Medicinal plant Potentilla fulgens and its eﬀ ect in vitro against Fasciola gigantica

Fascioliasis is a one of the most important serious parasitic zoonotic disease which caused by trematode giant liver ﬂ uke Fasciola hepatica and F. gigantica among cattle’s and humans. The infection of Fasciola can be control by the use of phytochemicals as anthelmintic components. The anthelmintic activities of dried root powder of medicinal plant Potentilla fulgens and their diﬀ erent preparations (organic extracts and column puriﬁ ed fraction) are uses in vitro against liver ﬂ uke F. gigantica . The dried root powder, diﬀ erent organic extract, and column fractions were time and concentration-dependent. Among all the organic extracts, ethanol extract was high toxic than other organic extracts. The toxic eﬀ ect of ethanolic extract of P. fulgens after 2h exposure the LC 50 value is 5.22 mg/ml against F. gigantica. The column puriﬁ ed fraction of dried root powder of P. fulgens shows more toxicity. The 2h LC 50 of column puriﬁ ed fraction was 3.25 mg/ml whereas in 8h exposure the LC 50 is 1.24 mg/ml. The phytochemicals of the P. fulgens may be used as anthelmintic components against liver ﬂ uke F. gigantica .


Preparation of crude plant products
The freshly dried root of P. fulgens washes through fresheater and dry under sunlight till to dry and cut into small pieces. The dried root pieces of P. fulgens were pulverized in the electric grinder machine and the crude powders thus obtained were used for the in vitro anthelmintic activity against F. gigantica.

Organic solvent extracts
Five gram dried powder of P. fulgens were extracted with 500 ml each of 98.1% acetone, 97.8% ether, 98.6% chloroform, and 94.7% ethanol at room temperature for 48h. The solvents were removed under a vacuum machine and the following remaining dried parts were used for the determination of in vitro anthelmintic activity of P. fulgens and extract obtained 350 mg-ethanol, 360 mg-chloroform, 385 mg-ether, and 395 mg-acetone.

Column purifi ed-fractions
One thousand milliliters of 94.7% ethanol were uses for the fractions of dried root powder of P. fulgens were subjected to silica gel through 5 × 45 cm column chromatography (60-120 mesh, Qualigens Glass, Purchases from Precious Electrochemidus Private Limited, Bombay, India) for different fractions. One hundred milliliter fractions eluted with ethanol were collected. Ethanol from the column puri ied fractions was evaporated under a vacuum machine and the remaining solids column extracts were used for the determination of in vitro anthelmintic activity against Fasciola.

In vitro toxicity determination
In vitro toxicity of dried root powder, organic extract (acetone, ether, chloroform, methanol, and ethanol), and column puri ied-fraction of P. fulgens were performed in the petri dish by the method of Kumar and Singh, [19]. Six Petri dishes were set for each concentration of different preparations of the P. fulgens against F. gigantica. Ten F. gigantica were kept in each Petri dish (10 cm × 1.5 cm) containing 50 ml H-F solution. Flukes were exposed to different concentrations of the different preparations ( Table 1). The mortality of adult Fasciola was observed after 2h, 4h, 6h, and 8h exposure. The control group of the experiment was kept in an equal volume of H-F solution in a Petri dish under similar laboratory conditions but without treatment. The mortality of Fasciola was established by the opening of the sucker and contraction of the body. Usually in H-F solution in in vitro F. gigantica can survive up to 48h. Mortality of luke was observed after 2h up to 8h were counted in treated and control groups.
In vitro, anthelmintic toxicity data were observed every 2h up to 8h. The Lethal Concentration (LC 50 ) values, lower and upper con idence limits (LCL and UCL), slope values, and t-ratio value were calculated by the POLO computer program [20].

Results
In vitro anthelmintic activity of dried root powder of P. fulgens and their different preparations against F. gigantica was concentration and time-dependent. The lethal concentration (LC 50 ) values of dried root powder at 2h, 4h, 6h, and 8h were 8.35, 7.12, 6.45, and 5.78 mg/ml, respectively (Table 1). Whereas, among all the organic extract (acetone, ether, chloroform, methanol, and ethanol) the ethanol extract were more toxic. The 2h, 4h, 6h, and 8h LC 50 of ethanol extract of dried root powder of P. fulgens against F. gigantica were 5.22, 5.02, 4.88, and 3.43 mg/ml, respectively. The column puri ied fractions of dried root powder of P. fulgens were highly toxic. The 2h, 4h, 6h, and 8h exposure the LC 50 value of the column puri ied-fractions were observed 3.25, 2.65, 1.94, and 1.24 mg/ml, respectively ( Table 1). The slope values given in table 1 were steep and the separate estimates of LC 50 values based on each of the six replicates of the experiments were found to be within the 95% con idence limits of lethal concentrations. The t-ratio value is greater than 1.96 that indicates the signi icant anthelmintic ef icacy of the various treatments (Table 1).

