In vitro evaluation of the composition and acaricidal efficacy of Urtica fissa leaf ethyl acetate extract against Sarcoptes scabiei mites

In veterinary medicine, natural products provide an alternative to chemical agents for mite management. In the present study, the acaricidal efficacy of Urtica fissa leaf ethyl acetate extract against Sarcoptes scabiei mites was examined. The chemical composition of the extract was determined using liquid chromatography-mass spectrometry (LC-MS) analysis. The ethyl acetate extract was found to be extremely toxic to mites at a concentration of 100 mg/ml (m/v), killing all S. scabiei within two hours. The median lethal time (LT50) values for ethyl acetate extract concentrations of 25, 50, and 100 mg/ml against S. scabiei were 1.706, 1.204, and 0.750 h, respectively. The median lethal dosage (LC50) for S. scabiei was 19.14 mg/ml at two hours. The chemical composition of the ethyl acetate extract was evaluated using LC-MS, showing that the major components were schaftoside (8.259%), carnosol (6.736%), prostaglandin A2 (5.94%), 13(S)-HpOTrE (4.624%), nandrolone (4.264%), 1H-indole-3-carboxaldehyde (4.138%), 9-oxoODE (3.206%), and stearidonic acid (2.891%). In conclusion, these findings indicate that Urtica fissa contains promising new acaricidal compounds capable of successfully controlling animal mites.

Sarcoptic mange, a skin condition caused by the mite Sarcoptes scabiei, can decimate wild animal populations (Montecino-Latorre et al. 2019).These ectoparasitic infestations have been observed to be highly contagious and often fatal in rabbits (Singh et al. 2022).The mite can also survive offhost for some time while remaining contagious.As the mite has no free-living growth stages or intermediate hosts in its life cycle, its morphology and ecology are well adapted to living in close proximity to the host.As a result, this infectious, host-specific, burrowing, and astigmatic mite acts as an obligatory parasite throughout its life cycle (Pasipanodya et al. 2021).As shown by Lynar et al. (2017), the mites secrete complement inhibitors into burrows, potentially promoting the development of secondary staphylococcal and streptococcal infections and, subsequently, invasive bacterial infections.Ivermectin, avermectin, and other pharmacological drugs are used frequently in veteri-https://doi.org/10.17221/6/2023-VETMEDnary clinics to treat and control psoriasis and have produced positive therapeutic benefits (Liao et al. 2014).However, due to improper drug usage, together with drug resistance and treatment failure, the management of mange mites remains a very difficult task (Gopinath et al. 2018).Therefore, there is an urgent need to develop a new generation of effective acaricides that can simultaneously kill all developmental stages of the scabies mite (adult, nymphal, larval, and egg), which would improve the effectiveness and efficiency of treatment and reduce the rate of scabies recurrence.
Plant-derived acaricides are preferred due to their various modes of action, low cost, simplicity of manufacture in countries with few manufacturing units, minimal non-target impact, high efficiency, and environmental friendliness (Benelli et al. 2017).The stinging nettle, Urtica fissa, grows widely in China.There are about 35 Urtica species in the world, of which 23 are found in China, mainly distributed in the northern and southwestern regions of the country (Wang et al. 2022).Most Urtica species have medicinal properties and have long been used in traditional medicine.In traditional Chinese medicine, Urtica extracts have the effects of expelling wind, dredging collaterals, activating blood circulation, and relieving pain and are mainly used to treat rheumatic pain, infant convulsions, and postpartum convulsions, among other applications.Western herbalists have also used stinging nettle extracts, juice, tea, and freeze-dried products, for instance, as blood-replenishing tonics and for the treatment of periodic rhinitis (Upton 2013).Studies have shown that Urtica leaves contain flavonoids, lignans, coumarins, triterpenoids, steroids, alkaloids, polysaccharides, organic acids, and other chemical components (Li et al. 2009;Wang et al. 2016;Wang et al. 2018).Studies have found that U. fissa plants have a wide range of pharmacological activities, and have been used to treat hyperglycaemia (Mao et al. 2020), as well as having antioxidant (Wang et al. 2019), lipid-regulatory, antinociceptive, anti-inflammatory (Hajhashemi and Klooshani 2013), antibacterial, antitumor, and anti-prostatic hyperplasia (Zhang et al. 2022) effects.
However, to the best of our knowledge, the acaricidal activity of U. fissa has not yet been investigated.Here, we assessed the effectiveness of ethyl acetate extracts of U. fissa against S. scabiei in vitro and investigated the main active components of the extract.

