CN116508787B

CN116508787B - Urtica thunbergii leaf extract and preparation method and application thereof

Info

Publication number: CN116508787B
Application number: CN202310490329.XA
Authority: CN
Inventors: 廖飞; 何洁; 包涛涛; 陶光耀; 慕江静; 彭昭权
Original assignee: Guizhou Vocational College Of Agriculture
Current assignee: Guizhou Vocational College Of Agriculture
Priority date: 2023-05-04
Filing date: 2023-05-04
Publication date: 2025-02-18
Anticipated expiration: 2043-05-04
Also published as: CN116508787A

Abstract

The invention relates to the technical field of plant source mite killing, and discloses a nettle leaf extract, a preparation method and application thereof, wherein the nettle leaf extract is 94-96% ethanol extract, and the preparation method specifically comprises the following steps of sun-drying and crushing nettle leaf, extracting with 94-96% ethanol by ultrasonic, filtering, extracting with ethyl acetate, drying to obtain the nettle leaf extract, and using the nettle leaf extract in preparing mite killing medicines. The invention prepares the ethanol extract of the nettle leaf 94-96% and proves that the extract has acaricidal activity on scabies in vitro for the first time, has remarkable acaricidal effect, has the acarid killing rate of 100.0%, 76.7% and 60.0% respectively when the doses of 100, 50 and 25mg/mL are acted for 2 hours, has strong toxicity on acarids, has the activity influenced by concentration and time, is used for preparing acaricidal drugs, and provides a novel botanical acaricide.

Description

Urtica thunbergii leaf extract and preparation method and application thereof

Technical Field

The invention belongs to the technical field of botanical acaricides, and particularly relates to a nettle leaf extract and a preparation method and application thereof.

Background

Mange is a skin disease caused by scabies (Sarcoptesscabiei) resulting in scabies that destroy a wild species population and is one of the highly contagious and deadly ectoparasite infections of rabbits. Scabies 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, since there is no developmental stage of free life in its life cycle, nor intermediate host, and thus such infectious, host-specific, cavitating and astigmatic mites remain obligatory parasites throughout their life cycle. Lynar et al report that scabies secrete complement inhibitors into the cavity, which may promote secondary streptococcal and staphylococcal infections and subsequent development of invasive bacterial disease. At present, chemical medicines are widely applied to veterinary clinics for treating and controlling scabies, and good treatment effects including ivermectin, abamectin and the like are achieved. Effective control of scabies remains a significant challenge due to improper administration, with reports of drug resistance and treatment failure. Therefore, there is an urgent need to develop new generation of effective acaricides, and at the same time kill all stages of development of scabies (adults, nymphs, larvae and eggs), thereby improving the therapeutic effect and efficiency and reducing the recurrence rate of scabies. Plant-derived acaricides are favored for their high efficiency and environmental protection, including low non-target effects, many mechanisms of action, low cost and ease of use in countries where industrial facilities are limited.

The nettle has abundant plant resources in China, about 35 kinds of nettle are produced in China worldwide, and 23 kinds of nettle are mainly distributed in northern and southwest areas. Most nettle species can be used as medicine, and the folk application history is long. Has effects of dispelling pathogenic wind, dredging collaterals, promoting blood circulation, and relieving pain. Is mainly used for treating rheumatalgia, infantile convulsion, puerperal convulsion, etc. Nettle is the staple food for western traditional Chinese medicine pharmacists and is widely used as a vegetable green, fruit juice, tea and freeze-dried product, mainly for replenishing blood and treating seasonal rhinitis. Researches show that nettle contains chemical components such as flavonoid, lignan, coumarin, triterpene, steroid, alkaloid, polysaccharide, organic acid and the like. However, in the prior art, when preparing the leaf extract of nettle with split leaves, 70% ethanol is adopted, only 6 chemical components (reference Yang Wenjuan and the like, i.e. the external hypoglycemic active chemical components of nettle with split leaves [ J ]. The food industry technology, vol.41, no.08,2020.) are extracted, the extracted components are too few, and the utilization rate is too low.

Disclosure of Invention

The invention discloses a nettle leaf extract with split leaves, a preparation method and application thereof, and solves the problems that when the nettle leaf extract with split leaves is prepared, only 6 chemical components are extracted by adopting 75% ethanol, the extracted components are too few, and the utilization rate is too low.

