Assessment of Anthelmintic Property and Insilico study of phytocompounds in roots of Dechaschistia crotonifolia Wight & Arn.

Background:Worm infections in developing countries were reported high. Phytoconstituents have been a vital role for the treatment of many ailments. The current study was aimed assess for anthelmintic activity of different root extracts of Dechaschistia crotonifolia Wight & Arn. belongs to the family Ebanaceae against Pheretima posthuma. Further Insilico study was carried out for phytocompounds present in Dechaschistia. Results: The chloroform, ethylacetate and ethanol extract of Dechaschistia crotonifolia Wight & Arn. were considered for the study of anthelmintic property on earthworms at concentrations 20 mg/ml, 40 mg/ml and 60 mg/ml. During this study, the parameters paralysis time (Pt) and Death Time (Dt) of adult Indian earthworms was observed. As a standard and control Albendazole 10 mg/ml and 2% Tween 80 in distilled water were taken respectively. The study resulted that ethanolic extract was signicant when compared with the Albendazole 10 mg/ml. Docking studies revealed the all phytocompounds in Dechaschistia shown binding anity, however comparatively scopoletin and stigmasterol had shown a good binding anitiy about -7.7 Kcal/mol and -7.6 Kcal/mol compared to standard drug Albendazole which was shown about -8.7 Kcal/mol. Conclusion: The study revealed that the ethanol extract of Dechaschistia crotonifolia Wight & Arn. at a concentration of 60mg/ml exhibited a stronger anthelmintic property compared to Albendazole 10mg/ml. A dose dependent anthelmintic activity is exerted by all the extracts in an ascending manner Chloroform<Ethyl acetate<Ethanol. These observations were made evidenced by docking studies of phytocompounds in Dechaschistia as the phytocompounds were shown excellent docking score when compared with standard Albendazole.


Background
Diseases caused by helminths are chronic. Helminthiasis is infested to human beings with worm's likely pinworm, round worm or tapeworm [1]. The diseases caused by parasites results in morbidity and leads to the condition onchocorciasis and Schistosomiasis. The more number of worm infections has been reported in developing countries due to lack of proper hygienic conditions. By considering the affordability and various side effects of synthetic compounds, a preferability towards herbal medicines were choosen. An adult Indian earthworm Pheretima posthuma is selected for assessment of anthelmintic property as it shows similarity in anatomy and physiology of round worm parasites resides in intestine of human beings.
Dechaschistia crotonifolia Wight & Arn. is a shrub consists of dense whitish wooly on stems and branches [2]. The leaves are in ovate lance shaped measures 3-6 cm long, 2-4 cm width. The base of leaf is heart shaped or rounded, pointed apex with coarsely toothed margins. Leaves are velvety, bears 1.5cm long stalks. It represents with Yellow owers with dark maroon centered in single leaf axils. The Sepal cup is bell in shape, 1-1.5cm long cup encloses capsules and seeds. The seeds are kidney-shaped. It is most common in the deciduous forests of peninsular India. Flowering takes place in the month of March to June.
Earlier preliminary phytochemical assessment was made [3,4]  to remove extraneous matter. The extract at concentration of 30mg/ml, 60mg/ml & 80mg/ml was used to examine the time of paralysis (Pt) and Death (Dt). The selected earthworms are categorized into 11 groups of 6 each viz., control group treated with 2% Tween 80 in distilled water, 9 Test groups treated with concentrations of 30mg/ml, 60mg/ml & 80mg/ml of each Chloroform, Ethylacetate and Ethanol extract of Dechaschistia crotonifolia Wight & Arn. and standard group treated with 10mg/ml concentration of Albendazole. Earthworms are treated with volume of 10ml of each concentration of standard, control and test solutions respectively. The time taken for Paralysis (Pt) and Death (Dt) was noted.

In-silico study
For molecular docking study [10,11,12] Autodock vina is used for prediction of potent phytocompounds of Dechaschistia viz., Parvi oral, Syriacusin A, Syriacusin B, Syriacusin C, Scopoletin and Stigmasterol against active site of β-tubulin. The chemical structures of phytoconstituents Parvi oral, Syriacusin A, Syriacusin B, Syriacusin C, Scopoletin and Stigmasterol were obtained from Pubchem Project Database shown in Fig. 3. They were structurally plotted in Discovery Studio Biovia. The 3D structure of protein βtubulin (PDB ID: 1oj0) is collected from Protein Data Bank (www.rcsb.org/pdb) shown in Fig. 2. The x, y & z attributes along with radius is noted. Further the structure is prepared by removing water, adds up polar hydrogen bond and made torsion free.

Statistical Analysis
The values were represented as mean ± S.D; via one-way ANOVA. The analysis was carried out by using Graph pad Prism (Version 3, U.S.A.) software program. P < 0.05 was taken into statistically signi cant.

Results
Anthelmintic activity Table 1

In-silico Study
Docking revealed that out of 6 phytocompounds Parvi oral, Syriacusin A, Syriacusin B, Syriacusin C, Scopoletin and Stigmasterol with protein β-tubulin had shown docking scores of -6.3 kcal/mole, -6.9 kcal/mole, -6.0 kcal/mole, -6.7 kcal/mole, -7.7 kcal/mole, -8.7 kcal/mole and standard drug Albendazole shown at -7.6 kcal/mole. The phytocompounds had shown hydrogen bond interactions with aminoacid and the results discloses the hydrogen bond interactions are associated with aminoacids in each ligand & protein complex except with Syriacusin C. The outcomes are depicted in table 3 and the complexes are made visualized in Fig no 4.

