CN116492404A

CN116492404A - Menghua musk extract and preparation method and application thereof

Info

Publication number: CN116492404A
Application number: CN202310787190.5A
Authority: CN
Inventors: 施宇茹; 张晓倩; 王趁; 王雨华; 裴盛基
Original assignee: Kunming Institute of Botany of CAS
Current assignee: Kunming Institute of Botany of CAS
Priority date: 2023-06-30
Filing date: 2023-06-30
Publication date: 2023-07-28
Anticipated expiration: 2043-06-30
Also published as: CN116492404B

Abstract

The invention relates to a Meng hair musk extract, a preparation method and application thereof, belonging to the technical field of natural extracts. The invention takes Menghua musk as a raw material, and extracts are obtained by extraction and extraction with an organic reagent. The Meng hair musk extract provided by the invention can achieve good anti-inflammatory and anti-itching effects by inhibiting the inflammation-related signal path, and the preparation method is simple and controllable. The invention is beneficial to fully utilizing the musk Meng Mao resource and provides theoretical basis for the development of related medicaments.

Description

Menghua musk extract and preparation method and application thereof

Technical Field

The invention belongs to the technical field of natural extracts, and particularly relates to a Meng hair musk extract and a preparation method and application thereof.

Background

Inflammation and itching are common skinSymptoms of the disease often affect each other. The provision of a medicament that can alleviate both itch and an inflammatory response is of great value for the treatment and alleviation of inflammation and itch related disorders. Menghuamaosheng (Menghuamaosheng)Adenosma buchneroidesBonati) is a plant of the genus Adenophora of the family ScrophulariaceaeAdenosma) In the Hani nationality of the Xishuangbanna area, the plant is often ground with fresh grass or hay to repel insects such as fleas and mosquitoes, or the fresh Menghai musk is smashed and the resulting juice is applied to the skin or the skin is scrubbed after the plant is boiled to relieve skin swelling and itching. Through further research on pharmacological actions of the Meng horseradish musk, particularly anti-inflammatory and antipruritic actions of the Meng horseradish musk, the method is helpful for exploring more potential values of the Meng horseradish musk, and provides theoretical basis for development of related medicaments.

Disclosure of Invention

The invention aims to provide a musk extractive of Menghuamao, a preparation method and application thereof, and simultaneously provides application of the prepared extractive in anti-inflammatory and anti-itching aspects.

In order to achieve the above purpose, the invention is realized by the following technical scheme:

the invention provides a preparation method of a musk extractive of Meng's hair, which comprises the following steps:

1) Pulverizing dried Meng Caoshan musk inflorescence into coarse powder;

2) Adding 95% methanol into the coarse powder, performing ultrasonic extraction for 10-120min, and filtering to obtain filtrate; repeating the operation for 3-5 times, and mixing the filtrates;

3) Concentrating the filtrate obtained in the step 2) into thick paste by rotary evaporation, and dissolving the thick paste with distilled water to obtain uniform suspension; adding petroleum ether, fully and uniformly mixing, standing in a separating funnel until obvious layering exists, and collecting a lower layer solution; repeating the operation for 3-6 times, and combining to obtain a solution I;

4) Adding ethyl acetate into the solution I, fully and uniformly mixing, and standing in a separating funnel until obvious layering exists, so that an upper layer solution can be collected; repeating the operation for 3-6 times; combining to obtain a solution II;

5) Concentrating the solution II by rotary evaporation to obtain thick paste, and obtaining the musk extract of Menghua.

Further preferably, in step 2), the coarse powder is prepared by weight/volume ratio: 95% methanol=1:5.

It is further preferred that in step 2), the ultrasound time is 30 minutes in order to achieve a better extraction in a short time.

Further preferably, in step 3), the suspension is homogenized in a volume ratio: petroleum ether=1:1.

Further preferably, in step 4), the solution I: ethyl acetate=1:1.

Further preferably, in step 3) or 4), the holding time of the separating funnel is 5 to 10 minutes.