Ethical statement
This study was reviewed and approved by the Animal Ethics and Welfare Committee of Sichuan Agricultural University (Approval No.: 2012-024).Institutional ethical and animal care guidelines were adhered to during sampling, and all procedures were conducted in accordance with the China Guide for the Care and Use of Laboratory Animals.Furthermore, all procedures involving animals and their care were conducted in accordance with ARRIVE guidelines [PLoS Bio 8(6), e1000412,2010] and all plant experiments and collections were done with relevant permissions and complied with the relevant institutional, national, and international guidelines and legislation.

Plant material and preparation of extracts
Fresh plant material was harvested from Zunyi (27°38'N; 107°46'E, 754 m), Guizhou Province, Southwest China, in July 2022.The U. fissa specimens were identified at the College of Veterinary Medicine, Sichuan Agricultural University, P.R. China.The U. fissa leaves were dried and crushed in a knife mill.Subsequently, 0.2 kg of the plant material was immersed in 75% ethanol (2 l of 75% per 100 g of plant material) for ultrasonic extraction at 50 °C and 500 W for 1 hour.The extracts were then mixed and concentrated by evaporation using a rotary evaporator.Extractions were performed three times using the same volume of ethyl acetate.After concentration on the rotary evaporator, the extracts were vacuum-dried for 24 h at 60 °C to guarantee thorough elimination of any remaining ethyl acetate that could impact the experimental outcomes.

Isolation of S. scabiei from rabbits
Institutional ethical and animal care standards were followed during the sampling exercise, and all procedures were performed following the Guidelines for the Care and Use of Laboratory Animals.Scabs obtained from the infected legs of naturally infected rabbits were used to isolate S. scabiei.Scabs were placed on Petri dishes and cultured in an incubator for 30 min at 35 °C.
To ensure consistency of the results, only recently fed adults were used; this was assessed by the colour of the mites; the darker ones were assumed to have fed more recently than paler individuals (Alimi et al. 2021).The rabbits were given treatment immediately after the materials had been collected.

Acaricidal activity of extracts in vitro
A contact toxicity bioassay was conducted as previously described (Nong et al. 2013) with some modifications.The ethyl acetate extracts were diluted in 10% glycerine to concentrations between 25 and 100 mg/ml (25, 50, 100 mg/ml).Filter paper chips were used to absorb the liquid after pouring the 0.1-ml sample into the Petri dishes (5 cm in diameter and 2 cm high), and 10 mites were introduced to each dish.Liquid paraffin was used as a negative control and ivermectin (1%) as a positive control.Six replicates of each concentration of extract were used.All plates were incubated for 0.5, 1, 1.5, and 2 h at 25 °C with 75% relative humidity before using evaluation under a stereomicroscope.A needle was used to periodically stimulate mites to test their vitality; if no response was observed, the mites were recorded as dead.

Statistical analyses
SPSS v20.0 (IBM Corp., Armonk, NY, USA) was used for all calculations (Zhou et al. 2019b).The significance of changes in mean mite mortality between various concentrations was determined using the probability technique.The complementary log-log (CLL) model was used to determine the median lethal concentration (LC50) values and median lethal time (LT50) (Liao et al. 2014).

Acaricidal activity of the extracts
The acaricidal efficacy of the U. fissa ethyl acetate extracts against S. scabiei was evaluated in vitro.The results showed that acaricidal activity depended significantly on the extract concentration.The mortality rates of mites treated with the highest concentration of extract (100 mg/ml) and those in the positive control group reached 100% within 2 hours (Figure 2).Significant effects (P < 0.05) on S. scabiei were also observed over a range of dosages, with 100.0%, 76,7%, and 60.0% of mites killed after treatment with 100, 50, and 25 mg/ml of extract, respectively, within 2 hours (Table 2).Tables 3 and 4 illustrate the lethal con-     The toxicity of the ethyl acetate extracts to mites was both concentration-and time-dependent.The LC50 result for S. scabiei was 19.14 mg/ml after 2 hours.

