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Anthelmintic Efficacy of Aqueous-Alcoholic Extracts Formulations of Embelia ribes Fruits and Vernonia anthelmintica Seeds against Ovine Gastrointestinal Nematodes

Vinod Sinha1 Amit Singh V. K. Pal and S. K. Maurya
Vol 10(3), 60-66

Aqueous alcoholic (70% ethanol) extracts mixture of Embelia ribes fruits and Vernonia anthelmintica seeds was evaluated for anthelmintic efficacy in sheep having natural infection of gastrointestinal nematodes. Twelve infected sheep were randomly divided into two groups, Group I and Group II comprising six animals each. Group I sheep were treated with Embelia ribes fruits and Vernonia anthelmintica seeds extract (1:1) at dose rate of 100mg /kg. b.wt. orally whereas G II was kept as untreated infected control. Faecal and blood samples from each sheep were collected on 0,3,7 and 10-day post-treatment to record egg per gram of faeces (EPG) and haematological (Hb, PCV and TEC) and biochemical indices (Total plasma protein, serum albumin, ALP, ALT, AST & serum creatinine). On day 10 PT, decrease in egg per gram, improvement in haematological values and increase in serum protein level suggests promising anthelmintic values of aqueous alcoholic extract mixture of Embelia ribes fruits and Vernonia anthelmintica seeds.

Keywords : Aqueous-alcoholic Embelia ribes Gastrointestinal Nematodes Sheep Vernonia anthelmintica

Gastrointestinal nematodes pose a major challenge to the health and productivity of small ruminants causing unaccountable economic losses to small and marginal farmers. Control of gastrointestinal nematodes is largely based on the use of chemotherapeutic drugs but improper and injudicious use of anthelmintics has led to the emergence of resistant strain of the gastrointestinal parasites particularly, Haemonchus contortus (Coles,1997). The parasite has been documented to be resistant to all three broad spectrum families of anthelmintics viz. benzimidazole, imidazothiazole and ivermectin (Yadav et al., 1995; Yadav and Garg, 2004; Singh et al., 2010 ; Rathod et al., 2019; Vohra et al., 2019) and against drugs with narrow spectrum of activity such as salicylanilides (Swarnkar et al.,1999).

The emergence of resistance to anthelmintic drugs which is now a worldwide phenomenon (Jackson and Coup,1997) and the increased awareness of consumers about drug residues that potentially enter the food chain have stimulated investigation into other alternative options. Therefore, as an alternative to the allopathic drugs, there are efforts for the development of eco-friendly, economical and efficacious anthelmintics of herbal origin. Keeping above information in mind, present study was planned to evaluate the efficacy of Embelia ribes fruits and Vernonia anthelmintica seeds in sheep naturally infected with gastrointestinal nematodes.

Extract Preparation

Embelia ribes fruits and Vernonia anthelmintica seeds were procured from local market. Moisture was evaporated by keeping them in hot air oven at 40oC for 8hrs and then pulverized to fine powder using electric grinder, sieved and kept in air tight container at 4 oC. Extract of Embelia ribes fruits and Vernonia anthelmintica seeds powder were prepared using 70% aqueous ethanol in a Soxhlet apparatus. Powder of Embelia ribes fruits and Vernonia anthelmintica seeds were placed in thimble and the Soxhlet apparatus was run for 9-12 hours (22-28 cycles). The extracts so obtained were evaporated at room temperature, weighed and then stored in air tight sterile container at 4oC.  

In -vivo Experiments

Aqueous alcoholic (70% ethanol) extract of Embelia ribes fruits and Vernonia anthelmintica seeds was used for in- vivo study. For the trial, twelve (12) naturally infected adult female sheep, weighing around 16-30kg and having EPG>500, were selected and randomly divided into two different groups (G I and G II), comprising six animals each. Group I animals were treated with Embelia ribes + Vernonia anthelmintica (1:1) extract at dose rate of 100mg /kg. b.wt. orally whereas G II was kept as untreated infected control.

Interpretation of Efficacy of Drug

Per-rectal faecal samples from individual sheep from each group was collected in sterile container using rubber glove on day 0, 3, 7 and 10 post- treatment. Egg per gram was calculated by modified McMaster technique (MAFF,1971). Efficacy was calculated using following formula-

% FECR=100 {1-}

FEC – Fecal egg count, FECR – Faecal egg count reduction)

Coproculture was also carried out on collected faecal samples for the larvae composition before and after treatment.