The invention also provides an application of the Meng hair musk extract prepared by the method, which comprises the following steps:

the application of the Menghuamao musk extract in preparing anti-inflammatory drugs,

in particular to the application of the Meng's musk extract in preparing medicines for inhibiting 5-LOX enzyme activity and COX-2 enzyme activity;

in particular to application of a Menghua musk extract in preparing a medicament for inhibiting NO generation of RAW264.7 cells caused by LPS induction, wherein the safety concentration range of the extract on the RAW264.7 cells is 5-15 mu g/mL, and the extract has NO influence on cell viability;

in particular to the application of the musk Menghua extract in preparing medicines for inhibiting the production of IL-6, TNF-alpha and IL-10, the concentration of the extract is preferably 4-12 mug/mL, more preferably 12 mug/mL, the inhibition rates of IL-6, TNF-alpha and IL-10 production are respectively 70%, 59% and 35%, and the inhibition effects on IL-6 and TNF-alpha are more obvious than that of positive medicines dexamethasone.

In particular to application of the Menghua musk extract in preparing medicines for inhibiting TLR4, myD88, NF-kappa B P-P65/P65 and iNOS protein expression, wherein the concentration of the extract is preferably 12 mug/mL.

In particular to the application of the Meng's musk extract in preparing the medicine for inhibiting nuclear translocation of NF-kappa B p protein in cytoplasm, wherein the concentration of the extract is preferably 4-12 mug/mL.

In particular to the application of the Meng's musk extract in preparing medicines for inhibiting acute inflammation caused by carrageenan or croton oil.

The application of the Menghuamao musk extract in preparing anti-itch drugs,

in particular to the application of the Meng's musk extract in preparing a medicament for relieving acute itch caused by histamine or chloroquine.

The invention has the beneficial effects that:

the invention provides a preparation method and application of a Meng Caoshanense extract, belonging to the technical field of natural extracts. The invention takes Menghua musk as a raw material, and extracts are obtained by extraction and extraction with an organic reagent. According to the invention, through in vitro enzyme activity experiments, cell level experiments and in vivo experiments of mice, the musk extract of the Meng horsehair has good anti-inflammatory and anti-itching activities, can inhibit the TLR4/MyD88/NF- κB signal path, has a simple and controllable preparation method, and can be better applied to development of related products.

Drawings

FIG. 1 shows the inhibitory effect of Meng Caoshanense extract on 5-LOX enzyme activity;

FIG. 2 shows the inhibition of COX-2 enzyme activity by Meng Caosbeckia extract;

FIG. 3 is an effect of Meng Caoshanense extract on RAW264.7 cell viability, wherein A represents the effect of extracts SYM, ST and JC on cell viability in the concentration range of 25-100 μg/mL; b represents the effect of extract YSYZ on cell viability in a concentration range of 5-15 μg/mL (.P<0.05, ns stands for no significant difference);

FIG. 4 is a graph showing the effect of Meng Caosbeck extract on LPS-induced NO production by RAW264.7 cells, wherein A represents the effect of extract SYM on NO production; b represents the effect of extract ST on NO production; c represents the effect of extract JC on NO production; d shows the effect of extract YSYZ on NO production (compared to the blank, # # #) P<0.001; comparison with model groupP<0.05，*** P<0.001)；

FIG. 5 is a graph showing the effect of the musk Meng's extract SYM on LPS-induced RAW264.7 cell inflammatory factor production, wherein A represents the effect of SYM on IL-6 production; wherein B represents SYM vs TNF-αThe effect of the generation; wherein C represents the effect of SYM on IL-10 production (compared to the blank, # # #) P<0.001; comparison with model groupP<0.01，*** P<0.001, ns stands for no significant difference);

FIG. 6 is a graph showing the effect of ST of a Menghatti musk extract on LPS-induced RAW264.7 cell inflammatory factor production, wherein A represents the effect of ST on IL-6 production; wherein B represents ST against TNF-αThe effect of the generation; wherein C represents the effect of ST on IL-10 production (compared to the blank group, # # # #) P<0.001; comparison with model groupP<0.05，** P<0.01，*** P<0.001, ns stands for no significant difference);

