Evaluation of in vivo Anthelmintic Efficacy of Ethanolic Extract of Butea frondosa Seed against GI Parasites in Goats

Gastrointestinal parasites (GIPs) infestation is one of the major economic problems in small ruminants worldwide, since it causes major health problems and loss of productivity. Many GIPs such as Haemonchous contortus, Oesophagostomum spp., Strongyloides spp., Trichostrongylus  spp., Moniezia spp., Paramphistomum etc. are prevalent in goats of northeastern region. Among the various GI nemotodes, H. contortus is the most prevalent and highly pathogenic parasite in small ruminants causing fatal anaemia (Das et al., 2018). Efficacy of synthetic anthelmintic drugs has been reduced due to its indiscriminate uses and lead to the development of anthelmintic resistance (AR). Therefore, it is important to establish alternative, safer anthelmintic agents for control of GIPs. Butea frondosa belonging to the family Fabaceae, popularly known as Palas, flame of the forest, Dhak, Keshu, Khakara and bastard teak is widely distributed throughout Indian subcontinents and Southeast Asia (Patil et al., 2006). Different parts and extract of B. frondosa showed various biological and pharmacological activities and used as antimicrobial, antifungal, antibacterial, anticonvulsive, antidiabetic, anthelmintic, antidiarrhoeal, antifertility, wound healing, anti-inflammatory, antihepatotoxic, antistress, antioxidant, memory and behaviour stimulant property (Singh et al., 2015 and Hedau et al., 2018). The seed of B. frondosa showed excellent anthelmintic property, especially for roundworms and tapeworm. Earlier, Iqbal et al., (2006); Khanolkar et al., (2018) and Saiyam et al., (2021) also reported anthelmintic activity B. frondosa.

The Goat Research Station (GRS), Assam Agricultural University, Byrnihat, is located in a low-lying area and Haemonchus spp. is a serious problem encountered in that farm. In the past, studies on traditional medicines have been undertaken by research workers to find out the best herbal product. Since Butea frondosa was not tested in the farm so far, therefore, the present investigation was planned to evaluate the in vivo anthelmintic efficacy of Butea frondosa seed extract against GIPs in goats.

Study area
 
The present investigation was conducted at Goat Research Station (GRS), Assam Agricultural University Byrnihat, from January, 2022 to March, 2022.
 
Preparation of extracts
 
Seeds of B. frondosa were washed, shade dried, powdered and about 250 g of powder was soaked in 1000 ml of ethanol for 3 days in a beaker and stirred using a sterile glass rod every day. The filtrate was obtained after passing through muslin cloth and was concentrated at 45-50°C in a rotary evaporator (Equitron, Roteva). The extract was further dried over a water bath at 37°C to get a semi-solid consistency and transferred to an airtight container at 4°C until use.

Selection and treatments of animals
 
A total of 124 goats were screened for parasitic infection by fecal sample examination and 18 numbers of goats positive for parasitic infestation were randomly divided into three groups viz. A, B and C. Ethanolic extract of seeds of Butea frondosa @ 100 mg/kg body weight was administered to the infected goats of Group A (n=6 goats), @ 200 mg/kg body weight to Group B (n= 6 goats) and combination of fenbendazole and praziquantal (fentasplus) @ 5 mg/kg body weight to Group C (n=6 goats) p.o.; two doses were administered on 0th day and 14^th day. Group C was considered as positive control. Approximately, 20-30 gm faecal samples of animals were individually collected in sterile zip lock bags from all the infected goats on 0th day (pretreatment) to 42^nd day post treatment at weekly intervals and EPG count by modified McMaster technique (Soulsby, 1982). The study was approved by IAEC no. 770/03/ac/CPCSEA/FVSC,AAU/IAEC/06/20.
 
Haemato-biochemical analysis
 
Exactly 5 ml of whole blood was collected from the infested goats at weekly intervals for 42 days. Out of which, 2 ml was transferred into EDTA vacutainer for haematological study and 3 ml in clot activator for serum biochemical analysis. Haematological study was done as per the standard methods (Jain, 1986) and biochemical analysis was done by spectrophotometric method using commercial kits as per manufacturers’ protocol.
 
Statistical analysis
 
The statistical analysis was performed by using SPSS software and parameters were analyzed by two way analysis of variance (ANOVA).

Extraction yield (%, yield) of the Butea frondosa seed extract
 
The percentage yield (w/w) of the solid residue of ethanolic extract of seeds of Butea frondosa (BFE) was found 14% (w/w).
 
Prevalence of GIPs
 
During the present study different eggs of GIPs were found in the faecal samples screened (Fig 1, 2).





Overall prevalence of GIPs in goats was found to be 78.22%, out of which prevalence of Haemonchus spp, Oesophagostomum spp, Strongyloides spp., Moniezia spp, Trichostrongyloid spp. was found 69.35%, 47.58%, 43.54%, 30.64% and 21.77% respectively. Among the GI nematodes, the prevalence of H. contortus was predominant. In this study, overall high prevalence of GI parasites might be due to the low immunity of the host due to malnutrition, the prevailing agro-climatic conditions of Assam and development of anthelmintic resistance.
 
Egg per gram
 
The results (Table 1) revealed that the values of EPG of faeces significantly (P≤0.05) deceased from day 7 till day 28 post treatment when compared with day 0 (pre treatment) in both the treated groups A and B. On day 28^th post treatment, maximum reduction (P≤0.05) was recorded in EPG of faeces in goats treated with a dose of 200 mg/kg body weight (Group B), followed by 100 mg/kg body weight (Group A) of BFE.