Discussion
The present study is demonstrated that the dried root powder, organic extracts, and column puri ied fractions of P. fulgens are potent sources of anthelminthic components against F. gigantica. It may be possible that active phytochemicals of the medicinal plant, P. fulgens are entering the body of luke F. gigantica and cause mortality. In the treated group, all the preparations of the P. fulgens are cause signi icant toxicity in vitro against the F. gigantica (Table 1). But no mortality was observed in the control group. It indicates that the active phytochemicals of P. fulgens are entering through the tegument layer which caused paralysis and mortality of the luke. The toxicity of different preparations of P. fulgens is time as well as concentration dependant as evident from the LC 50 values and exposure period (Table 1). Higher toxicity of ethanol extract was observed among all organic extracts which indicate that the anthelmintic components P. fulgens are more soluble in ethanol organic solvent.
In vitro toxicity of the root powder of the P. fulgens and their different extract preparations are signi icant and cause antilarvicidal activities against sporocyst, redia, and cercaria larva of the F. gigantica [21]. In in vitro treatment at 2h exposure, the highest toxicity was noted against sporocyst, redia, and cercaria the LC 50 value of column extract were 62.42, 59.25, and 45.11 mg/ml, respectively. It may be due to the uptake of the active moiety which progressively increases in the luke body with an increase in the exposure period. It may be possible that the phytochemicals of the P. fulgens in the F. gigantica could change the different enzyme activity and cause effects. Many numbers of medicinal plants are have been used for the treatment of parasitic infection in animals and humans [22]. Kumar and Singh, [19] have been reported that in in vivo the common species Allium sativum, Ferula asafoetida, Syzygium aromaticum and their active components like allicin, ferulic acid, umbelliferone, and eugenol have anthelmintic activities against F. gigantica. The binary combinations (1:1 ratio) of the active components the allicin, ferulic acid, umbelliferone, and eugenol are more effective in vitro against the liver luke F. gigantica [23]. The ethanolic root extract of P. fulgens is preventing gastric ulcers in rats due to H + K + -ATPase inhibitory and antihihistaminic activities [24]. The dried root powder of P. fulgens and their different organic extracts, column puri ied fractions are a potent source of the molluscicides which cause signi icant mortality against liver luke vector snails Lymnaea acuminata and Indoplanorbis exustus [15][16][17]. Roy, et al. [25] have been reported that the alcoholic extract of dried root powder of P. fulgens has signi icantly reduced the vital tegumental enzymatic activity of the alkaline phosphatase, acid phosphatase, and adenosine triphosphatase (ATPase) in cestodes parasite Raillietina echinobothrida and trematodes Gastrothylax crumenifer. The root extract of P. fulgens has lavonoids and tannin in high amounts [26]. In vitro and in vivo studies have been evidence to support the anthelmintic effect which feeds the tannins and other polyphenols against abdominal and intestinal parasitic nematodes [27,28]. Athanasiadou, et al. [29] have been evaluated that tannin shows anthelmintic activities against infected sheep with Trichostrongylus colubriformis and causing larval mortalities. The compound of tannin in in vitro inactivates the enzyme activities in Trichostrongylus colubriformis larvae which are responsible for hatching and development [30]. Previously in different studies have been evaluated that the root extract of P. fulgens possesses antitumor [31], antioxidant [26], anthelmintic [25], and gastroprotective [24]. The root extract of P. fulgens in in vitro inhibit enzyme activities of amylase, α-glucosidase, β-glucosidase, and lipase in the liver, kidney, and eye lens of the diabetic mice [32].

Conclusion
It can be concluded from the present study that the dried root powder of P. fulgens and their different organic extract and column puri ied fractions may be used as potent anthelminthic components for the control of liver luke F. gigantica. This study also revealed a further study that the phytochemicals of the P. fulgens at the molecular level elucidate in the parasitic life of the liver luke F. gigantica.