (n = 5) was placed in reference drugs, praziquantel (PZQ) (1 mg/ml) and albendazole (ABZ) (5 mg/ml). Observations for parasites paralysis and mortality were made at regular time intervals [20].

In vivo study
For in vivo testings in H. diminuta, rats were orally fed with 5 cysticercoid larvae to induce infection. Animals were divided into 5 groups (n = 5). Group 1 served as negative control and was administered only the vehicle. Group 2 to Group 4 of animals were administered with 125 mg/kg, 250 mg/kg, and 500 mg/kg doses of extract to animals. Group 5 of animals served as positive control and were administered PZQ at 5 mg/kg concentration. Eggs per gram (EPG) counts were done counts were done 3 days prior to and after the dosings. Animals were sacrificed on the 29 th day post-inoculation to calculate the worm burden.
For in vivo testings in S. obvelata, mice were kept in infected bed for 15 days. Establishment of infection was confirmed by cellophane tape test. Animals were divided into 5 groups, with 5 animals in each group. Group 1 served as negative control and was administered only the vehicle. Group 2 to 4 of animals were given 125 mg/ kg, 250 mg/kg, and 500 mg/kg doses of extract. Group 5 of animals were administered ABZ (20 mg/kg). EPG count was done for 3 days prior to and after dosing. The animals were dosed for 5 days and they were sacrificed for worm recovery count on day 11. The percentage reductions in EPG and worm counts were done as described by Kozan et al. (2006) [21].
The prescribed dose administered by practitioners to their clients was taken as the median dose i.e., 250 mg/ kg body weight (b.w.) and two doses, one exponentially lower (125 mg/kg b.w.) and the other higher (500 m g/ kg b.w.) to the median dose were selected.

Statistical analysis
The experimental data are represented as mean ± standard error of mean (SEM). The in vitro data were analyzed by student's t-test and the p value ≤ 0.05 was considered to be statistically significant between the values from control and treated groups. The data from in vivo tests was analyzed by one-way analysis of variance (ANOVA), followed by Tukey's test. The p value ≤ 0.05 was considered to be statistically significant. Statistical calculations were done using OriginPro 8.

Ethnomedicinal survey
In ethnomedicinal study, A. racemosus was found to be the third most commonly used anthelmintic plant in the study area, after two other plant species, Sesbania sesban (L.) and Cyperus compressus Linn. Of 300 respondents interviewed in this work, about 80 % individuals were recorded to use the roots of this plant as a deworming remedy. It was also observed that about 72 % of respondents consume one dose of the root infusion of this plant for about a week to cure worm infections. However, only 56 % of the interviewed persons considered it to be highly efficacious as a deworming remedy. Over and above, 82 % respondents had a view point that using this plant preparation does not cause any adverse effect to the user. An infusion of A. racemosus is prepared by grinding fresh roots which is then fed to patients or they are either made into pellets and administered (Fig. 1).

Anthelmintic assays In vitro study
The extract showed dose-dependent effects (p < 0.0001) against H. diminuta. Notably, 30 mg/ml concentration of extract revealed almost a comparable efficacy with that of reference drug PZQ at 1 mg/ml concentration. However, the in vitro efficacy of extract against S. Fig. 1 Fresh roots of Asparagus racemosus obvelata was comparatively lower than 5 mg/ml concentration of reference drug, ABZ. Against H. diminuta, at the highest dose, parasites showed mortality in 5.03 ± 0.05 h, whereas parasites placed in PZQ showed mortality a little later, i.e. in 5.96 ± 0.01 h (Fig. 2). Control worms survived till 42.15 ± 0.20 h. Against S. obvelata, at the same concentration of extract, the parasites showed mortality in 11.70 ± 0.04 h, whereas parasites placed in ABZ showed mortality much earlier (7.23 ± 0.01 h) (Fig. 3). Herein, the control worms showed physical activity until 34.16 ± 0.007 h.

In vivo study
The administration of A. racemosus extract to rats, harboring H. diminuta infections, showed a significant reduction (p < 0.001) in EPG counts and worm recovery rate, in a dose-dependent manner. The animals treated with 500 mg/kg dose of extract, for 5 days, showed 53.88 % reduction in EPG counts, compared to EPG counts of pre-treatment period, while, the reduction in worm count was recorded as 24 %. The treatment with PZQ (5 mg/kg) showed 87.08 and 84 % reductions in EPG counts and worm counts, respectively, while, EPG and worm counts in control group maintained a uniform trend throughout the observation period ( Table 1). The administration of extract to mice, carrying S. obvelata infections, showed a significant decrease in EPG counts of animals during the post-treatment period. During the first 3 days (pre-treatment EPG), eggs were detected in the cellophane test of all animals. However, after 5 days of treatment, 26.61 and 25.93 % reduction in EPG counts and worm counts were recorded in animals treated with 500 mg/kg dose of extract. On the other hand, ABZ (20 mg/kg) showed greater decrease in EPG counts (78.57 %) and worm counts (94.11 %) of animals, at necropsy. There was no noticeable variation observed in EPG counts of animals in control group, between the pre-and-post treatment periods ( Table 2).

