A kind of nettle leaf extract and its preparation method and application

Abstract

The invention relates to the technical field of plant-derived acaricide, and discloses a split-leaf nettle leaf extract and a preparation method and application thereof. The split-leaf nettle leaf extract is a 94-96% ethanol extract; the preparation method specifically comprises the following steps: drying and crushing the split-leaf nettle leaf, ultrasonically extracting with 94-96% ethanol, filtering, extracting with ethyl acetate, and drying to obtain the split-leaf nettle leaf extract; and using the extract in the preparation of acaricide drugs. The invention prepares a 94-96% ethanol extract of split-leaf nettle leaves, and proves for the first time that the extract has acaricide activity against scabies in vitro, and the killing effect is significant. The killing rates of mites at the dosages of 100, 50 and 25 mg / mL for 2 hours are 100.0%, 76.7% and 60.0% respectively, and the toxicity to mites is relatively strong, and the activity is affected by concentration and time, and the extract is used for the preparation of acaricide drugs, providing a novel plant-derived acaricide.

Description

Technical Field

[0001] The invention belongs to the technical field of plant-based acaricides, and particularly relates to a stinging urtica leaf extract, a preparation method and an application thereof. Background Art

[0002] Scabies is a skin disease caused by Sarcoptes scabiei, which leads to the formation of scabies, can devastate populations of wild species and is one of the highly contagious and fatal ectoparasitic infections of rabbits. Scabies mites can survive outside the host and remain infectious for a specific period of time. Their morphology and ecology are highly adapted to life in close contact with the host, as there are no free-living developmental stages and no intermediate hosts in their life cycle. Therefore, this infectious, host-specific, burrowing and scabiei mite remains an obligate parasite throughout its life cycle. Lynar et al. reported that scabies mites secrete complement inhibitors into the burrows, which may promote the formation of secondary streptococcal and staphylococcal infections and subsequent invasive bacterial diseases. At present, chemical drugs have been widely used in veterinary clinics to treat and control scabies, and have achieved good therapeutic effects, including ivermectin, avermectin, etc. Due to improper use of drugs, effective control of scabies remains a huge challenge, and there are reports of drug resistance and treatment failure. Therefore, there is an urgent need to develop a new generation of effective acaricides that can kill all developmental stages of scabies mites (adults, nymphs, larvae and eggs) simultaneously, thereby improving the efficacy and efficiency of treatment and reducing the recurrence rate of scabies. Botanical acaricides are favored due to their high efficiency and environmental friendliness, including low non-target effects, many mechanisms of action, low cost and ease of production in countries with limited industrial facilities.

[0003] There are abundant plant resources of split-leaf nettle in China. There are about 35 species in the world, 23 of which are produced in China, mainly distributed in the north and southwest. Most species of nettle can be used as medicine, and folk applications have a long history. It has the effects of dispelling wind, unblocking meridians, promoting blood circulation, and relieving pain. It is mainly used to treat rheumatism, infantile convulsions, postpartum convulsions, etc. Nettle is a staple food for Western Chinese medicine practitioners and is widely used as vegetable greens, juices, teas, and freeze-dried products. It is mainly used to replenish blood and treat seasonal rhinitis. Studies have shown that nettle contains chemical components such as flavonoids, lignans, coumarins, triterpenes, steroids, alkaloids, polysaccharides, and organic acids. However, the prior art uses 70% ethanol to extract only 6 chemical components when preparing split-leaf nettle leaf extract (reference Yang Wenjuan et al., Chemical components of split-leaf nettle in vitro hypoglycemic activity [J]. Food Industry Science and Technology, Vol. 41,. No. 08, 2020.), the extracted components are too few and the utilization rate is too low. Summary of the invention

[0004] The invention discloses a nematode urtica leaf extract and a preparation method and application thereof, which solves the problem that when preparing the nematode urtica leaf extract, 75% ethanol is used to extract only 6 chemical components, the extracted components are too few and the utilization rate is too low.

