Efficacy of aqueous and methanol extracts of Euphorbia wallichii Hook. f. against larval toxocariasis

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Efficacy of aqueous and methanol extracts of Euphorbia wallichii Hook. f. against larval toxocariasis

https://doi.org/10.21203/rs.3.rs-2433679/v1

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The ever increasing anthelmintic resistance and low efficacy of conventional anthelmintics against larval toxocariasis has created an interest in evaluating plants as an effective source of anthelmintics. The aqueous and methanol extracts of Euphorbia wallichii were evaluated for efficacy against larvae of Toxocara canis both in vitro and in vivo, using an embryogenesis inhibition assay and a larval recovery assay respectively. In the in vitro study (1000) eggs were treated with 0.5, 1, and 2 mg/mL of plant extracts and the positive control was treated with same dose of albendazole. Crude methanol extract of E. wallichii (LC₅₀ 1.09 mg/mL) exhibited higher embryogenesis inhibition compared to its crude aqueous extracts (LC₅₀ 1.89 mg/mL). In the in vivo study, embryonated eggs (1000) of Toxocara canis were orally inoculated to wistar rats followed by treatment with crude methanol and aqueous extracts of Euphorbia walliachii at concentrations of 100, 200 and 400 mg/Kg and their liver and lungs were examined for the presence of T. canis larvae on the 7th day post infection (dpi). Maximum efficacy (64.75%) in larval count reduction was recorded for crude methanol extract in liver at dose of 400 mg/Kg in liver. Least efficacy (6.25%) in larval count reduction was recorded for crude aqueous extract E. wallichii at dose of 100 mg/Kg in lungs. The results reveal that Euphorbia walliachii extracts has an antitoxocaral effect and warrants further study as to suitability as an alternative anthelmintic.

Anthelmintics

Toxocara canis

toxocariasis

Euphorbia wallichii

zoonoses and larval migrans

Helminth infections are a big challenge around the world because of their epidemiological characteristics such as the continuous contamination of environment. One such helminth is Toxocara canis (Werner, 1782), the most common roundworm of dogs and other canids, has detrimental public health consequences due to its zoonotic potential (Shalaby et al., 2009). Adult female T. canis excrete thousands of eggs into the environment with defecations of the primary infected host. Eggs become embryonated and infective within two to five weeks after excretion, they are unembryonated and uninfective when excreted (Magnaval et al. 2001). These can survive in the environment for years, owing to their highly resistant shell (Gillespie, 1993). Human infection with larvae of Toxocara canis is known as larval toxocariasis, caused by ingestion of the embryonated eggs usually after consuming contaminated raw vegetables, water or by eating infected raw meat containing encapsulated larvae (Baixench et al., 1992; Nagakura et al., 1989; Salem et al., 1992; Schantz, 1989 and Sturchler et al., 1990). Larvae penetrate the small intestines, through the portal circulation system, to visceral tissues (Asenbauer et al., 2001). Throughout the migration, they cause an inflammatory response and eosinophilia (Asenbauer et al., 2001). Many known drugs are effective against adult Toxocara canis living in the gastrointestinal tract of dogs. However, larval migrans, the disease in humans caused by the migration of nematode larvae in to tissue, are usually difficult to cure. The drugs presently used for treatment against visceral toxocariasis are limited to tinidazole, albendazole, diethylcarbamazine, and thiabendazole (El-Sayed and Ramadan 2017). One of the shortcomings of these drugs is their low water solubility, which can cause a low and/or variable bioavailability after oral administration (Torrado et al. 1996). Besides, albendazole is metabolized in the liver giving rise to the bioactive albendazole sulphoxide and then the inactive albendazole sulphone (Gottschall et al., 1990).

