Evaluation of Ficus racemosa (Moraceae), Cassia fistula (Fabaceae) and Syzygium cumini (Myrtaceae) as a potential source of anticoccidial agents

Abstract

Coccidiosis has a serious impact on the poultry business, which is the most economically significant sector in the world. The causative agent of the avian coccidiosis is protozoan parasite of the genus Eimeria. This lethal disease caused economic loss of almost $3 billion worldwide. Due to development of resistance against available drugs and vaccines there is need of alternate strategy for the control. Therefore, this study's main goal was to evaluate the anticoccidial and antioxidant properties of the crude methanolic and aqueous leaf extract of Syzygium cumini, Ficus racemosa, and Cassia fistula. Coccidiosis suspected samples were collected from Veterinary Research Institute (VRI), Peshawar. Different species of Eimeria were identified on the basis of oocyst morphology, size and site of infection in gut. The in vitro anticoccidial activity of extracts different concentration was evaluated using different assays on Eimeria spp oocysts that inhibit sporozoite viability and oocyst sporulation. After 48 hours of incubation, the setup was examined. The antioxidant potential of the extracts was evaluated by total antioxidant capacity estimation, ferric reducing antioxidant power and DPPH radical scavenging assay. On phytochemical screening, anti-oxidant components such as carbohydrates, phenols, saponins, alkaloids, flavonoids etc were found in different plant extracts. C. fistula methanolic leaf extract showed maximum oocyst sporulation inhibition (86.81±2.35%) and sporozoites viability inhibition (86.73±1.67) against E. mitis and E. tenella, respectively, at a concentration of 30mg/ml. C.fistula and F. racemosa methanolic leaf extract had the greatest reduction power (2.17±0.01) and DPPH radical scavenging capacity (67.82±0.00). The highest levels of total antioxidant activity were found in the methanolic leaf extracts of C. fistula (30.95±0.35) and F. racemosa (21.93±1.41). Maximum flavonoids (40.00±1.00) and phenols (32.50±0.00) g/ml were found in the methanolic extracts of C. fistula. It is concluded that because of the medicinally significant phytochemicals, certain plant methanolic extracts have the greatest anticoccidial and antioxidant activity. The methanolic leaves extract of selected plants were fractionated by column chromatography and thin layer chromatography. Preliminary quantitative phytochemical screening, anticoccidial and antioxidant potential of eluted fractions was xviii evaluated by different assays. A total of 18 fractions from F. racemosa, 23 fractions from C. fistula, and 19 fractions from S. cumini were eluted from the crude extracts. Among the F. racemosa fractions, fraction 19 at a dose of 30 mg/ml against E. tenella showed the greatest oocyst sporulation inhibition (67.11±2.18%). Fraction 7 showed the lowest effectiveness (3.31±2.37%) against E. mitis at a dosage of 2.5mg/ml. F-22 of C. fistula had the highest sporulation inhibition efficacy (69.11±2.18) and (69.56±1.14) % against E. tenella and E. necatrix respectively. F-4 showed the lowest effectiveness (4.562.05%) against E. mitis at a dosage of 2.5 mg/ml. F-15 of F. racemosa had the greatest DPPH radical scavenging inhibition with the lowest IC50 value (10.571.08) g/ml. This IC50 value's range (6.47–2.78) g/ml is quite close to that of standard ascorbic acid. F-15 of F. raccemosa has the highest total antioxidant capacity (45.41±1.55) gAAE/mg. Because their TAC is near to the antioxidant power of standard ascorbic acid (45.32±3.01). These fractions are considered to be powerful antioxidants. S. cumini F-19 (3.72.5) gAAE/mg was found to have the maximum ferric reducing capacity at a concentration of 600g/ml. The highest levels of the tested phytochemicals are present in various fractions of all selected plants. The highest total phenolic (120.00±2.13) and flavonoid (125.57±1.56) contents among all the eluted fractions of the selected plants were found in C. fistula F-22. It is suggested that particular plant methanolic extract bioactive fractions have the highest anticoccidial and antioxidant activity because they include medicinally significant phytochemicals. Selected plants methanolic extract and their fractions different concentrations in comparison with anticoccidial drug amprolium were evaluvated for their anticoccicidial potential in broiler chicks (n=315). The experimental birds were divided into 21 groups labelled A-U on day 14. There were five chicks in triplicate in each group. With the exception of negative control group T, all experimental groups chicks received an oral infection from 7000 to 10,000 sporulated oocysts of a mixed Eimeria species. All groups, with the exception of negative control group T and medicated group S, were given with various supplements of plant extracts and their fractions orally at a dose of 1 ml/chick daily for 5 days beginning on day 4 post inoculation (PI), as the first evidence of infection occurred. Amprolium (1.25mg/ml) was administered to group S, the medication group. The efficiency of experimental dosages was assessed for 36 days on the basis of oocyst shedding, mean weight gain, diarrhea, feed consumption, feed conversion ratio, mortality, blood chemistry, histology etc. The acute toxicity of xix extracts was examined in one-day-old broiler chicks (n=50). Total of ten groups, each containing 5 chicks. All groups, with the exception of the control group, received a maximum oral dosage of 2,000 mg kg-1 b.wt of different extracts of selected plants and their anticoccidial fractions. For 14th days, all the chicks were monitored for any clinical symptoms of toxicity. The anticoccidial index (ACI), anticoccidial sensitivity test (AST) and the percentage of optimal anticoccidial activity (POAA) was used to measure the drug resistance of Eimeria species. The maximum weight gain was observed in group N (447±4.04) and group M (444±3.05) treated with 20% and 10% of F-22 of C. fistula, respectively, on the fifth week. This weight gain was in similar range to that of group treated with amprolium (443±3.07) gm and the uninfected non medicated control group (456±2.51) gm. Maximum mean feed consumption was observed in groups N (761±0.57) and M (759±0.57) treated with 20% and 10% of F-22 of C. fistula, respectively, at the fifth week of age. This was higher than the mean feed consumption in groups treated with amprolium (725±1.52)gm and within the range of the uninfected non-medicated control group S(762±1.15)gm. Group N, FCR results were the best. Among all experimental groups, group M treated with 10% of F-22 of C. fistula showed the greatest decrease in oocyst shedding. In the groups who received the best treatment, there was no death or diarrhea. Different concentrations of anticoccidial fractions from chosen plants were administered to groups, and normal histology, serum biochemistry, and hematological were observed in these groups. Chemical characterization of the most active fractions was done by using UV Vis, FTIR, and GC-MS analyses. The anticoccidial fractions UV-vis spectroscopy examination was carried out between 200 and 800 nm, and several absorption peaks were noted at various wavelengths. Based on typical peak values, the FTIR analysis was carried out to identify various functional groups. The spectrum was recorded by an FTIR spectrometer with a wavelength scan range of 400 to 4000cm-1. Each fraction identified compounds were docked with target S-Adenosylmethionine synthetase by in silico molecular docking. Each fraction possesses amino acids, alkanes, alkynes, amines, aldehydes, carboxylic acid, and other compounds confirmed by FTIR analysis. Various common compounds, such as oleic acid, n-hexadecanoic acid, octadecanoic acid, trichloromethane, and etc were discovered by GC-MS analysis. Cyclododecane methanol of F-19 and oleic acid of F-15 of F. racemosa exhibit the greatest binding xx affinities for the target S-Adenosylmethionine synthase among all docked compounds. These substances are perfect for the synthesis of plants based anticoccidial drugs.