Helminths are parasitic worms. They are the most common infectious agents of humans in developing countries and produce a global burden of disease. Helminth infections are commonly found in villages of developing countries and are being recognized as cause of much acute as well as chronic illnesses among humans (Peter et al., 2008; Nisha et al., 2012). Many helminth infections occur in poverty-stricken and developing countries with warm, moist environments and poor sanitary conditions (Peter et al., 2007). Although helminth infections can affect anyone, children in developing nations are mostly at risk (Peter et al., 2008). The World Health Organization reports a 35% infection rate for roundworm, which is a common parasitic worm (Perry et al., 2002; Krishnamurthi, 2003). These infections are usually transmitted through contaminated soil, food, or water; feces; and unwashed hands or contact with a contaminated object.
Chemical control of helminthes coupled with improved management has been the important worm control strategy throughout the world. However, increasing problems of development of resistance in helminthes (Gilbert, 1996; Coles,1997). Hence, the best alternative is plant-based medicine. Plants continue to be an important source for identification of new drug. Important plant-derived drugs are still obtained commercially by extraction from their whole plant sources. Plant produces a large number of secondary metabolites that may serve as future reservoir for novel drugs and therapeutic agents. Screening and proper evaluation of the claimed medicinal plants could offer possible alternatives that may be both sustainable and environmentally acceptable. Development of anthelmintic resistance and high cost of conventional anthelmintic drugs led to the evaluation of medicinal plants which are an alternative source of anthelmintics.
Before evaluating the anthelmintic activity, knowledge about the yield of extract is important. Lower extract yielding plants are not commonly preferred by the pharmaceuticals industry. Ishnava et al. (2015) reported that the same plant methanol extract showed maximum extraction yield (3.28%); compared to our study, the maximum extraction yield (%) of extract was obtained in ethyl acetate (4.46%). This may be due to improper selection of solvent system and method of extraction, using ethyl acetate and collection of our sample from semi deciduous forest of Gujarat. Environmental factors of soil and the location of plant also play significant roles.
Methanolic extract of C. epigaeus plant in concentrations 25, 50, 75, and 100 mg/ml showed paralysis of worms within 1.27 to 2.06 min and death within 3.26 to 4.38 min depending on the concentration (Table 2). Nisha et al. (2012) reported that the methanol extract of Pyrostegia venusta at the concentration of 5 mg/ml showed time of paralysis and death at 65 and 88 min, respectively. For concentrations at 7.5, 10, and 12.5 mg/ml, paralysis was shown at 70, 66, and 34 min, respectively. and death occurred at 94, 91, and 78 min, respectively. Compared to this result, methanol extract of C. epigaeus showed better activity, and in short time, death was observed because concentration is very high.
Chloroform extract of C. epigaeus plant of concentrations 25, 50, 75, 100 mg/ml showed paralysis of worms within 4.75 to 6.19 min and death within 10.06 to13.7 min depending on the concentration (Table 2). Vidyadha et al. (2010) reported the chloroform extract of Enicostemma littorale at the concentration of 25 mg/ml showed the time of paralysis and death at 45 and 59 min, respectively. Compared to this study, chloroform extract of C. epigaeus showed better result.
Ethyl acetate extract of C. epigaeus plant of concentrations 25, 50, 75, and 100 mg/ml showed paralysis of worms within 1.27 to 2.06 min and death within 3.84 to 4.38 min (Table 2). Vidyadha et al. (2010) reported that the ethyl acetate extract Enicostemma littorale at the concentrations of 25, 50, and 100 mg/ml showed the time of paralysis and death at 35, 27, 20 and 49, 39, 27 min, respectively. Compared to this study, ethyl acetate extract of C. epigaeus showed paralysis and death at 2, 1.5, 1.2 and 4.3, 4.1, 3.26 min respectively. This result of C. epigaeus shows good activity compared to Enicostemma littorale.
More time was taken by hexane extract; at concentrations 25, 50, 75, and 100 mg/ml, it showed paralysis of worms within 6.08 to 7.77 min and death at 10.28 to 12.94 min (Table 2). Vidyadha et al. (2010) reported the hexane extract of Enicostemma littorale at the concentration of 25 and 100 mg/ml showed the time of paralysis and death at 50, 36 and 64, 49 min, respectively. Compared to this study, C. epigaeus showed paralysis and death at 7.7, 6.08 and 12.94, 10.28 min, respectively. This result shows C. epigaeus’ good activity compared to Enicostemma littorale. It can be tested for use as an alternative drug.
The standard drug, albendazole showed paralysis at 15.30 min and death after 34.18 min at 20 mg/ml concentration (Table 2). From the above result, it is clear that all the extracts of C. epigaeus plant have significant anthelmintic activity in a dose-dependent manner when compared with standard anthelmintic drug. But ethyl acetate extract showed great results as time taken for death is comparatively very low as compared to standard drug. Vijaya et al. (2011) reported that the same plant using the solvent of ethanolic and aqueous extract compared to the ethyl acetate extract showed great results as time taken for death is comparatively very low. The best solvent is ethyl acetate extract since it gives the best response for a short time and at a minimum dose.