The information about herbs and their collective and respective roles in promoting health is modest (Ballantine et al., 1999). Herbs have been used for medicinal purposes for centuries, however, the use of herbs as medicine has increased and research interest has focused on various herbs that possess antitumor, immune stimulating, antiplatelet, or hypolipidemic properties that may be useful to reduce the risk of cancer and cardiovascular disease (Craig, 1999). Euphorbia species are known for medicinal properties and used for treating various human disease like migraine, skin diseases, gonorrhea, viral disease, intestinal parasites, (Ramezan et al., 2008) and antimicrobial (Papp et al., 2004; Al-Younis et al., 2009). Euphorbia wallichii is used in Chinese folk medicine for the curing of skin disease and edema (Pan et al., 2006). Its leaves are used as purgative and indigestion treatment. The juice of the plant is used externally on warts and is used for dermatological infection (Farooq et al., 2014). Seeds and whole plant are used as remedy for edema, cutaneous disease, exanthema, asthma and anthrax (Haq et al., 2012). They possess antitumor activity and thus are recommended as anticancer remedies (Ahmad et al., 1988; Duarte et al., 2006). The development of a drug for larval toxocariasis is a pressing need. To find a new anthelmintic against the Toxocara canis larvae, both in vitro and in vivo screening assays to define the anti-Toxocaral activity of E. walliachii extracts, was used.

Morhology of Plant

Euphorbia wallichii is an erect perennial herb up to 60 cm tall with several stems arising directly from a stout woody stock, forming dense clumps. Leaves are sessile or sub-sessile, alternate, elliptic-lanceolate to ovate-lanceolate. Smaller scale leaves at the base of the stem are sub-acute, acute, rounded or tapered at the base. The inflorescence cyathia is sessile, except for the pedunculate one terminating the main stem. Fruits are trilobate sub-globose, smooth glabrescent, bright green. Seeds are shiny and pale green, ovoid smooth (Hassan et al., 2016).

Collection of Plant Material

The present plant material Euphorbia wallichii (locally known as ‘junglee gur sochal’) a plant species of the family Euphorbiaceae, growing mainly in the Himalaya, including Qinghai-Tibetan Plateau, India, Nepal (Haq et al., 2012; Muzafar et al., 2017). The plant material (shoot) was collected from Gulmarg, Kashmir during July, 2017. The mature plant at peak of flowering was collected in polythene bag and was processed by standard techniques adopted by KASH (Kashmir University Herbarium). The plant was identified and authenticated at Center for Biodiversity and Plant taxonomy. A voucher specimen (2680-KASH) was deposited in KASH.

Preparation of Extracts

Euphorbia walliachii were washed with distilled water then shade dried in a well ventilated room at Department of Zoology, University of Kashmir. The dried plant was powdered by an electric blender and stored in air tight container. Methanol extracts were prepared by dissolving 100g of the powdered plant material in 500 mL of methanol in a Soxhlet extractor. The plant matter was allowed to percolate for 24 hrs. at 40-50^oC, the extract was filtered (Iqbal et al., 2004). The filtrate was evaporated under reduced pressure of 22-26mmHg at 40^oC in a vacuum rotary evaporator (R-201, Shanghai Shenshen). The final crude extract was transferred to an airtight container for storage at 4^oC until further use. Aqueous extracts were prepared by dissolving 100g of the powdered plant material with 500 mL of distilled water in a Soxhlet extractor. The plant matter was allowed to percolate for 24 hrs. at 90-100^oC, the extract was filtered. The filtrate was evaporated under reduced pressure of 22–26 mmHg at 40^oC in a vacuum rotary evaporator (R-201, Shanghai Shenshen). The final crude extract was transferred to an airtight container for storage at 4^oC until further use.

Recovery of eggs and preparation of inoculums

Toxocara canis were collected from fecal samples of stray dogs in Kashmir valley. Eggs of T. canis were collected by dissecting the uteri of female T. canis as described by Bowman et al., (1987). The obtained eggs were suspended in distilled water and purified by straining through a sieve of 0.5 mm size and washed with saline several times by centrifugation at 500 g for 3 min. Then the sediment was decanted and saline was added. The number of eggs was determined using the hemocytometer to obtain a concentration of 1000 eggs/ mL. The eggs were incubated for four weeks at 27ºC to allow the eggs to develop to infectivity (Zibaei et al., 2007).