FIG. 7 is a graph showing the effect of an extract of Meng Caosbeckia amara JC on LPS-induced production of RAW264.7 cell inflammatory factors, wherein A represents the effect of JC on IL-6 production; wherein B represents JC to TNF-αThe effect of the generation; wherein C represents the effect of JC on IL-10 production (compared to the blank group, # # #) P<0.001; comparison with model groupP<0.01，*** P<0.001, ns stands for no significant difference);

FIG. 8 is a graph showing the effect of YSYZ of Meng Caosbeck extract on LPS-induced production of RAW264.7 cell inflammatory factor, wherein A represents the effect of YSYZ on IL-6 production; wherein B represents YSYZ vs. TNF-αThe effect of the generation; wherein C represents the effect of YSYZ on IL-10 production (compared to the blank, # # #) P<0.001; comparison with model groupP<0.01，*** P<0.001, ns stands for no significant difference);

FIG. 9 shows the extract YSYZ of Meng Caosbeck versus TLR4, myD88, NF-κB P-P65/P65, wherein A represents the effect of immunoblotting on TLR4, myD88, NF-κB P-P65/P65, iNOS protein expression; wherein B represents the relative expression level of each histone relative to GAPDH (# compared to the blank group) P<0.001; comparison with model groupP<0.01，*** P<0.001, ns stands for no significant difference);

FIG. 10 shows the extract YSYZ of Meng Caosbeck versus NF-κB p65 effects of nuclear translocation;

FIG. 11 shows a musk extract YS of Meng CaoEffect of YZ on carrageenan-induced acute inflammation of murine foot swelling (P<0.05，** P<0.01, ns stands for no significant difference);

fig. 12 is the effect of YSYZ of the musk Meng's extract on acute inflammation of ear swelling in mice induced by croton oil (x)P<0.05, ns stands for no significant difference);

FIG. 13 is a graph showing the effect of YSYZ from Meng Caosbeck extract on histamine-induced acute pruritus scratching behavior in mice (compared to the blank group, # # # P<0.001; comparison with model groupP<0.05， ** P<0.01, ns stands for no significant difference);

FIG. 14 is a graph showing the effect of YSYZ from Meng Caosbeck extract on chloroquine-induced scratching behavior in mice with acute pruritus (compared to the blank group, # # # P<0.001; comparison with model groupP<0.05， ** P<0.01)。

Description of the embodiments

In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, preferred embodiments of the present invention will be described in detail below to facilitate understanding by the skilled person.

Example 1:

pulverizing dried Meng Caoshan musk inflorescence into coarse powder, adding 95% methanol into the coarse powder, performing ultrasonic extraction for 10-120min, repeating for 3-5 times, and filtering residues with filter paper to collect filtrate. Concentrating the filtrate into soft extract by rotary evaporator, dissolving with distilled water, adding petroleum ether at volume ratio of 1-3:1-3, mixing, standing in separating funnel for 2-20min until there is obvious delamination, and collecting upper layer solution. The operation was repeated 3 to 6 times. Concentrating the collected upper layer solution under reduced pressure with rotary evaporator to obtain Meng Cao Musk extract, which is named SYM.

Example 2:

crushing inflorescences of fresh Menghuamao musk plants into coarse powder, and adding distilled water 5-15 times to submerge the samples. Heating to boiling with heating jacket, adjusting temperature to 100deg.C, and heating for 1-3 hr. Filtering the boiled juice, and rotary evaporating under reduced pressure to obtain thick paste, to obtain Meng Caoshan Musk extract, denoted ST.

Example 3:

pulverizing dried Meng Caoshan musk inflorescence into coarse powder, adding 95% methanol into the coarse powder, performing ultrasonic extraction for 10-120min, repeating for 3-5 times, and filtering the residue with filter paper. Adding 65% methanol into the residue, and repeating for 3-5 times. Filtering the residue with filter paper, collecting filtrate, concentrating into soft extract by rotary evaporation to obtain Meng Caoshan Musk extract, and recording as JC.

