Submitted: May 03, 2021 | Approved: May 12, 2021 | Published: May 13, 2021
How to cite this article: Kumar P. Medicinal plant Potentilla fulgens and its effect in vitro against Fasciola gigantica. Insights Clin Cell Immunol. 2021; 5: 004-007.
Copyright: © 2021 Kumar P. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Keywords: Potentilla fulgens; Fasciola gigantica; Fascioliasis; Anthelmintic activity
Fascioliasis is a one of the most important serious parasitic zoonotic disease which caused by trematode giant liver fluke 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 different preparations (organic extracts and column purified fraction) are uses in vitro against liver fluke F. gigantica. The dried root powder, different 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 effect of ethanolic extract of P. fulgens after 2h exposure the LC50 value is 5.22 mg/ml against F. gigantica. The column purified fraction of dried root powder of P. fulgens shows more toxicity. The 2h LC50 of column purified fraction was 3.25 mg/ml whereas in 8h exposure the LC50 is 1.24 mg/ml. The phytochemicals of the P. fulgens may be used as anthelmintic components against liver fluke F. gigantica.
The liver fluke Fasciola hepatica and F. gigantica is the causative agent of fasciolosis in cattle and human populations . These diseases are parasitic in livestock with over 700 million production animals at risk of infection . Fascioliasis is mainly related to plant-borne trematode digenetic zoonotic disease. The definite host of liver fluke is cattle, sheep, buffaloes, and goats which have an impact on the development, growth rate, the productivity of the ruminants and it’s considered economically significant [3,4]. Fasciolosis is very common in the cattle population of the eastern part of Utter Pradesh, India [5-10]. In humans, the adult fluke of Fasciola causes a variety of symptoms such as long-standing fever, malaise, weight loss, eosinophilia, pain under the right costal margin, and anemia due to feeding on the blood .
Anthelmintic synthetic drugs can be used for the control of liver fluke infection, but it causes adverse effect on the host as well as develops resistant/residual effect in the fluke. Synthetic drugs are not easily available in some of the remote rural areas of developing countries . The plant products or active phytochemicals are may be an effective anthelmintic component. Therefore, the use of the common medicinal plant as anthelmintic offers an alternative source that can solve these problems and it may be more acceptable due to eco-friendly and easily available for the users . The medicinal plant Potentilla fulgens are commonly used as antihyperglycemic, antioxidant, antitumor, anti-hyperlipidemic, antiulcerogenic, anti-inflammatory, anthelmintic larvicidal, and molluscicidal [14-17]. The present study aims to evaluate the different preparations of medicinal plant P. fulgens in vitro treatments and their anthelmintic efficacy against the giant liver fluke F. gigantica.
Collection of liver fluke (F. gigantica)
The adult fluke F. gigantica (3.2 ± 0.18 cm in length) were collected from the infected bile ducts of the freshly slaughtered buffaloes from slaughterhouse district Gorakhpur (UP), India. The live fluke was kept in freshly prepared Hedon-Fleig (H-F) solution at 37 ± 2 0C in a BOD incubator until use.
The dried root of the Potentilla fulgens was procured from the local market in Muhammadabad Gohna, district Mau (UP), India, and authenticity by Dr. A. K. Singh, Department of Botany, S.G.N. Government P.G. College, Muhammadabad Gohna, Mau, Uttar Pradesh.
Preparation of crude plant products
The freshly dried root of P. fulgens washes through fresh-eater 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.
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 purified 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.
Hedon-Fleig (H-F) solution
The Hedon-Fleig (H-F) solution is used in this study which was prepared by the method of Hajare, et al. . H-F solution contain NaCl-119.82 mM, MgSO4-0.29 mM, KCl-4.01 mM, CaCl2-0.40 mM, NaHCO3-17.8 mM, Glucose-22.3 mM, Streptomycin sulphate-6900 unit 10 mg/l and Benzyl pencillin-9900 units/l at 38 ± 2 0C in BOD incubator.
In vitro toxicity determination
In vitro toxicity of dried root powder, organic extract (acetone, ether, chloroform, methanol, and ethanol), and column purified-fraction of P. fulgens were performed in the petri dish by the method of Kumar and Singh, . 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 fluke 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 (LC50) values, lower and upper confidence limits (LCL and UCL), slope values, and t-ratio value were calculated by the POLO computer program .
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 (LC50) 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 LC50 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 purified fractions of dried root powder of P. fulgens were highly toxic. The 2h, 4h, 6h, and 8h exposure the LC50 value of the column purified-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 LC50 values based on each of the six replicates of the experiments were found to be within the 95% confidence limits of lethal concentrations. The t-ratio value is greater than 1.96 that indicates the significant anthelmintic efficacy of the various treatments (Table 1).
|Table 1: In vitro anthelmintic activity of dried root powder of P. fulgens and their different organic extract, column purified against F. gigantica at different exposure periods.|
|Values||In vitro exposure of Helminthicides (mg/ml)|
|Potentilla fulgens dried root powder||Acetone extract||Ether extract||Chloroform extract||Methanol extract||Ethanol extract||Column purified|
|Slope-value||1.30 ± 0.21||1.12 ± 0.20||1.27 ± 0.11||1.18 ± 0.19||1.23 ± 0.27||1.19 ± 0.17||1.25 ± 0.16|
|Slope-value||1.14 ± 0.18||1.15 ± 0.21||1.26 ± 0.29||1.27 ± 0.11||1.19 ± 0.24||1.11 ± 0.23||1.31 ± 0.25|
|Slope-value||1.35 ± 0.20||1.33 ± 0.19||1.27 ± 0.22||1.18 ± 0.35||1.26 ± 0.16||1.39 ± 0.26||1.35 ± 0.21|
|Slope-value||1.26 ± 0.19||1.22 ± 0.24||1.26 ± 0.17||1.34 ± 0.29||1.17 ± 0.24||1.34 ± 0.26||1.32 ± 0.26|
|Ten Fasciola gigantica in six batches were exposed in vitro (H-F solution) on different concentrations of the above anthelmintic preparations. Mortality of Fasciola was determined after every 2 h exposure period. LCL: Lower Confidence Limits; UCL: Upper Confidence Limits|
The present study is demonstrated that the dried root powder, organic extracts, and column purified 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 fluke F. gigantica and cause mortality. In the treated group, all the preparations of the P. fulgens are cause significant 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 fluke. The toxicity of different preparations of P. fulgens is time as well as concentration dependant as evident from the LC50 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 significant and cause antilarvicidal activities against sporocyst, redia, and cercaria larva of the F. gigantica . In in vitro treatment at 2h exposure, the highest toxicity was noted against sporocyst, redia, and cercaria the LC50 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 fluke 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 . Kumar and Singh,  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 fluke F. gigantica . The ethanolic root extract of P. fulgens is preventing gastric ulcers in rats due to H+ K+-ATPase inhibitory and antihihistaminic activities . The dried root powder of P. fulgens and their different organic extracts, column purified fractions are a potent source of the molluscicides which cause significant mortality against liver fluke vector snails Lymnaea acuminata and Indoplanorbis exustus [15-17]. Roy, et al.  have been reported that the alcoholic extract of dried root powder of P. fulgens has significantly 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 flavonoids and tannin in high amounts . 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.  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 . Previously in different studies have been evaluated that the root extract of P. fulgens possesses antitumor , antioxidant , anthelmintic , and gastroprotective . 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 .
It can be concluded from the present study that the dried root powder of P. fulgens and their different organic extract and column purified fractions may be used as potent anthelminthic components for the control of liver fluke 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 fluke F. gigantica.
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