Discussion:
Helmenthiasis is considered as disease in south Asia including India. Hence and investigation in larger no on alternative sources are made for their anthelmintic acitivity [13][14][15][16][17]. The considerations of anthelmintic activity due to avonoids and steroids were stated earlier. The avonoids biochanin A and genistein was shown effective anthlemintic activity against Aspiculuris tetraptera. Anthelmintic tests according to the procedure of Hounzangbe Adote et al were conducted for the phytocompounds against Haemonchus contortus. The best activity was obtained with avonoids [18].
Aqueous extract of whole plant of Amaranthus spinosus had exerted anthlemintic activity against Pheritima posthuma in dose dependent manner due to presence of steroids and avonoids [19]. The study aimed to evaluate anthelmintic activity of chloroform ethylacetate and ethanolic root extract of Dechaschistia crotonifolia. The pharmacognositical investigations were carried out. The qualitative chemical screening of Dechaschistia was studied and revealed the presence of steroids, avonoids and tannins more in ethanolic extract. In earlier studies Trinorcadalenes, parvi orals A, Syriacusin A, B & C, Scopoletin and Stigmasterol were isolated and their structures along with resonance were elucidated by 1 H and 13 C NMR spectroscopy [4]. It is possible to learn the mechanism of action of phytoconstituents in virtual screening methods. These methods make to design phytoremedies for various diseases. A various phytocompounds for antihelmintic activity was investigated [20,21,22]. Docking studies signify the fact that out of 6 phytochemicals stigmasterol and scopoletin shown -8.7 kcal/mole and syriacusin B with least among 6 phytochemicals was -6 kcal/mole. The docking score of scopoletin and stigmasterol had shown at -7.6 kcal/mole and -8.7 kcal/mole representing good binding a nity between phytocompound and β-tubulin than between the protein (β-tubulin) and standard drug albendazole, shown the docking score at -7.6 kcal/mole. ADME analysis of phytocompounds and standard revealed that zero violation of drug likeness and obeyed the Lipinski rule.

Discussion:
Helmenthiasis is considered as disease in south Asia including India. Hence and investigation in larger no on alternative sources are made for their anthelmintic acitivity [13][14][15][16][17]. The considerations of anthelmintic activity due to avonoids and steroids were stated earlier. The avonoids biochanin A and genistein was shown effective anthlemintic activity against Aspiculuris tetraptera. Anthelmintic tests according to the procedure of Hounzangbe Adote et al were conducted for the phytocompounds against Haemonchus contortus. The best activity was obtained with avonoids [18].
Aqueous extract of whole plant of Amaranthus spinosus had exerted anthlemintic activity against Pheritima posthuma in dose dependent manner due to presence of steroids and avonoids [19]. The study aimed to evaluate anthelmintic activity of chloroform ethylacetate and ethanolic root extract of Dechaschistia crotonifolia. The pharmacognositical investigations were carried out. The qualitative chemical screening of Dechaschistia was studied and revealed the presence of steroids, avonoids and tannins more in ethanolic extract. In earlier studies Trinorcadalenes, parvi orals A, Syriacusin A, B & C, Scopoletin and Stigmasterol were isolated and their structures along with resonance were elucidated by 1 H and 13 C NMR spectroscopy [4]. paralyze and comparatively with standared drug Albendazole it is mere the same. All the extracts at 20mg/ml were taken too long to paralyze or to kill the adult earthworms.
It is possible to learn the mechanism of action of phytoconstituents in virtual screening methods. These methods make to design phytoremedies for various diseases. A various phytocompounds for antihelmintic activity was investigated [20,21,22]. Docking studies signify the fact that out of 6 phytochemicals stigmasterol and scopoletin shown -8.7 kcal/mole and syriacusin B with least among 6 phytochemicals was -6 kcal/mole. The docking score of scopoletin and stigmasterol had shown at -7.6 kcal/mole and -8.7 kcal/mole representing good binding a nity between phytocompound and β-tubulin than between the protein (β-tubulin) and standard drug albendazole, shown the docking score at -7.6 kcal/mole. ADME analysis of phytocompounds and standard revealed that zero violation of drug likeness and obeyed the Lipinski rule.

Conclusions
The current study aimed in evaluating anthelmintic activity of Dechaschistia crotonifolia. The test revealed a signi cant anthelmintic activity of ethanolic root extract and the remaining extracts were also shown but it is considered as dose dependent manner. This activity is supported by docking studies.
Docking studies shown that binding poses and distance measurement of β-tubulin complexes parvi orals A, Syriacusin A, Syriacusin B & Syriacusin C, Scopoletin and Stigmasterol reveals that the lead phytocompounds were in near proximity associated with most active site of aminoacids. This con rms the phytocompounds present in Dechaschistia need to investigate for the discovery of new generation of drugs as they will be remedies against organisms causing helminths.

Declarations
Ethics approval and consent to participate This study did not take include samples from humans. So no content was required.

Consent for publication
Not applicable Availability of data and materials All data generated or analyzed during the study are included in this article.

Competing interests
Authors declare that they have no competing interest.