Example 4:

1) Pulverizing dried Meng Caoshan musk inflorescence into coarse powder;

2) Adding 95% methanol into the coarse powder, performing ultrasonic extraction for 10-120min, filtering the residue with filter paper, and collecting filtrate; repeating the operation for 3-5 times, and mixing the filtrates;

3) Concentrating the filtrate obtained in the step 2) into thick paste by using a rotary evaporator, dissolving the thick paste by using distilled water to obtain uniform suspension, adding petroleum ether according to the volume ratio of 1-3:1-3, fully and uniformly mixing, standing in a separating funnel for 2-20min until obvious layering exists, and collecting a lower solution; repeating the operation for 3-6 times, and combining to obtain a solution I;

4) Adding ethyl acetate into the solution I according to the volume ratio of 1-3:1-3, fully and uniformly mixing, standing for 2-20min in a separating funnel until obvious layering exists, and collecting the upper layer solution; repeating the operation for 3-6 times; combining to obtain a solution II;

5) Concentrating the solution II under reduced pressure with rotary evaporator to obtain Menghua Moschus extract, which is denoted as YSYZ.

Leukotriene generated by the action of 5-LOX and arachidonic acid is an important inflammation regulating medium, promotes the progress of inflammation, and can block signal paths by inhibiting the activity of 5-LOX enzyme so as to achieve the anti-inflammatory effect. To examine the inhibition of 5-LOX enzyme activity by the musk extract of Menghua, a blank group, a control group, a sample group and a sample background well group were set, 4 wells per group, and 3 independent replicates were performed.

The reaction system is shown in Table 1, wherein the 5-LOX concentration is 700U/well, the concentration of the musk extract from Mentha arvensis is 250. Mu.g/mL, and the concentration of positive NDGA is 75. Mu.M.

Table 1 5-Experimental procedure for inhibition of LOX enzyme Activity

Inhibition ratio = 1- (OD) _{Sample well} -OD _{Sample background hole} ）/（OD _{Control wells} -OD _{Blank hole} ）×100%

As shown in Table 2 and FIG. 1, the obtained musk extract ST, JC and YSYZ at 250. Mu.g/mL showed remarkable inhibitory effect on 5-LOX enzyme activity, wherein the inhibition rate of YSYZ was 102.15%.

TABLE 2 inhibition of 5-LOX enzyme Activity by Musk Meng Mao extract

EXAMPLE 3 inhibition of COX-2 enzymatic Activity by Meng Caosbeck extract

COX-2 is a pro-inflammatory enzyme, and free arachidonic acid is subjected to COX-2 action to produce Prostaglandins (PGs), prostacyclin (PGI 2), thromboxane (TXs) and the like, and by up-regulating prostaglandin production, angiogenesis and metastasis are caused, resulting in tissue damage and inflammation.

To test the inhibition of the COX-2 enzyme activity by the musk extract of Menghua, a blank group, a control group, a positive inhibitor group and a sample group of musk extract of Menghua were set, 4 duplicate wells per group, and 3 independent replicates were performed. The reaction system is shown in Table 3, wherein the concentration of the positive inhibitor is 10. Mu.M, and the concentration of the extract is 50. Mu.g/mL.

TABLE 3 COX-2 enzyme Activity inhibition assay procedure

Inhibition ratio= (OD _{100% enzyme Activity} -OD _{Sample well} ）/（OD _{100% enzyme Activity} -OD _{Blank hole} ）×100%

As shown in Table 4 and FIG. 2, the 50. Mu.g/mL concentration of the musk extract SYM, ST, JC and YSYZ had a remarkable inhibitory effect on the COX-2 enzyme activity, wherein the inhibition rate of the YSYZ extract was 96.33%.