An extract of nettle leaf, wherein the extract of nettle leaf is 94-96% ethanol extract;

The compounds in the nettle leaf extract comprise schaftoside, carnosol, 13 (S) -HpOTrE, 1H-Indole-3-carboxaldehyde, 9-OxoODE, linoleic acid, gamma-aminobutyric acid,

(13E) -11a-Hydroxy-9,15-dioxoprost-13-enoic acid, pelargonidin-O-beta-D-sambubioside, methoxyisobutylamine, bz-Arg-OEt, neocembrene, L-tryptophan, genistein, 9,12,13-TriHOME, cyanidin-3-O-rutinoside, phenyl ester pro-praline, indole, aspartic acid, anethole, acetoaspartyl glutamic acid, vitexin-2 "-O-rhamnoside, compact She Xin lignin, m-hydroxybenzoic acid, indoxyl acid, guanidyl succinic acid, alpha-octadecadienoic acid.

The second purpose of the invention is to protect the preparation method of the nettle leaf with split leaves, which comprises the following steps:

sun drying herba Urticae Cannabinae, pulverizing, ultrasonic extracting with 94-96% ethanol, filtering, extracting with ethyl acetate, and drying to obtain herba Urticae Cannabinae leaf extract.

Preferably, the nettle leaf is split with 95% ethanol=100g:2l.

Preferably, the ultrasonic condition is that ultrasonic extraction is carried out for 1h under the condition of 50 ℃ and 500W.

Preferably, the compound in the nettle leaf extract comprises schaftoside, carnosol, 13 (S) -HpOTrE, 1H-Indole-3-carboxaldehyde, 9-OxoODE, linoleic acid, gamma-aminobutyric acid, (13E) -11a-Hydroxy-9,15-dioxoprost-13-enoic acid, pelargonidin-O-beta-D-sambubioside, methoxyisobutylamine, bz-Arg-OEt, neocembrene, L-tryptophan, genistein, 9,12,13-TriHOME, cyanidin-3-O-rutinoside, phenyl ester propiolate, indole, aspartic acid, anethole, acetoaspartyl glutamate, vitexin-2 "-O-rhamnoside, compact She Xin lignin, m-hydroxybenzoic acid, indolecarboxylic acid, guanidyl succinic acid, alpha-octadecadienoic acid.

The third purpose of the invention is to protect the application of the nettle leaf extract in preparing acaricide.

Preferably, the nettle leaf extract has an acaricidal effect on scabies in vitro.

Preferably, the dosage of the nettle leaf extract is 100mg/mL, and the mite killing rate is 100.0% when the nettle leaf extract acts for 2 hours.

Compared with the prior art, the invention has the following advantages:

1. The invention prepares the ethanol extract of the nettle leaf 94-96% and proves that the extract has acaricidal activity on scabies in vitro for the first time, has remarkable acaricidal effect, has the acarid killing rate of 100.0%, 76.7% and 60.0% respectively when the doses of 100, 50 and 25mg/mL are acted for 2 hours, has strong toxicity on acarids, has the activity influenced by concentration and time, is used for preparing acaricidal drugs, and provides a novel botanical acaricide. LC 50 value of 19.14mg/mL was effective against scabies within 2 h.

2. The analysis of the invention determines the chemical components of the extracts of the 27 nettle leaves.

Drawings

FIG. 1 is a total ion flow diagram of LC-MS/MS of nettle leaf extract;

FIG. 2 shows the results of microscopic observations (x 400) of the killed mites after 2 hours of treatment with ivermectin;

fig. 3 shows the results of microscopic observation (x 400) of the killed mites after 2 hours of treatment with nettle leaf extract.

Detailed Description

The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The main reagent sources used in the invention are as follows:

Name of the name	CAS	Purity of	Branding
				Acetonitrile	75-05-8	≥99.9%	Thermo
Formic acid	64-18-6	LC-MSgrade	TCI
				Ammonium formate	540-69-2	≥99.9%	Sigma
H₂O	/	/	Millipore

1 Materials and methods

1.1 Preparation of plant Material and extracts

Fresh plant material of nettle schizophyllum is collected from Zunyi (N27 DEG 38', E107 DEG 46', altitude 754 m), 7 months in 2022, guizhou province, southwest China. The nettle is identified as nettle plant nettle of nettle genus of nettle family by the veterinary medical college of Sichuan university of agriculture.

Drying herba Urticae Cannabinae in knife grinder until the water content is below 15%, grinding into 50 mesh powder, soaking 0.2kg powder in 95% ethanol, soaking 100g powder in 2L 95% ethanol, and ultrasonic extracting at 50deg.C and 500W for 1 hr. The extract was concentrated by rotary evaporator. Then, three extractions were performed with equal amounts of ethyl acetate. The extract was concentrated using a rotary evaporator. Subsequently, the concentrated extract was dried under vacuum at 50 ℃ for 24 hours to ensure complete removal of residual ethyl acetate that may affect the experimental results.