In-Vitro study

Inhibition of embryonation was conducted according to method described by Coles et al. (2006). Approximately 1000 eggs of T. canis were collected per tube in 1 mL of saline and 1 mL of increasing concentrations of plant extracts (0.5, 1 and 2g/mL) prepared with saline. In addition, Albendazole (0.5, 1 and 2mg/mL) was used as positive control and 1000 eggs in 2 ml saline as negative controls. The tubes were covered, and the eggs were incubated for 4 weeks at temperature of 27°C. Thereafter, the number of the embryonated eggs present per tube was counted using a microscope. This experiment was carried out in triplicates.

In-Vivo study

Wistar rats of (240–260 g) were obtained from Indian Institute of Integrative Medicine, Jammu. They were maintained on water and stock commercial pellet diet ad libitum. Rats were kept under 12 hours light/dark cycle. They were acclimatized to laboratory conditions for two weeks before the commencement of experiment. Handling, and sacrifice procedures followed were as per the guidelines of CPCSEA (Committee for the Purpose of Control and Supervision of experiments), India. All animal proceedings were approved by the Animal Ethics Committee, University of Kashmir (IAEC-Approval-KU/2017/7).

Inoculation of T. canis eggs to wistar rats.

Wistar rats were randomly divided to 5 groups (I, II, III, IV, and V) for each extract, with each group having twelve rats, six each for methanolic and aqueous extracts. Embryonated eggs were gavaged with the help of a flexible tube fitted to a graduated syringe at the rate of 1000 embryonated eggs of T. canis per rat. Subsequently, seven days after eggs inoculation, treatment was started and continued for a week before the rats were sacrificed. Rats of the control group (Group I) received only eggs and were left untreated during the course of experiment. Group V received albendazole as standard drug at the dose of 20 mg/Kg twice a day for seven days. Group II, III and IV received crude aqueous and methanolic extract at a dose of 100 mg/Kg, 200 mg/Kg and 400 mg/Kg respectively, twice a day for seven days.

Larval recovery

The larvae in the visceral tissue were collected by artificial digestive juice treatment, as described by Parsons and Grieve (1990). The tissue were artificially digested individually in pepsin-HCl solution (pH 1–2) (Sigma-Aldrich, India) and incubated for four hours at 37^OC with periodic agitation. The liquid was centrifuged at 1500 rpm for 2 minutes. The sediment was observed under microscope for larvae count. The brain was squashed between two slides to count the larvae.

Efficacy of extracts was determined by following formula.

Efficacy (%) = 100 × (x-y)/x

Where, x = mean larvae count in infected-untreated control group

y = larvae count in infected and treated group

Statistical analysis

Data of embryogenesis inhibition and larval recovery counts were presented as a percentage of inhibition/efficacy and mean ± standard deviation (M ± SD). The lethal concentration 50 (LC₅₀) of extract concentration required to prevent embryogenesis of 50% of the eggs (in case of embryonation inhibition assay) was calculated by Probit analysis. The means of embryogenesis inhibition and larval counts treated with extracts were analyzed by one-way ANOVA post-hoc Tukey test. Difference between means was considered statistically significant at p < 0.05. All statistical procedures were carried out using SPSS 16.0 (SPSS Inc., Chicago, IL, USA).

In-Vitro

The results from the embryogenesis inhibition experiment revealed that the methanol and aqueous extract of Euphorbia wallichii in the different concentrations (0.5, 1, 2 mg/mL) significantly inhibited embryogenesis of Toxocara canis eggs (Table 1). The best inhibitory effect was observed with 2 mg/mL methanolic concentration of E. wallichii, where the efficacy was 86.49%. Eggs treated with the concentration of 0.5, and 1 mg/mL of methanolic extract of E. wallichii showed embryogenesis inhibition activity of 31.16 and 60.49% respectively (Table 1). The LC₅₀ value of 1.09 mg/mL was found for the methanolic extracts of E. wallichii. However, lower inhibitory effect was recorded for the aqueous extract of E. wallichii compared to its methanolic extract. Eggs treated with 2 mg/mL concentration of aqueous extract of E. wallichii inhibited embryogenesis by 66.76%, and eggs treated with the concentration of 0.5, and 1 mg/mL of aqueous extract of E. wallichii showed embryogenesis inhibition activity of 19.58 and 41.02% respectively (Table 1). The LC₅₀ value of 1.89 mg/mL was found for the aqueous extracts of E. wallichii, higher to its methanolic extract. The effects of all extract concentrations showed statistically significant differences compared to the negative control (p < 0.05). However, positive control (albendazole treated) showed embryogenesis inhibition efficacy of 94.04, 81.02 and 61.29% with concentration of 2, 1 and 0.5 mg/mL, respectively (Table 2). LC₅₀ value of albendazole was found to be 0.55 mg/mL. Albendazole showed best embryogenesis inhibition activity followed by methanolic extracts of E. wallichii, and least embryogenesis inhibition activity was observed for aqueous extracts of E. wallichii.