TABLE 4 inhibition of COX-2 enzyme Activity by Musk Meng extract

EXAMPLE 4 inhibition of NO production by Meng Cao musk extract on RAW264.7 cells

1. Safe concentration range of Menghuamao musk extract to RAW264.7 cells

RAW264.7 macrophages purchased from Yu Punuo race company. Taking out the cells frozen in liquid nitrogen, putting the cells into a water bath kettle preheated to 37 ℃ to be rapidly melted, resuscitating and culturing for three generations, and after the cells recover to a good growth state, using the cells for experimental study.

The experiment was performed in a blank group and a group of Menghua musk extracts, 4 duplicate wells per group, and 3 independent replicates were performed. RAW264.7 cells were seeded in 96-well plates at 100. Mu.L per well at a cell mass of 2X 10 ⁴ Individual wells at 37 ℃, 5% CO ₂ The cells were incubated in a constant temperature incubator for 24h.24 After h, liquid exchange is carried out, and 100 mu L of complete culture medium is added into each hole of a blank group; the extract+LPS solution prepared in examples 1-4 is added into each hole of the musk Meng/Helianthus tuberosus extract group, the final concentration of LPS in each hole is 1 mug/mL, the final concentration of the extract prepared in examples 1-3 is 25 mug/mL, 50 mug/mL and 100 mug/mL, the final concentration of the extract prepared in example 4 is 5 mug/mL, 10 mug/mL and 15 mug/mL, and the culture is carried out for 24 hours. 24 After h, the stock medium was removed, 100. Mu.L of fresh complete medium and 10. Mu.L of CCK-8 solution were added to each well, incubated for 1.5h in the dark and the OD at 450 nm was measured with an ELISA reader. The measured data were analyzed using Graphpad Prism 7.0.

Cell viability= (OD _{Treatment group} -OD _{Blank group} ）/（OD _{Control group} -OD _{Blank group} ）×100%

The experimental results are shown in FIG. 3, which shows the effect of the musk extract of Mentha on the viability of RAW264.7 cells. The extracts prepared in examples 1-3 were 25-100 μg/mL, and the extract prepared in example 4 was not affecting cell viability in the concentration range of 5-15 μg/mL.

2. Inhibition of LPS-induced RAW264.7 cell NO production by Menghuamao musk extract

The experiment was performed in 3 independent replicates by setting a blank control group, an inflammation model group, a positive control group and a Menghua musk extract group. RAW264.7 cells were seeded in 24-well plates at 500. Mu.L per well at a cell mass of 2X 10 ⁵ Individual wells at 37 ℃, 5% CO ₂ After culturing 24h in a constant temperature cell incubator, the liquid is changed. The positive control group was added with 500 μl of dexamethasone solution at a concentration of 10 μΜ per well; the extracts prepared in examples 1-4 are respectively added into each hole of the musk Meng-Mao extract group, the final concentration of the extracts prepared in example 1 is respectively 12.5, 25 and 50 mug/mL, the final concentration of the extracts prepared in examples 2 and 3 is respectively 25, 50 and 100 mug/mL, the final concentration of the extracts prepared in example 4 is respectively 4, 8 and 12 mug/mL, LPS solution is respectively added into each hole after incubation for 2 hours, the final concentration is 1 mug/mL, and the culture is carried out for 22 hours. 500 μl of serum-free medium was added per well of the blank control group; the inflammatory model group was incubated with 500. Mu.L of LPS solution at a concentration of 1. Mu.g/mL per well for 24h. After the culture time, the cell supernatant was collected and centrifuged at 1000 r/min for 3min.

The supernatant was assayed for NO content according to the Griess method, 3 wells per group. Equal volumes of N- (1-naphthyl) ethylene-19 amine (component A) and sulfanilic acid (component B) were mixed to form a Griess solution, depending on the amount required for the experiment. And respectively adding 75 mu M cell supernatants subjected to different treatments into the 96-well plate, adding 10 mu L of Griess reagent and 65 mu L of deionized water, incubating for 30min in a dark place, and detecting an OD value at 548nm by using an enzyme-labeled instrument. The measured data were analyzed using Graphpad Prism 7.0.