In our study, the effective dose was estimated to be 12.5 mg/ml as the time of paralysis and death took 3.27 and 5.84 min, respectively. The rest of the concentration took more time for paralysis and death. It caused paralysis followed by death of the worms at all tested dose levels. Potency of the extracts was inversely proportional to the time taken for paralysis death of the worms. It is quite apparent from the studies that the ethyl acetate extract possesses significant anthelmintic activity. The above findings justify the use as an anthelmintic, as suggested in the folklore medicines.
In the qualitative phytochemical analysis of ethyl acetate extract of C. epigaeus tuber, various phytoconstituents were present. The ethyl acetate extract showed the presence of alkaloids, tannins, phenol, flavonoid, steroids, saponin, and glycosides. Gnananath et al. (2013) reported that the ethanolic extract of C. epigaeus rhizomes contains phenolic compounds, flavonoids, terpenoids, and phytosterols. Preliminary phytochemical analysis of ethyl acetate extract of C. epigaeus tuber indicated the mix of compounds gives better activity.
Phytochemical constituents of the ethyl acetate extract of C. epigaeus tuber were quantitatively analyzed for the presence of chemical constituents (%) in the plant under study. The results have been depicted in Table 4. The maximum phytochemical present is alkaloid (44.3 %) in the ethyl acetate extracts (Table 4). The phenol and flavonoid are in relatively less quantity in the ethyl acetate extracts (Table 4). The phenol and flavonoid contents in the standard gallic acid at various concentrations (μg/ml) were obtained (Table 4). The values obtained in the ethyl acetate extract are very less compared to the standard value. Alkaloids are also used as starting materials in the manufacture of steroidal drugs and carry out protective functions in animals, thus are medicinal especially steroidal alkaloids (Maxwell et al., 1995). Isolated pure plant alkaloids and their synthetic derivatives are used as basic medicinal agent for their anthelmintic effects (Gnananath et al., 2013). The presence of these secondary metabolites has contributed to its medicinal value as well as physiological activity (Lewis and Elvin-Lewis., 1977). For instance, phenols have been shown to have antibacterial, anthelmintic, and antineoplastic activities (Alan and Miller., 1996).
HPLC analysis of ethyl acetate extract of C. epigaeus tuber has showed six major peaks. The area of peak no. 2 is 56.00 and height is 20.8. The peak no. 2 is of the chemical constituent that is majorly present in the extract (Fig. 3). Thangavel et al. (2014) reported the same plant collected from Tamilnadu, India using the mobile phase (toluene:methanol (8:2)) separating 11 bands. In our study, a total of 9 bands were obtained in the sample. The plant material is collected from semi-deciduous forest of Gujarat. Environmental factors may have a significant effect on the production of secondary metabolites.
GC–MS analysis of ethyl acetate extract of C. epigaeus tuber identification confirmation was by comparison of their mass spectra with NIST Library version year 2005. Based on the mass spectrophotometer predication, two compounds majorly identified is n-hexadecanoic acid and octadecanoic acid (Fig. 5) present in the ethyl acetate extract of C. epigaeus tuber. Moonjit and Himaja (2014) reported that they investigated the insecticidal activity, anthelmintic activity, GC–MS analysis, and the phytochemical screening of the petroleum ether (IEP) and ethanol (IEE) extracts of the aerial parts of Ipomoea eriocarpa with the presence of hexadecanoic acid and octadecanoic acid. Alby and Regi (2016) reported the phytochemical studies portray the presence of several biologically active secondary metabolites and GC–MS profiling of B. phoenicea ethanolic extract revealed the presence of possible compounds in the plant are hexadecanoic acid and octadecanoic acid. Dubal et al. (2013) reported the components present in the methanolic extract of rhizome of T. coadunate were identified by GC–MS analysis as n-hexadecanoic acid (Palmitic acid) having antioxidants, hypochloresterolenic, nematicide, pesticide, lubricant, antiandrogenic flavor, and hemolytic properties. Bohm et al. (2016) reported that the anthelmintic activity of the methanolic extract might be attributed to the wide range of chemical classes including octadecanoic acid, oleic acid, n-hexadecanoic acid, eicosenoic acid, oleyl alcohol, cyclohexane, and heptadecanoic acid as per the results of GC–MS. Anthelmintic activity of ethyl acetate extract of C. epigaeus tuber might be attributed to the wide range of chemical classes including octadecanoic acid, n-hexadecanoic acid, and heptadecanoic acid as per the results of GC–MS.
The structure, chemical formula, and molecular weight of n-hexadecanoic acid and octa decanoic acid (Fig. 5) that may be the bioactive compound responsible for the anthelmintic activity are stated below:
IUPAC name: n-hexadecanoic acid, Chemical formula: C16H32O2, Molecular weight: 256 g/mol, Boiling point: 351 °C, Melting point: 62.9 °C and
IUPAC name: octadecanoic acid, Chemical formula: C18H36O2, Molecular weight: 284 g/mol, Boiling point: 361 °C, Melting point: 69.3 °C.