Study of Haematological and Biochemical Parameters 

Blood samples were also collected on day 0, 3, 7, and 10 post-treatment to record any haematological and biochemical changes. Haemoglobin (Hbg/dl), packed cell volume (PCV %), total erythrocyte count (TEC× 106/µl) were estimated by cyanomethaemoglobin, Wintrobe’s haematocrit and haemocytometer method, respectively. Estimation of total protein (g/dl), Albumin (g/dl), Serum creatinine (mg/dl), ALP (Alkaline phosphatase), ALT (Alanine amino transferase), AST (Aspartate amino tranferase) and serum creatinine was done using commercial kit (SPAN diagnostics).

Statistical Analysis

All the haematological and biochemical values were analyzed using SPSS software programmed one-way ANOVA.

Results and Discussions

Physical Characteristics of Extracts

Extract of Embelia ribes obtained was dark tan in colour, oily and molten mass like consistency. In case of Vernonia anthelimintica, the extract was dark brown and semi-solid in consistency. Yield obtained in Embelia ribes and Vernonia anthelimintica extract was 15.5% w/w and 27.9% w/w, respectively.

Reduction in Faecal Egg Count

In group I, treated with mixture of aqueous ethanolic extract of 1:1 mixture (g/g) of Embelia ribes fruits and Vernonia anthelmintica seeds, significant reduction in faecal egg count (72.97%) was noticed on day 10 post treatment (PT) (Table 1). Earlier,using similar combination ratio, 93% reduction in faecal egg count with methanolic extract was reported ( Javed and  Akhtar ,1990). High level of efficacy observed in that study may be due to the high dose (2g/kg. b.wt) of mixture used. Coproculture examination showed the presence of 99% larvae of Haemonchus contortus before and after treatment. Reduction in EPG and coproculture reports suggests that the herbal preparations used in the current study were able to reduce worm burden of Haemonchus contortus.

Haematological and Biochemical Responses

Haemato-biochemical values(Mean ± S.E ) before and after treatment with  ethanol extract mixture of Embelia ribes  and Vernonia anthelmintica  against Haemonchus contortus in sheep have been depicted in Table 1. Within infected treated group , Hb (g%) values improved significantly on day 7PT. When infected treated group were compared with infected control, Hb (g%) values were observed significantly different from day 7 PT onwards. Similarly, the PCV and total erythrocyte count values values (TEC x106//µl) improved significantly (p<0.05) from day 7 PT onwards within G. I. and comparison with control groups revealed values of treated groups significantly higher (p<0.05) from day 7 PT onwards. Significant improvement in these parameters following successful treatment of haemonchosis has been recorded earlier in goats (Jayraw and Raote, 2004) and in sheep (Qamar and Maqbool, 2012) The blood sucking habit of Haemonchus contortus causes continuous blood loss which might be responsible for decreased value of Hb, PCV, and TEC. The post- treatment increases in these values indicated elimination of the parasite and thereby improved health status of animals.

Table 1: Faecal egg count and haemato-biochemical values (Mean ± S. E.) before and after treatment with ethanol extract mixture of Embelia ribes and Vernonia anthelmintica against Haemonchus contortus in sheep

0 day 3-day PT 7-day PT 10-day PT
EPG GI 1723.33±30.84 943.33±31.26* 501.667±5.42* 525±28.137*
GII 1866.66±158.99 2000±146.62 1966.667±141.81 1941.667±25.15
FECR% GI 52.83 74.49 72.97
Hb(g%) GI 8.15±0.10 8.23±0.10 9.11±0.07*a 9.90±0.13*a
GII 8.60±0.29 8.61±0.21 8.35±0.18 b 8.35±0.14b
PCV(%) GI 24.45 ±0.32 24.33 ±0.33 28.58 ±0.27* a 31.28 ±0.51* a
GII 25.90 ±0.92 26.36 ±0.90 25.10 ±0.13* b 24.21 ±0.22* b
TEC(x106//µl) GI 4.88 ±0.19 5.39 ±0.18 6.90 ±0.17* a 7.83 ±0.14* a
GII 5.48 ±0.55 5.78 ±0.32 4.98 ±0.37 b 5.08 ±0.28 b
Total plasma protein GI 6.5 ± 0.21 6.63±0.19 6.78±0.16 6.90±0.17* a
GII 6.4±0.10 6.467±0.10 6.45±0.11 6.25±0.12 b
Serum albumin(g/dl) GI 3.01±0.06 3.05±0.05 3.10±0.03 a 3.21±0.031 a *
GII 3.10±0.03 2.933±0.04 2.933±0.04 b* 2.86±0.033 b *
AST(IU/L) GI 58.38±1.98 65.76±2.17* 63.01±2.36 61.26±2.09
GII 57.06±2.07 61.15±0.54 60.81±2.30 59.45±2.11
ALT(IU/L) GI 18.43±0.74 23.60±0.86* a 21.23±1.10 19.88±1.11
GII 17.26±0.67 19.06±0.53 b 19.66±0.72 18.78±0.67
ALP(IU/L) GI 105.71 ± 5.44 114.61±4.84 97.18±4.71 a 86.133±4.55* a
GII 107.26±3.88 105.88±2.50 111.45±3.40 b 108.05±3.53 b
Serum creatinine (IU/L) GI 1.20 ± 0.10 1.08 ±0.09 1.15 ±0.12 1.06 ±0.92
GII 1.06 ±0.06 1.11 ±0.03 1.08 ±0.07 1.18 ±0.07