In-Vivo

Results from in-vivo experiment showed promising efficacy of Euphorbia walliachii extracts to that of standard drug Albendizole. The mean larval count in group I was 122 ± 6.0663 and 48 ± 3.6878 in liver and lungs respectively. Present study indicated that all infected groups showed dose dependent depression in larval count. The methanolic extract of Euphorbia walliachii showed high larval reduction compared to similar doses of its aqueous extract. Methanol extract at 400 mg/Kg showed larval count of 43 ± 1.7889 in liver and 24 ± 2.00 in lungs with efficacy of 64.75% and 50% in respective organs (Table 3). Compared to methanol extract, aqueous extract at same dose (400 mg/Kg) showed larval count, 69 ± 2.8284 larvae in liver and 34 ± 2.4495 in lungs with lower efficacy of 43.44% and 29.16% in respective organs (Table 4). The effects of all methanolic concentrations of Euphorbia walliachii showed statistically significant differences on larval count within the group p < 0.05 (Table 3). Aqueous extract at 25 mg/ml showed least larval reduction, with larval count of 104 ± 3.0332 in liver and 45 ± 2.5298 in lungs with efficacy of 14.75% and 6.25% in respective organs (Table 4). The effects of all aqueous concentrations of Euphorbia walliachii showed statistically significant differences on larval count within the group (p < 0.05), except for Group I and II in lungs (p > 0.05) (Table 4). The highest larval count was recorded in infected-untreated group, with 122 ± 6.0663 larvae from liver, 48 ± 3.6878 from lungs and 2 ± 2.08 from brain.

Table 1

Mean count of embryonated eggs of *T. canis* treated with aqueous and methanolic extracts of *E. wallichii.*
Dose (Group treated with Aq.E)	Mean ± SD of Embryonated eggs	Efficacy (%)	LC₅₀	Dose (Group treated with Me.E)	Mean ± SD of Embryonated eggs	Efficacy (%)	LC₅₀
Negative Control (I)	878.33 ± 18.72^a		1.89 mg/mL	Negative Control (I)	878.33 ± 18.72^a		1.09 mg/mL
0.5 mg/mL (II)	706 ± 15.01^b	19.58		0.5 mg/mL (II)	604.67 ± 17.21^b	31.16
1 mg/mL (III)	518 ± 16.82^c	41.02		1 mg/mL (III)	347 ± 31.00^c	60.49
2 mg/mL (IV)	292 ± 2.64^d	66.76		2 mg/mL (IV)	118.67 ± 16.62^d	86.49

Superscripts a,b,c, d and e : Means within the same columns with different letters are statistically significant (p < 0.05).

Table 2

Mean count of embryonated eggs of *T. canis* treated with albendazole.
Dose (Group treated with Albendazole)	Mean ± Standard Deviation of Embryonated eggs	Efficacy (%)	LC₅₀
Negative Control (I)	887.33 ± 18.72^a		0.56 mg/mL
0.5 mg/mL (II)	340 ± 5.00^b	61.29
1 mg/mL (III)	166.67 ± 11.50^c	81.02
2 mg/mL (IV)	52.33 ± 21.18^d	94.04

Superscripts a,b,c, and d : Means within the same columns with different letters are statistically significant (p < 0.05).