The positive control (Group C) exhibited the highest faecal egg count reduction (P≤0.05) on day 21 post treatment and later a non-significant increase in EPG values were found in Group C from 28^th day onwards. Similarly Group A and B, also showed a non-significant (P>0.05) increase in EPG values from 35^th day onwards. Earlier workers, Iqbal et al., (2006) reported that the crude powder of B. monosperma seeds showed maximum reduction of EPG in sheep on day 10 and Saiyam et al., (2021) reported that methanolic and aqueous extract of B. frondosa showed significant reduction in GI nematodes in goats on 14^th and 21^st day post treatment. In this study, the anthelminthic effect in group A and B might be due to presence of high concentration of phenolic, flavonoids, tannin and other secondary metabolites present in the seeds of B. frondosa which might inhibit the hatching of eggs. Besides palasonin may act by inhibition of energy metabolism/alteration in motor activity of the parasite (Saiyam et al., 2021).
 
Body weight
 
The mean±SE values of body weight of goats (Table 2) in all the groups found to be significantly (P≤0.05) reduced on 0^th day pre treatment. A significant (P≤0.05) gradual increase was observed in all the goats of group A, B and C in the present study from 7^th day post treatment till 42^nd day post treatment when compared to 0th day pre treatment.



The increase in body weight in the treated animals could be co-related to the improved appetite of the animal after elimination of different GIPs. Similar observations of gain in body weight was also reported by Khanolkar et al., (2018) after administrating polyherbal anthelmintic tablet to the infested goats.
 
Hematological parameters
 
The mean values of haematological parameters are presented in Table 3.



Hemogram revealed that the levels of Hb, PCV, TEC and lymphocytes were significantly (P≤0.05) reduced on 0th day pre treatment. On the other hand, the levels of ESR, TLC, neutrophils, eosinophils, monocytes count were significantly (P≤0.05) elevated prior to treatment. Similar findings were reported by Ahmed et al., (2015) and Shashank et al., (2019) in goats infested with GIPs. The lower values of Hb, TEC, PCV in goats infested with GIPs is attributed to acute loss of blood by their blood sucking activity and leakage through the damaged GIT caused by the parasites (Sunder et al., 2022). The decrease in lymphocyte count may be due to malnutrition, stress condition and ultimately leading to lymphoid atrophy, thereby hampering lymphopoiesis (Estaben, 1968). Increase in TLC level may be because of increased local immune response of body against the parasites by eosinophils (Ahmed et al., 2015). Eosinophils are considered to be important elements that respond against parasitic infestations (Soulsby, 1982). The increase in monocytes counts may be due to stress in the infected animals or the phagocytic activity of the cell, digesting the particulate matter and debris of parasites as observed in cell-mediated immune response (Ahmed et al., 2015).
 
Biochemical parameters
 
The mean±SE values of biochemical parameters are presented in Table 4.



Biochemical study revealed that the values of total serum protein, albumin, sodium, potassium, calcium, chloride and phosphorus were found to be significantly (P≤0.05) reduced on 0^th day pre treatment. The decreased level of total serum protein, albumin in our study corroborated with the earlier report of Ahmed et al., (2015) and Shashank et al., (2019). The hypoproteinemia and hypoalbuminemia in GI parasitism could be attributed to protein-losing enteropathy (Soulsby, 1982) and malabsorption of proteins from damaged intestinal mucosa (Ahmed et al., 2015). A significant decrease in sodium, potassium, calcium, chloride and phosphorus in the study was similar with the previous finding of Vijay et al., (2010) in sheep affected with GIPs. A decrease in sodium, potassium, calcium, chloride and phosphorus level could be attributed to rapid loss of faecal water and electrolyte as a result of parasitic diarrhoea. On the other hand, the values of AST, ALT, ALP were found to be significantly (P≤0.05) elevated on 0^th day pre treatment. Significant increases in the serum enzymes in our study were similar to Shashank et al., (2019). Increased activity of serum enzymes observed in the study may be due to damage of abomasal and intestinal lining mucosa by the parasites (Sharma et al., 2001).

A significant (P≤0.05) gradual increase in the mean±SE values of Hb, PCV, TEC, lymphocytes, TSP, albumin, sodium, potassium, calcium, chloride and phosphorus were observed in all the goats of group A, B and C in the study from 7^th day post treatment onwards. Moreover, a significant (P≤0.05) gradual decrease in the mean±SE values of ESR, TLC, neutrophils, eosinophils, monocytes, AST, ALT, ALP were found in all the group A, B and C from 7^th day post treatment upto 28^th day post treatment. Improvement in hematological and biochemical parameters in group A and B may be due to elimination of parasites from the host through wormicidal activity of Butea spp. and presence of high concentration of bioactive molecules like phenolics, flavonoids, tannins and high nutritional value (Cu, Zn, protein etc) which have immune boosting and gastro protective effect that reduces inflammatory changes, improve blood parameters and reduce oxidative stress to cell membrane lipid, protein and nucleic acid (Singh et al., 2015).

The present study can be concluded that the ethanolic seed extract of Butea frondosa possesses anthelmintic activity next to the commercial anthelmintic drug Fenbendazole and Praziquantel (Fentasplus) and might contribute to the field of plant anthelmintic in order to develop a safe and effective alternative to the chemical anthelmintic. Higher dose of B. frondosa @ 200 mg/kg body weight was found to be effective in elimination of common GIPs in goats. In addition, post treatment improvement in body weight and hemato-biochemical parameters were also recorded and we have also found its activity against cestodes, which was not reported earlier by other workers.

The first authoress wishes to express her gratitude to ICAR, Govt of India, New Delhi for financial support of the All India Net work project on Ethnoveterinary medicine and the Director of Research, College of Veterinary Science, AAU, Khanapara, Assam, India for his continuous support and providing the necessary facilities for smooth conduct of the study.

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