DISCUSSION
To the best of our knowledge, the above results present the first demonstration of the acaricidal effects of ethyl acetate extracts of U. fissa against S. scabiei.The toxicity of the extract was both time-and concentration-dependent (Table 2).Comparable in vitro and in vivo effects have been observed for Eupatorium adenophorum ethanol extracts against S. scabiei and Psoroptes cuniculi (Hu et al. 2014;Liao et al. 2014) whereas an investigation of the effects of Ligularia virgaurea extracts against S. scabiei found that concentrations of 2 g/ml killed all S. scabiei within 2 h and concen-(A) (B) https://doi.org/10.17221/6/2023-VETMEDtrations of 1 g/ml killed all S. scabiei within 6 hours (Luo et al. 2015).Acacia nilotica ethanol extracts were found to have an LC50 value of 0.218 g/ml at 6 h against S. scabiei (Khan et al. 2022), and Laurus nobilis essential oil was observed to be effective against Dermanyssus gallinae (Alimi et al. 2021).Organic solvents such as methanol, alcohol, ethyl acetate, petroleum ether, and acetone have been used to solubilize the acaricidal component(s) (Luo et al. 2015).
The present study revealed that ethyl acetate extracts of U. fissa including acaricidal component(s) at a concentration of 100 mg/ml killed all S. scabiei within 2 h, with an LC50 value of 19.14 mg/ml at 2 hours.Compared with other plant extracts, the acaricidal activity of U. fissa thus has certain advantages.
Many studies have observed that schaftoside has a variety of pharmacological effects, including the prevention of cholesterol gallstone disease, inhibition of glycosidases, and regulation of autophagy, as well as antioxidant, anti-obesity, anti-melanogenic, and anti-human respiratory syncytial virus activity (De Melo et al. 2005;Zhou et al. 2019a;Yi et al. 2022).Apigenin C-glycosides and other flavonoid glycoside chemicals, such as apigenin C-glycosides, can alter the feeding behaviour of the brown planthopper, resulting in its demise.Hao et al. (2018) demonstrated that schaftoside is markedly toxic to brown planthoppers.
Carnosol is an active polyphenolic component of Rosmarinus officinalis L., Salvia fruticosa Mill, and Salvia officinalis L. (Yang et al. 2022) with anti-inflammatory, anti-oxidant, anti-microbial, and anticancer properties (Alsamri et al. 2021).1H-indole-3-carboxaldehyde is a key intermediate in the preparation of biologically active compounds and indole alkaloids (El-Sawy et al. 2017).The results of these studies show that the U. fissa extract contained a variety active ingredients.It is likely that the acaricidal activities of U. fissa may be attributed to the presence of terpenoids and flavonoids.Flavonoids and phenolics have been found to have multiple bioactivities (Alimi et al. 2021).However, the specific acaricidal components of the extract require further investigation.These preliminary results suggest that U. fissa may be a source of novel acaricidal substances capable of successfully controlling livestock mites.Additional systematic research is required to determine the active ingredients in U. fissa and assess them in clinical trials, acute toxicity tests on animals, and safety tests.
In conclusion, we assessed the effectiveness of U. fissa ethyl acetate extracts as an acaricide against S. scabiei and investigated the primary active components of the extract.The results have shown that the ethyl acetate extracts of U. fissa leaves have marked acaricidal activity in vitro and that the activity was both concentration-and timedependent.The chemical composition of the ethyl acetate extract was determined by LC-MS analysis.This showed that the extracts contained significant amounts of active components.
Thus, ethyl acetate extracts of U. fissa offer a more cost-effective, safe, and environmentally friendly alternative to commercial medications for the management of arthropods hazardous to both human and animal health.
These extracts can create innovative biocides to protect crops and livestock.However, the precise active ingredients, clinical efficacy, and safety evaluations of these extracts require further investigation.

Figure 1 .
Figure 1.Ion flow diagram extracted from LC-MS/MS samples Figure 2. Dead S. scabiei (A) Observed by microscopy (× 400) following treatment with ivermectin after 2 hours; (B) Observed by microscopy (× 400) following treatment with ethyl acetate extract of U. fissa leaves after 2 hours In each line, different lowercase letters indicate significant differences in the same treatment concentrations at different time intervals (P < 0.05).In each column, different capital letters indicate significant differences in the same time intervals at different treatment concentrations (P < 0.05) SD = standard deviaton centrations and times for the killing of S. scabiei.

Table 1 .
Chemical composition of ethyl acetate extract from U. fissa leaves

Table 4 .
Probit regression analysis of toxicity (LC50) of the extract against mites in vitro

Table 2 .
The acaricidal activity of the extract against S. scabiei in vitro