Table 3

In vivo antitoxocaral activity of crude methanolic extracts of *Euphorbia walliachii* in rat
Group (dose)	Mean ± Standard Error of larvae count in Liver	Efficacy in liver (%)	Mean ± Standard Error of larvae count in Lungs	Efficacy in lungs %	Mean ± Standard Error of larvae count in Brain
I (negative control)	122 ± 6.07^a		48 ± 3.69^a		2 ± 2.08
II (100 mg/Kg)	84 ± 1.79^b	31.14	39 ± 1.41^b	18.75	0
III (200 mg/Kg)	69 ± 3.40^c	43.44	32 ± 1.79^c	33.33	0
IV (400 mg/Kg)	43 ± 1.79^d	64.75	24 ± 2.00^d	50	0
V Albendizole treated: Positive control (20 mg /Kg)	32 ± 1.79^e	73.77	17 ± 1.79^e	64.58	0

Superscripts a,b,c, d and e : Means within the same columns with different letters are statistically significant (p < 0.05).

Table 4

In vivo antitoxocaral activity of crude aqueous extracts of *Euphorbia walliachii* in rat
Group (dose)	Mean ± Standard Error of larvae count in Liver	Efficacy %	Mean ± Standard Error of larvae count in Lungs	Efficacy %	Mean ± Standard Error of larvae count in Brain
I negative control	122 ± 6.0663^a		48 ± 3.6878^a		2 ± 2.280
II (100 mg/Kg)	104 ± 3.0332^b	14.75	45 ± 2.5298^a	6.25	0
III (200 mg/Kg)	87 ± 2.3664^c	28.68	39 ± 2.0976^b	18.75	0
IV (400 mg/Kg)	69 ± 2.8284^d	43.44	34 ± 2.4495^c	29.16	0
V Albendizole treated: positive control (20 mg/Kg)	32 ± 1.7889^e	73.77	17 ± 1.7889^d	64.58	0

Superscripts a,b,c, d and e : Means within the same columns with different letters are statistically significant (p < 0.05).

An effective drug against larval toxocariasis is yet to be developed (Lescano et al., 2005). For the effective chemotherapy of larval Toxocara canis, drugs with high activity are required. Promising results were obtained from inhibition of embryogenesis of T.canis eggs at concentration of 2 mg/mL for both E. wallichii. However, at lower concentration of 0.5 and 1 mg/mL lower inhibition was found compared to 2 mg/mL treated group. Embryogenesis was recorded in as much as 50% of eggs treated with lower doses of plant extracts. Our findings are in accordance with the results of Orengo et al. (2016) who reported ethanol extracts of Allium sativum and Allium cepa possesses antitoxocaral activity as they affect the viability of T.canis larvae. Furthermore, Verocai et al. (2010) found that the eggs of T. canis treated with sodium hypochlorite were damaged. El-Sayed and Ramadan (2017) reported the embryogenesis inhibition activity of Zingiber officinale ethanolic extracts with an efficacy of 99.64% at the concentration of 100 mg/mL. The high inhibition of embryogenesis of T. canis eggs may be attributed to diffusion of the extracts through egg shell and destruction of the cells by bioactive compounds (El-Sayed and Ramadan, 2017). Besides larvicidal potential of some herbs has been reported against different helminths (Ketzis et al., 2002; Assis et al., 2003; Hordegen et al., 2006; Bizimenyera et al., 2006; Maciel et al., 2006; Eguale et al., 2007; Shaibani et al., 2009), attributed to secondary metabolites of plants. Plants provide a range of natural compounds, belonging to diverse molecular families, having medicinal properties. Ethno-botanical information revealed that the plant used in the current study is used for various medicinal conditions (Hassan et al., 2016). The present study revealed that the crude extracts of E. wallichii are effective against larvae of Toxocara canis. In-vivo antitoxocaral activity of methanolic extract were more effective than its aqueous extracts, this could be due to the presence of a higher concentration of the alcohol-soluble active molecule(s). Euphorbia walliachii showed a significant dose dependent effect on the viability of T. canis larvae. The best effect was observed at the concentration of 400 mg/Kg, larval count of 43 ± 1.7889 in liver and 24 ± 2.00 in lungs with efficacy of 64.75% and 50% respectively were found. Antitoxocaral property of Euphorbia walliachii extracts may be attribute to its photochemical constituents such as, saponins flavonoids, tannins, alkaloids, glycosides that were held responsible for the varying inhibition against the microorganisms (Riaz et al. 2015). Euphorbia hirta reduced fecal egg output when used to treat dogs infected with Ancylostoma caninum, Toxocara canis, Dipylidium caninum and Echinococcus spp., attributed to excessive phenolic compounds (Adedapo et al., 2005). The reduction of worm load observed with the extracts of E. hirta in experimental animal was attributed to the presence of phenol compounds C₈H₁₈O₅ in the plant extracts (Rice, 1965). Nitroxynil niclofolan, and disophenol and other substituted phenols are established anthelmintics. They act by uncoupling the reaction involved in electron transport-associated events for ATP generation in mitochondria. This is very fatal to blood sucking helminths (Behnke et al., 1991). The phenols present in the plant extract could have caused reduction in larval count through the mechanism that leads in exhaustion and death of larvae. Besides Euphorbia walliachii possesses tannins, chemically which are polyphenol compounds (Bate-Smith, 1962). It is also possible that tannins present in the extracts of E. walliachii could act in a similar manner to that of E. helioscopia which caused death of H. contortus as reported by Kane et al. (2009). The anthelmintic activity of tannins is attributed to their binding to free proteins in the gastrointestinal tract of the host (Athnasiadou et al., 2001; Hoste et al., 2006) or glycoprotein on cuticle and affecting physiological processes like feed absorption, reproduction, and motility (Thompson and Geary, 1995; Aerts et al., 1999; Githiori et al., 2006). Tannins possess anthelmintic activity which is attributed to connecting to the cutricle of larvae, rich in glycoprotein’s. (Cala et al. 2012). Moreover secondary plant metabolites are bioactive against various pathogens. Flavonoids which inhibit the formation of DNA /RNA or inhibit the reproduction of the infectious agents (Jassim and Naji, 2003). Saponins affect permeability of cell membrane of the helminths, besides it causes disintegration and vacuolization of the teguments (Wang et al. 2010). Alkaloids intercalate with DNA synthesis and interfere with the metabolism of aromatic amino acids in the parasite (Shaibani et al. 2009).

