This medicinal plant is a rich source of phytochemical constitutes of secondary metabolites like phenol, alkaloid, terpanoid, and glycosides. These metabolites jointly or separately may act by inhibition of tubulin polymerization and blocking glucose uptake and any damage to the mucopolysaccharide membrane of worms which will expose the outer layer restricting their movement which finally may cause paralysis and ultimately death of parasite.
Extractive percentage yields and weight of extract are shown in Fig. 2. Maximum percentage yield of extract was obtained in methanolic extract (20%), and minimum percentage yields of extract were obtained in petroleum ether extract (9.46%). Shweta et al. (2011) reported the P. fistulosus preparation of plant fruit extract material defatted with petroleum ether (60–80 °C) and then extracted with methanol in a Soxhlet apparatus. The percentage yield is 2.6% w/w and 4.7% w/w with respect to dried plant material in methanol. In our study, hot extraction method observed the percentage yield of methanolic extract has five times more yield.
Praecitrullus fistulosus Pangalo fruit plant material was extracted using different solvent (methanol, petroleum ether, and chloroform) systems. Normal saline solution was taken as a control treated to the earthworms (Pheretima posthuma) which remained active with whole body movements (Table 1). Using 1% albendazole drug as a standard treatment, the earthworms become shrunken and remain motile only in some body parts, paralyzed at 6.13 min, and then finally dead after 7.26 min. In case of 2% albendazole drug solution, the earthworms become paralyzed at 3.08 min and dead after 5.01 min (Table 1). In case of 5% albendazole drug solutions, the earthworms become slender, shrunken, paralyzed at 2.00 min, and dead after 4.03 min (Table 1).
In the earthworms tretaed with 1% methanolic extract of Praecitrulus fistulosus pangalo, slow movement, paralysis at 8.00 min, and death after 10.02 min were observed (Table 1). In the earthworms tretaed with 2% methanolic extract of Praecitrulus fistulosus Pangalo fruit, slight movement, paralysis at 5.06 min, and death after 6.17 min were observed (Table 1). In the earthworms treated with 5% methanolic extract of Praecitrulus fistulosus Pangalo fruit, strong paralysis at 3.00 min and death after 4.05 min (Table 1) were observed.
Shweta et al. (2011) reported the P. fistulosus methanol extract of 1% and 2% time taken for death 393.75 ± 25.769 min and 398.75 ± 29.324 min, respectively. The crude extract in comparison with Soxhlet our result of methanol extracts of 1%, 2% and 5% time taken for death 10.02 ± 0.027, 6.17 ± 0.017, and 4.05 ± 0.011 min respectively. This result shows all the extracts of methanol have better response to the hot extraction method compared to the Soxhlet extraction method. Shweta et al. (2011) collected the fruit from Bhopal, and our sample was collected from Gujarat. The effect of the different geographical areas also affect the yield and better activity of the extracts, and for that reason, the increase in secondary compounds gives more activity. For this reason, the collected sample of Gujarat has better activity. The Gujarat state is also a semi arid region which might be the reason the active secondary compound produce more and give more activity. Similar type of the study is also reported from other authors. The methanol extracts of A. caudatus and A. viridis also exhibited dose-dependent anthelmintic activities that caused paralysis at 19.21, 14.33 min (at 60 mg/ml); 12.16, 10.2 min (at 80 mg/ml); and 5.75, 7.8 min (at 100 mg/ml), and death at 27.7, 26.6 min (at 60 mg/ml); 18.6, 18.6 min (80 mg/ml); and 8.5, 12.7 min (100 mg/ml) post-treatment. The earthworms were more sensitive to the extracts of A. spinosus, A. caudatus, and A.viridis at 60, 80, and 100 mg/ml concentrations as compared to the reference drug piperazine citrate (10 mg/ml). All the three plants’ methanol extracts were more effective in causing the death of the worms as well as promoting paralysis (Ashokkumar et al. 2010). Our results in comparison with other authors proved better anthelmintic activity. This extract also showed very short time for the death of the earthworms. This different author reported different plant species compared to our result which give better activity of anthelmintic activities in the methanolic extract of 1%, 2%, and 5%. These extracts also take very short time for the death of the earthworms.