[0005] A nematode urtica leaf extract, wherein the nematode urtica leaf extract is a 94-96% ethanol extract;

[0006] The compounds in the nettle leaf extract include schaftoside, carnosol, 13(S)-HpOTrE, 1H-Indole-3-carboxaldehyde, 9-OxoODE, linoleic acid, γ-aminobutyric acid,

[0007] (13E)-11a-Hydroxy-9,15-dioxoprost-13-enoic acid, Pelargonidin3-O-beta-D-sambubioside, Methoxyisobutrazol, Bz-Arg-OEt, Neocembrene, L-tryptophan, Genistein, 9,12,13-TriHOME, Cyanidin-3-O-rutinoside, Phenylpropionic acid, Indole, Aspartic acid, Anethole, Acetylaspartylglutamate, Vitexin-2"-O-rhamnoside, Hydroxyprost-13-enoic acid, m-Hydroxybenzoic acid, Indolecarboxylic acid, Guanidinosuccinic acid, α-Octadecadienic acid.

[0008] The second object of the present invention is to protect the preparation method of the split-leaf urtica leaf, which specifically comprises the following steps:

[0009] The split-leaf urtica leaf is dried and crushed, ultrasonically extracted with 94-96% ethanol, filtered, extracted with ethyl acetate, and dried to obtain the split-leaf urtica leaf extract.

[0010] Preferably, stinging urtica leaves: 95% ethanol = 100 g: 2L.

[0011] Preferably, the ultrasonic conditions are ultrasonic extraction at 50° C. and 500 W for 1 h.

[0012] Preferably, the compounds in the nettle leaf extract include schaftoside, carnosol, 13(S)-HpOTrE, 1H-Indole-3-carboxaldehyde, 9-OxoODE, linoleic acid, γ-aminobutyric acid, (13E)-11a-Hydroxy-9,15-dioxoprost-13-enoic acid, Pelargonidin3-O-beta-D-sambubioside, methoxyisobutrazol, Bz-Arg-OEt, Neocembrene, L-tryptophan, genistein, 9,12,13-TriHOME, cyanidin-3-O-rutinoside, phenylpropionic acid, indole, aspartic acid, anethole, acetylaspartylglutamate, vitexin-2"-O-rhamnoside, octadecadienoic acid, m-hydroxybenzoic acid, indolecarboxylic acid, guanidinosuccinic acid, and α-octadecadienoic acid.

[0013] The third object of the present invention is to protect the use of the Urtica dioica leaf extract in the preparation of acaricide drugs.

[0014] Preferably, the Urtica dioica leaf extract has a miticidal effect on scabies mites in vitro.

[0015] Preferably, the dosage of the Urtica dioica leaf extract is 100 mg / mL, and the killing rate of mites is 100.0% after 2 hours of action.

[0016] Compared with the prior art, the present invention is beneficial in that:

[0017] 1. The present invention prepares a 94-96% ethanol extract of Urtica dioica leaves, and proves for the first time that the extract has acaricidal activity against scabies in vitro, and the killing effect is significant. The killing rates of mites at doses of 100, 50 and 25 mg / mL for 2 hours are 100.0%, 76.7% and 60.0%, respectively. The toxicity to mites is relatively strong, and the activity is affected by concentration and time. The extract is used to prepare acaricidal drugs, and a new botanical acaricide is provided. The LC 50 value is 19.14 mg / mL, which is effective against scabies within 2 hours.

[0018] 2. The present invention analyzes and determines the chemical components of 27 kinds of Urtica dioica leaf extracts. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the total ion chromatogram of LC-MS / MS of Urtica dioica leaf extract;

[0020] Figure 2 The results of microscopic observation (×400) of the killed mites after 2 hours of treatment with ivermectin;

[0021] Figure 3The results of microscopic observation (×400) after treatment with the extract of Urtica dioica leaves for 2 hours show the killing of mites. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] The main reagent sources used in the present invention are as follows:

[0024] name CAS purity brand Acetonitrile 75-05-8 ≥99.9％ Thermo Formic acid 64-18-6 LC-MSgrade TCI Ammonium formate 540-69-2 ≥99.9％ Sigma <![CDATA[H2O]]> / / Millipore

[0025] 1 Materials and methods

[0026] 1.1 Preparation of plant materials and extracts

[0027] Fresh plant materials of split-leaf urtica were collected from Zunyi (N 27°38′, E 107°46′, 754 m above sea level), Guizhou Province, southwestern China, in July 2022. Split-leaf urtica was identified as split-leaf urtica of the genus Urtica, Urticaceae, by the College of Veterinary Medicine, School of Medicine, Sichuan Agricultural University.