As shown in Table 5 and FIG. 4, SYM showed a NO inhibition of 74% at high dose (50. Mu.g/mL), ST extract showed a NO inhibition of 37% at high dose (100. Mu.g/mL), JC extract showed a NO inhibition of 55% at high dose (100. Mu.g/mL), YSYZ showed a NO inhibition of 67% at high dose (12. Mu.g/mL). The Meng's musk extracts prepared in examples 1-4 all significantly inhibited the NO content produced by LPS-induced RAW264.7 cell lines, and were concentration-dependent.

TABLE 5 inhibition of LPS-induced RAW264.7 cell NO production by musk extract of Meng Cao

EXAMPLE 5 ELISA method for detecting the content of inflammatory factors

The experiment was performed in 3 independent replicates by setting a blank control group, an inflammation model group, a positive control group and a Menghua musk extract group. RAW264.7 cells were seeded in 6-well plates at 2000. Mu.L per well at a cell mass of 5X 10 ⁵ Individual wells at 37 ℃, 5% CO ₂ After culturing 24h in a constant temperature cell incubator, the liquid is changed. 2000 μl of complete medium was added per well of the blank control group; 2000. Mu.L of LPS solution with the concentration of 1. Mu.g/mL was added to each well of the inflammation model group, and the mixture was cultured for 24 hours. Adding dexamethasone solution with final concentration of 10 mu M into each hole of the positive control group; the extracts prepared in examples 1-4 are respectively added into each hole of the musk extract group, the final concentration of the extracts prepared in example 1 is respectively 12.5 mu g/mL and the final concentration of the extracts prepared in examples 2 and 3 is respectively 25 mu g/mL and 100 mu g/mL, the final concentration of the extracts prepared in example 4 is respectively 4 mu g/mL and 12 mu g/mL, LPS solution is respectively added into each hole, the final concentration of the extracts is respectively 1 mu g/mL, and the extracts are cultured for 24 hours. After the culture time, cell supernatants were collected, centrifuged at 300 Xg for 10min, and the inflammatory factor content was measured by ELISA. The measured data were analyzed using Graphpad Prism 7.0.

The results of the detection are shown in Table 6 and FIGS. 5-8.

SYM treated cells at a concentration of 12.5. Mu.g/mL inhibited the production of IL-6, IL-10 and TNF- α at 22%, 12% and 27%, respectively. The SYM treated cells at the concentration of 50 mug/mL have inhibition effects on the production of IL-6, IL-10 and TNF-alpha, the inhibition rates are 52%, 36% and 44% respectively, and the inhibition effect on IL-6 is similar to that of positive medicament dexamethasone.

ST has inhibition effect on the production amount of IL-6, IL-10 and TNF-alpha at the concentration of 25 mug/mL, and the inhibition rates are 23%, 27% and 18%, respectively. However, ST had no significant inhibitory effect on the levels of IL-6, TNF- α and IL-10 at a concentration of 100. Mu.g/mL.

The JC treatment of cells at the concentration of 25 mug/mL has inhibition effect on the generation amount of IL-6, IL-10 and TNF-alpha, and the inhibition rates are respectively 50%, 18% and 38%, wherein the inhibition effect on IL-6 is strongest and is equivalent to that of positive control Dexamethasone (DEX); at a concentration of 100. Mu.g/mL, the inhibitor only has inhibition effect on IL-6 and TNF-alpha, and the inhibition rates are 31% and 23%, respectively.

The YSYZ treated cells at the concentration of 4 mug/mL has inhibition effect on the production amount of IL-6, IL-10 and TNF-alpha, and has no obvious difference with the treatment effect of positive medicament dexamethasone, and the inhibition rates are 57%, 31% and 46% respectively. The production of IL-6, IL-10 and TNF-alpha under the action of 12 mug/mL YSYZ-1 has inhibition effect, the inhibition rates are respectively 70%, 35% and 59%, and the inhibition effect on IL-6 and TNF-alpha is more obvious than that of positive medicament dexamethasone.