1.2 LC-MS analysis of extracts

LC analysis was performed on ThermoVanquishU HPLC systems (Sieimer's Feishmanic sciences, USA). ChromatographyHSS T3 (150X 2.1mm,1.8 μm) (Waters, milford, mass., USA). The column was maintained at 40℃and the flow rate and injection were set at 0.25mL/min and 2. Mu.L, respectively, and the elution procedure is shown in tables 1 and 2.

TABLE 1LC-ESI (+) -MS elution procedure at analysis

TABLE 2 Mobile phase gradient parameters for LC-ESI (-) -MS analysis

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%

Mass spectrometric conditions Mass spectrometric detection of metabolites

Thermo Q Exactive (Thermo FISHER SCIENTIFIC, USA), ESI ion source. And simultaneously adopting MS1 and MS/MS (Full MS-ddms2 mode, data-dependent MS/MS) to collect. The parameters are sheath gas pressure, 30arb, auxiliary gas flow, 10arb, spray voltages of 3.50kV and-2.50 kV for ES I (+) and ES I (-), respectively, capillary temperature, 325 ℃, M S ranges, m/z 100-1000, MS1 resolution, 70000FWHM, scan number of each cycle dependent data, 10, MS/MS resolution, 17500FWHM, normalized collision energy, 30eV, dynamic exclusion time, and automatic.

1.3 Separation of Rabbit scabies

Scabies were isolated from scabies collected from the infected legs of naturally infected rabbits. The scab was placed in a petri dish and incubated in an incubator at 35 ℃ for 30min. The mites were then collected for detection, and the rabbits were treated immediately after the material was collected. Institutional ethics and guidelines for animal care were followed during sampling, and all procedures were performed in accordance with guidelines for laboratory animal care and use.

1.4 In vitro anti-mite Activity of the extracts

Ethanol extracts of nettle schizophyllum are diluted in 10% glycerol at concentrations of 25, 50, 100mg/mL, respectively. 0.1mL of the sample was added to a petri dish (diameter 5 cm, height 2 cm) with filter paper chip to absorb the liquid, and mites were placed in 10 samples per dish. Ivermectin (1%) was used as positive control and 10% glycerol was used as negative control. The extracts at each concentration were treated in 6 replicates, all dishes incubated at 25 ℃, 95% relative humidity, observed by stereomicroscope at 0.5, 1, 1.5, 2h, respectively, and periodically checked for mite viability with needle stimulation and the number of mites recorded as death if no reaction was present.

1.5 Statistical analysis

All calculations were performed using statistical software (SPSS, 20.0). And calculating the significance of the average mortality difference of mites between different concentrations by adopting a probability method. Median lethal time value (LT 50) and median lethal concentration value (LC 50) were calculated using a complementary log-log (CLL) model.

2 Results

2.1 Chemical composition of nettle leaf extract

275Mg of extract is extracted from 200g of nettle leaves with the extraction rate of 0.138 percent. The detection of small molecule metabolites of ethanol extract by LC-MS/MS, pre-treatment, which retains 886 peaks, is shown in figure 1, and mainly comprises small molecule organic acid, amino acid, sugar, lipid, sugar alcohol, phenolic compound, alkaloid, flavonoid, etc. The chemical components of the nettle leaves with split leaves are extracted by using 95% ethanol, and 27 chemical components are determined by analysis, and are shown in table 3. The prior art uses 70% ethanol to extract the chemical components of nettle leaves, and the analysis determines 6 chemical components (reference Yang Wenjuan, etc., the external hypoglycemic active chemical components of nettle leaves [ J ]. Food industry technology, vol.41, no.08,2020.).

TABLE 3 Main Compounds in 95% ethanol extract of nettle leaf

2.2 Acaricidal Activity of Urtica thunbergiana leaf extract

The in vitro acaricidal activity of the nettle leaf extract was evaluated. The results show that the mite-killing activity of the nettle leaf extracts with different concentrations is greatly different. The mortality rate of the high concentration group (100 mg/mL) and the positive control group within 2 hours is 100%, as shown in fig. 2 and 3, and compared with the control group, the effect of the nettle leaf extract on killing mites is remarkable (P < 0.05) under the condition of isolated culture, and the killing rates of 100, 50 and 25mg/mL doses on 2h mites are 100.0%, 76.7% and 60.0%, respectively, as shown in table 4. The lethal concentration and lethal time values of nettle leaf extract on mites are shown in tables 5 and 6. The nettle leaf extract has strong toxicity to mites, and the activity is influenced by concentration and time. LC 50 value of 19.14mg/mL was effective against scabies within 2 h.