Discussion
Numerous studies have been conducted to investigate the anthelmintic effects of medicinal plants used in traditional medicine, which have not only involved different parasite species, but have also used an array of experimental models to validate the therapeutic potentials of plant species [22]. While some researchers have used Fig. 2 In vitro anthelmintic effects of A. racemosus methanolic root extract against H. diminuta. Data is expressed as mean ± SEM. *p˂0.0001 compared with control group, student's t-test only in vitro assays [23,24], other workers have also employed a variety of in vivo models to validate the anthelmintic properties of numerous plant species [25,26]. Nonetheless, a review of literature also reveals that in vitro studies alone do not provide a solid scientific basis about the anthelmintic credentials of a substance in question [27]. Therefore, it is now widely recognized that to obtain a more authentic anthelmintic profile of a plant, in vitro studies should be followed-up by proper in vivo validation processes [18]. Therefore, to provide a better therapeutic profile of A. racemosus, in the present study, both the in vitro as well as in vivo studies on the plant were conducted. In addition, the helminth species used herein were also selected from two different major  *Administration of plant extract and praziquantel on days 21-25 post-inoculation with five cysticercoids/rat a p<0.001 as compared to control value, b p<0.001 as compared to pre-treatment period, one-way ANOVA, followed by Tukey's test groups, i.e. a cestode, H. diminuta and another nematode, S. obvelata, that were maintained in two different rodent models. The findings of ethnomedicinal study revealed that A. racemosus is considered as the third most commonly used remedy for intestinal-worm infections in the study area. Further, about 80 % of people in study apprised about the regular use of roots of this plant as a traditional anthelmintic. Likewise, 56 % of the interviewed persons considered it to be highly efficacious against intestinal helminths. However, 82 % respondents also had an opinion that using this plant preparation does not cause any adverse effect to them. Thus, findings of this field study provided a concrete basic lead to put this plant for proper evaluation of its anthelmintic potentials.
In the present study, both the in vitro and in vivo studies of A. racemosus root extract against H. diminuta and S. obvelata showed a dose-dependent efficacy (p < 0.001). The comparative in vitro and in vivo data analysis of extract revealed that the extract was comparatively more efficacious against cestode, H. diminuta than nematode, S. obvelata. The in vitro efficacy of extract against H. diminuta was also almost comparable with that of 1 mg/ml concentration of PZQ, the reference drug. However, in in vivo studies against H. diminuta, the extract showed a comparatively low efficacy than PZQ (5 mg/kg). On the other hand, the in vitro as well as in vivo efficacy of extract against the nematode, S. obvelata was noticed to be comparatively lower than cestode parasite.
The available published data reveal two more studies related to anthelmintic potentials of A. racemosus. Firstly, Kiranmayi et al. (2012) in their study on efficacy of root extract of A. racemosus showed that root extract of this plant possesses wormicidal activity and thus, may be useful as an anthelmintic [28]. Similarly, Gupta et al. (2012) also tried to prove the presence of anthelmintic efficacy of this plant based on their experiments on Pheretima posthuma (earthworm) [29]. It may however be mentioned here that both these studies were based on testing of extracts on adult Indian earthworms as model organisms, which as such do not bear any anatomical or physiological similarities with parasitic roundworms. Further, in the study by Kiranmayi et al. (2012), the concentration of extract tested was also considerably high, i.e., 100 mg/ml and 200 mg/ml [28]. In the light of these stated facts, it may be concluded that the findings of these two in vitro studies about anthelmintic potentials of A. racemosus have only limited relevance.
To date, only a few studies on anthelmintic potentials of medicinal plants have backed their in vitro assays with in vivo studies. For example, Gogoi and Yadav (2016) and Gogoi and Yadav (2017) have supplemented their in vitro anthelmintic studies of plants with in vivo studies, using H. diminuta-rat and S. obvelata-mice models [18,30]. The findings are in agreement with that of Gogoi and Yadav (2016), who in their in vitro and in vivo efficacy testing of Caesalpinia bonducella, against H. diminuta and S. obvelata, also observed a dosedependent efficacy of extract [18]. However, in this in vitro study, the 30 mg/ml concentration of extract caused mortality of H. diminuta in 2.5 ± 0.2 h and S. obvelata in 3.57 ± 0.16 h, taking almost similar time duration. However, in in vivo study, the same extract showed a comparatively better efficacy on S. obvelata, than against H. diminuta. Based on these findings, Gogoi and Yadav (2016) suggested that the leaf extract of C. bonducella possesses significant anthelmintic effects and supported its use as an anthelmintic in traditional medicine [18].
In another study by Gogoi and Yadav (2017), the in vitro and in vivo anthelmintic efficacy of Croton joufra leaf extract was investigated against H. diminuta. The in vitro study showed a dose-dependent effect on parasite, and the highest concentration of the extract (30 mg/ml) caused mortality of in worms 1.53 ± 0.12 h, p<0.001 as compared to control value, b p<0.001 as compared to pre-treatment period, one-way ANOVA, followed by Tukey's test compared to 3.46 ± 0.10 h shown by PZQ (1 mg/ml). The in vitro findings were corroborated by in vivo experiments, which showed a very high efficacy of extract against H. diminuta. The overall findings of this study suggest that C. joufra leaf extract possesses significant anthelmintic efficacy and support its traditional anthelmintic claims [30].

Conclusions
The findings of the present study scientifically validate the common belief of the local people of the Santhal tribe in Assam, that roots of Asparagus racemosus are highly effective as a deworming remedy. More precisely, this study suggests that the root extract of this plant species possesses a comparatively better efficacy against cestode than against the nematode parasite, as evidenced by in vitro and in vivo experimentations.