Efficacy was more for methanol than aqueous extracts, demonstrating methanol to be better solvent for extraction of bioactive ingredient. Besides, the total action is a sum of the activities of the bioactive constituents in the extract (Rates, 2001). It is likely that the compounds determining yield are more soluble in methanol than water. This finding calls for more studies to identify the underlying compound. The antitoxocaral activity could be attributed to the secondary plant metabolites that may act in additive, synergistic or antagonistic manner at single or multiple sites unlike the synthetic drugs as was recognized by Briskin (2000). Reduction of larval count by the extract of E. walliachii in this study is a positive and welcome development in our struggle to control larval toxocariasis, because the appropriate anthelmintic effects demonstrated by its ability for higher availability, rapid preparation, stronger effects at low concentrations, high efficacy in shorter time after exposure, and less toxic effects. E. walliachii is available throughout the Himalaya. The easy access to this plant and its availability might mean that the cost of medication would be reduced drastically.

The results confirmed the effectiveness of Euphorbia wallichii extracts in comparison to standard drug albendazole, against larvae of Toxocara canis. The study highlighted the potential of E. wallichii as a natural source of anthelmintic drug, alternative to conventional anthelmintics for the control of larval toxocariasis, should be subjected to quality controlled extracts or bioactive compounds isolation. Toxicity studies must be conducted in vivo to determine the minimum non-lethal concentrations needed for the treatment before processing for the use against human toxocariasis.

Acknowledgement

The authors are highly acknowledged to Council of Scientific and Industrial Research (CSIR), New Delhi, India for providing financial support in the form of Junior Research Fellowship (JRF) to the corresponding author. We are thankful to Plant Taxonomy Division, Department of Botany, University of Kashmir for identifying the plant material. Thanks are due to the HoD, Department of Zoology, University of Kashmir for providing the laboratory and animal house facilities for successful completion of the work.

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Efficacy of aqueous and methanol extracts of Euphorbia wallichii Hook. f. against larval toxocariasis

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