[0028] Dry the split-leaf nettle on a grinder until the water content is less than 15% by mass, grind it into 50 mesh powder, take 0.2kg of powder and soak it in 95% ethanol, soak it in 2L 95% ethanol for every 100g of powder, and ultrasonically extract it at 50℃ and 500W for 1h. The extract was combined and concentrated using a rotary evaporator. Then, it was extracted three times with an equal amount of ethyl acetate. The extract was concentrated using a rotary evaporator. Subsequently, the concentrated extract was vacuum dried at 50℃ for 24h to ensure that the residual ethyl acetate that may affect the experimental results was completely removed.

[0029] 1.2 LC-MS analysis of extracts

[0030] Liquid chromatography conditions: LC analysis was performed on a Thermo Vanquish U HPLC system (Thermo Fisher Scientific, USA). HSS T 3 (150×2.1 mm, 1.8 μm) (Waters, Milford, MA, USA) was used for the elution. The column was maintained at 40°C, the flow rate and injection volume were set to 0.25 mL / min and 2 μL, respectively, and the elution program was shown in Tables 1 and 2.

[0031] Table 1 Elution program for LC-ESI(+)-MS analysis

[0032]

[0033]

[0034] Table 2 Mobile phase gradient parameters for LC-ESI(-)-MS analysis

[0035] Time / min Acetonitrile (B3) Ammonium formate (A3) 0～1 2％ 98％ 1～9 2％～50％ 50～98％ 9～12 50％～98％ 50％～2％ 12～13.5 98％ 2％ 13.5～14 98％～2％ 2％～98％ 14～17 2％ 98％

[0036] Mass spectrometry conditions: mass spectrometry detection of metabolites

[0037] Thermo Q Exactive (Thermo Fisher Scientific, USA), ESI ion source. MS1 and MS / MS (Full MS-ddms2 mode, data-dependent MS / MS) acquisition were performed simultaneously. The parameters were as follows: sheath gas pressure, 30 arb; auxiliary gas flow, 10 arb; spray voltage, 3.50 kV and -2.50 kV for ESI (+) and ESI (-), respectively; capillary temperature, 325 °C; MS 1 range, m / z 100-1000; MS1 resolution, 70000 FWHM; number of data-dependent scans per cycle, 10; MS / MS resolution, 17500 FWHM; normalized collision energy, 30 eV; dynamic exclusion time, automatic.

[0038] 1.3 Isolation of rabbit scabies

[0039] Scabies mites were isolated from scabies collected from infected legs of naturally infected rabbits. Scabies were placed in a petri dish and incubated in an incubator at 35°C for 30 min. Adult mites were then collected for testing, and rabbits were treated immediately after material collection. Institutional ethics and animal care guidelines were followed during sampling, and all procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals.

[0040] 1.4 In vitro mite removal activity of extracts

[0041] The ethanol extract of Urtica dioica was diluted in 10% glycerol at concentrations of 25, 50, and 100 mg / mL. 0.1 mL of the sample was added to a culture dish (5 cm in diameter, 2 cm in height) with filter paper scraps to absorb the liquid, and mites were placed in each dish with 10 mites. Ivermectin (1%) was used as a positive control, and 10% glycerol was used as a negative control. Six replicates were treated with each concentration of the extract, and all culture dishes were incubated at 25°C and 95% relative humidity. They were observed under a stereo microscope at 0.5, 1, 1.5, and 2 hours, respectively. The viability of the mites was checked regularly with needle stimulation, and the number of mites was recorded. If there was no response, it was considered dead.

[0042] 1.5 Statistical analysis

[0043] All calculations were performed using statistical software (SPSS, 20.0). The probability method was used to calculate the significance of the difference in average mite mortality between different concentrations. The median lethal time value (LT 50) and median lethal concentration value (LC50) were calculated using the complementary log-log (CLL) model.