TABLE 4 investigation of in vitro acaricidal Activity of Urtica dioica leaf extract on scabies

Note that different lower case letters per row represent significant differences (p < 0.05) in the same treatment concentration at different time intervals. In each column, different capital letters indicate significant differences in different treatment concentrations over the same time interval (p < 0.05)

TABLE 5 half-life of Urtica dioica leaf extract against scabies mites (LT 50)

Time (h)	Regression line	LT50(h)(95%FL)	Chi square value
				25mg/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

Note that FL = confidence interval

TABLE 6 half lethal concentration of Urtica dioica leaf extract on scabies mites (LC 50)

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

Note that FL = confidence interval

Discussion 3

The invention proves that the nettle leaf extract has mite killing activity on scabies in vitro for the first time. The toxicity of the extract proved to be time and concentration dependent. Research results show that the nettle leaf extract contains mite-killing components. The acaricidal composition is soluble in organic solvents such as methanol, ethanol, ethyl acetate, petroleum ether, and acetone. The LC-MS analysis is used for determining the chemical components of the nettle leaf extract, and the total phenol, flavonoid and alkaloid content in the nettle leaf extract is high. The main compounds in the nettle leaf extract are bergamot glycoside (8.259%), carnosol (6.736%), 13 (S) -HpOTrE (4.624%), 1H-Indole-3-carboxaldehyde (4.138%), 9-OxoODE (3.206%), linoleic acid (2.891%), etc. The schaftoside is a flavonoid compound existing in various Chinese herbal medicines, such as arisaema tuber, common goosegrass herb, lysimachia, liquorice, dendrobium, and the like. A large number of researches show that the schaftoside has wide pharmacological activities, including melanogenesis resistance, autophagy regulation, obesity resistance, oxidative stress resistance and glycosidase inhibition, and can prevent cholesterol lithiasis. Some flavonoid glycoside compounds, such as apigenin c-glycoside, can affect feeding behavior of brown planthoppers, resulting in death. Studies show that the schaftoside has remarkable inhibition effect on the survival of brown planthoppers. It has also been reported that carnosol is an active polyphenol compound, which can be isolated from rosemary, red sage and sage, and has anti-inflammatory, antioxidant, antibacterial and anticancer properties. 1H-Indole-3-carboxaldehyde is a key intermediate for preparing bioactive compounds and indole alkaloids. Therefore, the result shows that the nettle leaf extract contains abundant active ingredients. According to our findings, the acaricidal properties of nettle leaf extract may be due to the involvement of terpenoids and flavonoids. Flavone and phenolic compounds have been reported to express various biological activities.

Conclusion 4

In the invention, we evaluated the acaricidal effect of nettle leaf extract on scabies and analyzed the main active ingredients of the extract. The results show that the nettle leaf extract has obvious in-vitro acaricidal activity, and the activity is influenced by concentration and time. The LC-M S method is used for determining the chemical components of the nettle leaf extract. The result shows that the nettle leaf extract contains rich active ingredients. Therefore, the nettle leaf extract can replace commercial medicines in the aspect of controlling arthropods harmful to human and livestock health, and provides a cheaper, safer and more environment-friendly method. Such extracts are useful in the construction of novel biological acaricides for crop and livestock protection.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. A nettle leaf extract is characterized in that nettle leaf extract is obtained by sun-drying and crushing nettle leaf, ultrasonic extracting with 94-96% ethanol, filtering, extracting with ethyl acetate, and drying;

The compounds in the nettle leaf extract comprise schaftoside, carnosol, 13 (S) -HpOTrE, 1H-Indole-3-carboxaldehyde, 9-OxoODE, linoleic acid, gamma-aminobutyric acid, (13E) -11a-Hydroxy-9,15-dioxoprost-13-enoic acid, pelargonidin-O-beta-D-sambubioside, methoxyisobutylamine, bz-Arg-OEt, neocembrene, L-tryptophan, genistein, 9,12,13-TriHOME, cyanidin-3-O-rutin, phenyl ester propiolic acid, indole, aspartic acid, anethole, acetoaspartylglutamic acid, vitexin-2' -O-rhamnoside, compact She Xin lignin, m-hydroxybenzoic acid, indolecarboxylic acid, guanidyl succinic acid and alpha-octadecadienoic acid.

2. A method for preparing an extract of nettle leaf, as claimed in claim 1, comprising the steps of:

3. The method for preparing an extract of nettle leaf according to claim 2, wherein the nettle leaf comprises 95% ethanol = 100 g/2 l.

4. The method for preparing nettle leaf extract according to claim 2, wherein said ultrasonic extraction is carried out for 1 hour at 50 ℃ and 500W.

5. The use of the nettle leaf extract according to claim 1 for the preparation of a miticidal medicament, characterized in that the nettle leaf extract has a miticidal effect on scabies in vitro.

6. The use of the nettle leaf extract for preparing a miticidal agent according to claim 5, wherein the dose of the nettle leaf extract is 100mg/mL, and the mite killing rate is 100.0% when the nettle leaf extract is used for 2 hours.