EPG- Egg per gram ; FECR% – Faecal egg count reduction percentage ; Hb- haemoglobin; PCV-Packed cell volume; TEC- Total erythrocytic count ; ALP -Alkaline phosphatase, ALT-Alanine amino transferase , AST – Aspartate amino transferase; Values marked * differ significantly within group with value at day zero (P< 0.05) , Values `with dissimilar superscript a,b differ significantly  between groups (P< 0.05).

Within the treated group, total plasma protein (TPP) levels and serum albumin were found to be significantly increased (p<0.05) on 10-day PT as compared to that of day zero values. Within control group, significant (p<0.05) decrease in total serum albumin (TA) level was recorded from day 7 PT onwards. Further comparison of treated group with infected control group revealed that albumin level in group I was significantly (p<0.05) higher than the control group from day 7 PT onwards. Increase in TPP and TA values following successful therapy have also reported earlier (Patil et al., 1993). So, increase in TPP and TA values indicates the effectiveness of herbal preparation used in the present study.

Within group I, AST values were found significantly (p<0.05) increased on day 3 PT but later on day 7 and 10 PT, the values decreased to non-significant level. Upon comparison with control groups, values in group-I were found non significantly different. ALT level values within GI got significantly elevated (p<0.05) on day 3 PT but later on values declined and eventually became non-significant on day 10 PT. Within control group, there was no significant change in ALT level. When compared with the control group, ALT values on day 3 PT was significantly (p<0.05) higher in GI only to be non-significantly different on day 10 PT. Rise in AST and ALT values in the treated group could be attributed to the consumption of f herbal extract and subsequent hyperactivity of liver, although rise were in ranged with standard upper limit values as mentioned in the standard book (Radiostites et al., 2007). This suggests no significant damage to the liver. Alkaline phosphatase (ALP) levels within the treated group was found significantly decreased on day 10 PT as compared to the values on day zero. However, within infected control group there were no significant changes. When compared with infected control group, the ALP levels in the treated group were significantly lower on day 10 PT. Decreasing trends from day zero indicated the reduction in the number of worms since presence of worms and subsequent damaged to abomasal mucosa induce production of enzyme alkaline phosphatase (ALP) in gastric mucosa which eventually pushes the serum ALP level up. Following treatment, no significant changes were noticed in the creatinine level in all treated groups and were in normal limit. This suggests that extract did not cause any harm to the kidney function.

In the present study, both Embelia ribes and Vernonia anthelmintica in combination induced moderate anthelmintic activity. The possible mechanism of Embelia ribes may be due to its effect on inhibition of glucose uptake in the parasites and depletion of its glycogen synthesis. It may also have activated nicotinic cholinergic receptor in the worms resulting in either persistent depolarization and hyperpolarisation (Tripathi, 2006; Swarnkar et al., 2009). The molecule which is said to be responsible for anthelmintic acitivity in Embelia ribes is embolic acid or embelin. This embelin is of small molecular weight and cell permeable and which alone or synergistic with other bioactive molecules exerts its anthelmintic activity. In Vernonia anthelmintica, the anthelmintic property may be attributed to the molecule vernolin, vernolic acid, vernodalol in addition to flavonoids and fatty acids (Javed and Akhtar,1990). However, the mechanism of action of these compounds is not fully understood. Despite having moderate efficacy, it is clear from the present investigation that Embelia ribes and Vernonia anthelmintica can very well be the alternative therapeutic approach and can provides an answer to the problem caused by widespread development of anthelmintic resistance. However, more research work needed to be done regarding standardization of dose in different species of animals for an effective in- vivo bioavailability of bioactive molecules, detection of new bioactive substances, mechanism of action of bioactive molecules, toxicity in animals before it can be put to large scale commercial use in animals.


Aqueous alcoholic extract of Embelia ribes fruits and Vernonia anthelmintica seeds reduced faecal egg count by 72%. Improvement in haematological and biochemical values suggested the reduction in number of worm loads. Coprological examination showed Haemonchus contortus as predominant strongyles. Hence the formulation showed promising efficacy against Haemonchus contortus which is the most pathogenic worm of small ruminants and has exhibited resistance to number of anthelmintics. However, further study is needed regarding dose standardization so as to further increase the efficacy of the formulation.


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