[0044] 2 Results

[0045] 2.1 Chemical composition of Urtica dioica leaf extract

[0046] 275 mg of extract was extracted from 200 g of Urtica dioica leaves, and the extraction rate of the extract was 0.138%. The small molecule metabolites of the ethanol extract were detected by LC-MS / MS, and 886 peaks were retained after pretreatment. Figure 1 , mainly including small molecule organic acids, amino acids, sugars, lipids, sugar alcohols, phenolic compounds, alkaloids, flavonoids, etc. The present invention uses 95% ethanol to extract the chemical components of the split-leaf urtica leaves, and analyzes and determines 27 chemical components, as shown in Table 3. The prior art uses 70% ethanol to extract the chemical components of the split-leaf urtica leaves, and analyzes and determines 6 chemical components (reference Yang Wenjuan et al., Chemical components of split-leaf urtica with hypoglycemic activity in vitro [J]. Food Industry Science and Technology, Vol. 41,. No. 08, 2020.).

[0047] Table 3 Major compounds in the 95% ethanol extract of Urtica dioica leaves

[0048]

[0049]

[0050] 2.2 Acaricidal activity of Urtica dioica leaf extract

[0051] The in vitro acaricidal activity of Urtica dioica leaf extract was evaluated. The results showed that there were significant differences in the acaricidal activity of Urtica dioica leaf extract at different concentrations. The mortality rate within 2 hours in the high concentration group (100 mg / mL) and the positive control group was 100%, as shown in Table 1. Figure 2 and Figure 3 Compared with the control group, under in vitro culture conditions, the extract of urtica dioica leaf had a significant killing effect on mites (P<0.05), and the killing rates of 100, 50 and 25 mg / mL for mites within 2 hours were 100.0%, 76.7% and 60.0%, respectively, as shown in Table 4. The lethal concentration and lethal time values ​​of the extract of urtica dioica leaf to mites are shown in Tables 5 and 6. The extract of urtica dioica leaf has a strong toxicity to mites, and its activity is affected by concentration and time. The LC 50 value is 19.14 mg / mL, which is effective for scabies mites within 2 hours.

[0052] Table 4 Study on the in vitro acaricidal activity of Urtica dioica leaf extract against scabies

[0053]

[0054]

[0055] Note: Different lowercase letters in each row indicate significant differences in the same treatment concentration at different time intervals (p<0.05). Different uppercase letters in each column indicate significant differences in different treatment concentrations at the same time interval (p<0.05).

[0056] Table 5 The median lethal time (LT50) of the extract of Urtica dioica leaves against scabies

[0057] Time (h) Regression line LT50(h)(95%FL) Chi-square value 25 mg / mL y=-1.412+0.828x 1.706(1.413～2.303) 4.772 50mg / mL y=-1.171+0.956x 1.226(0.942～1.498) 5.213 100mg / mL y=-1.116+1.489x 0.750(0.471～0.934) 8.954

[0058] Note: FL = confidence interval

[0059] Table 6 LC50 of Urtica dioica leaf extract against scabies

[0060] Time (h) Regression line LC50(mg / ml)(95%FL) Chi-square value 0.5h y=-2.074+0.018x 115.110(84.362～379.800) 3.345 1h y=-1.479+0.022x 66.892(45.117～100.326) 6.659 1.5h y=-0.465+0.013x 34.973(-19.643～55.368) 3.265 2h y=-0.553+0.029x 19.14(-16.887～31.573) 3.417