One percent petroleum ether extract of Praecitrullus fistulosus (Stocks) Pangalo fruit was treated to the earthworms which resulted to their slow movement, paralysis at 13.21 min, and death after 16.02 min (Table 1). Two percent petroleum ether extract of Praecitrullus fistulosus (Stocks) Pangalo fruit was treated to the earthworms in which slight movement, paralysis at 9.18 min, and death after 11.01 min were observed (Table 1). Five percent petroleum ether extract of Praecitrullus fistulosus (Stocks) Pangalo fruit was treated to the earthworms which resulted to strong paralysis at 7.05 min and death after 8.04 min; in this case, death takes more time (Table 1). The results of our study showed 5% decrease in the death time of earthworms. Out of the three percentage solvents, best results and death of the earthworms within a short time were observed in 5% petroleum ether extracts compared to other percentage solvents. Shweta et al. (2011) reported the same plant fruit extract of petroleum ether extract 1% and 2%. In this time taken, death takes more time. Our study shows the result compared to five times less for taken death time for earthworms. Even our study in 5% exract times less for taken death for earthworms.
One percent chloroform extract of Praecitrullus fistulosus (Stocks) Pangalo fruit was treated to the earthworms resulting in their slow movement, paralysis at 16.16 min, and death after 18.4 min (Table 1). Two percent chloroform extract of Praecitrullus fistulosus (Stocks) Pangalo fruit was treated to the earthworms in which slight movement, paralysis at 12.12 min, and death after 15.04 min were observed (Table 1). Five percent chloroform extract of Praecitrullus fistulosus (Stocks) Pangalo fruit was treated to the earthworms which resulted to strong paralysis at 8.34 min and death after 9.08 min (Table 1).
All extract of (methanol, petroleum ether, and chloroform) Praecitrullus fistulosus (Stocks) Pangalo fruit showed the highest anthelmintic activity in 1%, 2%, and 5% methanolic extract compared with chloroform extract than petroleum ether extract and albendazole. So, all study groups show the good activity observed in the 5% methanolic extract of Praecitrullus fistulosus (Stocks) Pangalo fruit which give the best response. Based on these results, further detailed study on the methanol extract was needed to isolate the active compound which may be responsible for the anthelmintic activity.
Qualitative phytochemical analysis of methanolic extracts of P. fistulosus fruit showed the presence of terpenoid, alkaloids, tannins, phenol, flavanoid and glycoside present. The methanolic extracts showed the presence of terpenoid, alkaloids, tannins, phenol, flavanoid, and glycoside. In this extract, saponin tests negative. Shweta and Yogesh (2011) reported the same plant for the preliminary phytochemical screening of petroleum ether extract of Praecitrullus fistulosus showed the presence of alkaloids, tannins, and proteins, while methanolic extract revealed the presence of alkaloids, tannins, carbohydrates, and cardiac glycosides. Our study shows the similar type of compound present. The activities of the extract may be attributed to the presence of various secondary metabolites. Therefore, further works should be performed on the isolation and identification of the anthelmintic components in the methanoilc extract of Praecitrullus fistulosus fruit.
In the HPTLC analysis, two samples were selected for the study. The first sample is A1-isolated compound by column chromatography and A2-crude extract of methanolic extract. Both samples are run in the methanol to chloroform (11:0.5) which was used as the mobile phase. A2 sample band was observed under 254 nm light in TLC plate. A1 sample band was not observed under 254 nm. A1 and A2 sample blue fluorescence band was observed under 366 nm UV light in the TLC plate. In the sample of A1-isolated compound by column chromatography, the Rf value is 0.74.
Fourier-transform infrared (FTIR) spectroscopy analysis (functional group were identified) was used. We analyzed the A1 sample different peak which observed that different functional groups are present as shown in Table 3 and Fig. 3. A1 sample presents the functional group of secondary amine (=NH), H-bonded, C=C alkene stretch vibration, C=N amine of oxides, alkene bending vibration, acids, and anhydride group in the sample.
A1 sample analyses for the identification of the compound present in the sample were used with the help of gas chromatography and mass spectroscopy. The peak is showing the maximum percentage area at RT 32.93 in GC-MS analysis scan through mass spectrophotometer, with the presence of phenol, 3, 5bis(1,1-Dimethyl)-, and the molecular weight is 206, of which at peak is 32.93. GC-MS chromatogram is shown in Fig. 4. The present isolated compound from the methanolic extract of Praecitrullus fistulosus fruit is phenol, 3,5-bis (1,1-dimethyl) (Fig. 5). This active compound is responsible for the anthelmintic activity. Phytochemical screening of the crude extract of Praecitrullus fistulosus revealed the presence of tannins as one of the phytoconstituents. Reported literature indicated that tannins are polyphenolic compounds, which showed potent anthelmintic activity (Niezen et al. 1995; Ali and Wadekar 2008; Patel et al. 2010b). The anthelmintic effect of plants depends upon the content and type of tannins (Suleiman et al. 2005). The methanol extract showed potent anthelmintic activity.