[0061] Note: FL = confidence interval

[0062] 3 Discussions

[0063] The present invention proves for the first time that the extract of Urtica dioica leaf has acaricidal activity against scabies in vitro. The toxicity of the extract is proved to be time- and concentration-dependent. The research results show that the extract of Urtica dioica leaf contains acaricidal components. The acaricidal components are soluble in organic solvents such as methanol, alcohol, ethyl acetate, petroleum ether and acetone. The chemical components of the extract of Urtica dioica leaf are determined by LC-MS analysis, and the total phenol, flavonoids and alkaloids in the extract of Urtica dioica leaf are high. The main compounds in the extract of Urtica dioica leaf are schaftoside (8.259%), carnosol (6.736%), 13(S)-HpOTrE (4.624%), 1H-Indole-3-carboxaldehyde (4.138%), 9-OxoODE (3.206%), linolenic acid (2.891%) and the like. Schaftoside is a flavonoid compound existing in many Chinese herbal medicines, such as Arisaema consanguineum, Herba Lysimachiae, Glycyrrhizae uralensis, Dendrobium officinale and the like. Numerous studies have shown that schaftosides have a wide range of pharmacological activities, including anti-melanogenesis activity, regulation of autophagy, anti-obesity activity, anti-oxidative stress, glycosidase inhibitory activity, and prevention of cholesterol gallstone disease. Some flavonoid glycoside compounds, such as apigenin c-glycoside, can affect the feeding behavior of brown planthoppers, leading to death. Studies have shown that schaftosides have a significant inhibitory effect on the survival of brown planthoppers. It has also been reported that carnosin, an active polyphenolic compound that can be isolated from rosemary, salvia miltiorrhiza, and sage, has anti-inflammatory, antioxidant, antibacterial, and anticancer properties. 1H-Indole-3-carboxaldehyde is a key intermediate for the preparation of bioactive compounds as well as indole alkaloids. Therefore, the results showed that the extract of Urtica dioica leaf contains abundant active ingredients. According to our results, the acaricidal properties of the extract of Urtica dioica leaf may be due to the participation of terpenoids and flavonoids. Flavonoids and phenolic compounds have been reported to express various biological activities.

[0064] 4 Conclusion

[0065] In the invention, we evaluated the acaricidal effect of the nematode leaf extract on scabies mites and analyzed the main active ingredients of the extract. The results showed that the nematode leaf extract had obvious in vitro acaricidal activity, and its activity was affected by concentration and time. The chemical composition of the nematode leaf extract was determined by LC-MS method. The results showed that the nematode leaf extract was rich in active ingredients. Therefore, the nematode leaf extract can replace commercial drugs in controlling arthropods that are harmful to human and animal health, providing a cheaper, safer and more environmentally friendly method. Such extracts can be used to construct new biological acaricides for crop and livestock protection.

[0066] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0067] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An extract of Urtica dioica leaves, characterized in that Dry and crush the leaves of Urtica dioica, extract them with 94-96% ethanol by ultrasonic wave, filter, extract them with ethyl acetate, and dry them to obtain the extract of Urtica dioica leaves; The compounds in the nettle leaf extract include schaftoside, carnosol, 13(S)-HpOTrE, 1H-Indole-3-carboxaldehyde, 9-OxoODE, linoleic acid, γ-aminobutyric acid, (13E)-11a-Hydroxy-9,15-dioxoprost-13-enoic acid, Pelargonidin3-O-beta-D-sambubioside, methoxyisobutyrazine, Bz-Arg-OEt, Neocembrene, L-tryptophan, genistein, 9,12,13-TriHOME, cyanidin-3-O-rutinoside, phenylpropionic acid, indole, aspartic acid, anethole, acetylaspartylglutamate, vitexin-2"-O-rhamnoside, octadecadienoic acid, m-hydroxybenzoic acid, indolecarboxylic acid, guanidinosuccinic acid, and α-octadecadienoic acid.

2. A method for preparing the Urtica dioica leaf extract according to claim 1, characterized in that: The specific steps include: The split-leaf urtica leaf is dried and crushed, ultrasonically extracted with 94-96% ethanol, filtered, extracted with ethyl acetate, and dried to obtain the split-leaf urtica leaf extract.

3. The method for preparing an extract of Urtica dioica leaves according to claim 2, characterized in that: Stinging nettle leaf: 95% ethanol = 100g: 2L.

4. The method for preparing an extract of Urtica dioica leaves according to claim 2, characterized in that: The ultrasonic extraction was performed at 50° C. and 500 W for 1 h.

5. Use of the extract of Urtica dioica leaves according to claim 1 in the preparation of acaricides, characterized in that: The extract of Urtica dioica leaves has acaricidal effect on scabies mites in vitro.

6. The use of an extract of Urtica dioica leaves in the preparation of acaricides according to claim 5, characterized in that: The dosage of the Urtica dioica leaf extract is 100 mg / mL, and the killing rate of mites is 100.0% after 2 hours of action.

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