Table 6 inhibition ratio of Menghua musk extract to RAW264.7 cell inflammatory factor production

EXAMPLE 6 Western blot detection of expression of iNOS, TLR4, myD88, P-P65 and P65 proteins

The experiment was performed 3 times in a blank control group, an inflammation model group, a positive control group and a Menghua musk extract groupThe experiment was repeated independently. RAW264.7 cells were seeded in 6-well plates at 2000. Mu.L per well at 4X 10 cells ⁵ Individual wells at 37 ℃, 5% CO ₂ After culturing 24h in a constant temperature cell incubator, the liquid is changed. 2000 μl of complete medium was added per well of the blank control group; 2000. Mu.L of LPS solution with the concentration of 1. Mu.g/mL was added to each well of the inflammation model group, and the mixture was cultured for 24 hours. Adding dexamethasone solution with final concentration of 10 mu M into each hole of the positive control group; the extract prepared in example 4 was added to each well of the musk extract group at final concentrations of 4 and 12. Mu.g/mL, and LPS solution was added to each well to give final concentrations of 1. Mu.g/mL, and the mixture was cultured for 24 hours.

Cell culture supernatants from 6-well plates were discarded, cells were washed 2-3 times with PBS, and the remaining PBS was blotted off with a pipette and the cell plates were placed on ice. Adding cell lysate to cells, standing for 3-5 min, transferring cell lysate to a centrifuge tube, centrifuging at 12000 rpm at 4deg.C for 5min, and collecting protein supernatant.

Protein denaturation: the extracted protein supernatant was diluted with protein loading buffer at a 4:1 ratio and subjected to a boiling water bath for 10min, the sample was clear and non-viscous indicating that protein denaturation was complete, and after denaturation was complete, cooled to room temperature.

And (3) electrophoresis separation: an electrophoresis gel (prepared as shown in Table 7) was placed in the electrophoresis tank, and denatured protein and Marker were added thereto, with the total protein content of the sample being 40. Mu.g. After the sample is added, the concentrated glue is run at constant pressure of 80V, and then the separation glue is run at constant pressure of 120V.

TABLE 7 SDS-PAGE electrophoresis gel preparation system

Transferring: washing the target strip with distilled water, soaking PVDF film in methanol to form homogeneous semi-transparent film, soaking the film together with film transferring clamp, sponge and filter paper in film transferring buffer solution, and eliminating air bubbles with rollers. The transfer conditions are as follows: myD88 and GAPDH 200mA-90min; TLR4, iNOS 200mA-120min and then 300mA-30min; P-P65 and P65 mA-120min.

Immunoblotting color development: after completion of transfer, the membrane was shaken in a 1 XTBE solution for 5min with the front side facing upwards, and then transferred to a blocking solution (TBST of 5% skim milk powder) for 2h in a room temperature shaker. The corresponding primary antibody was diluted with blocking solution (Table 8) and the PVDF membrane was immersed in this incubation solution for 24h.

TABLE 8 dilution ratio of anti-antibody

As shown in FIG. 9, the effect of the Meng's musk extract YSYZ on TLR4, myD88, NF- κ B P-P65/P65, iNOS protein expression. After LPS stimulated RAW264.7 cells 24h, TLR4, myD88, NF-κThe expression level of B P-P65/P65 and iNOS proteins is obviously increased, and the 12 mu g/mL of the musk extract can obviously inhibit the expression level of all the proteins.

EXAMPLE 7 immunofluorescence assay of NF-κB/p65 Nuclear in Condition

The experiment was performed 3 times with a blank control group, an inflammation model group, a positive control group and a Menghua musk extract group. RAW264.7 cells were seeded into 6-well plates with slides placed therein at a cell count of 4X 10 at 2000. Mu.L per well ⁵ Individual wells at 37 ℃, 5% CO ₂ After culturing 24h in a constant temperature cell incubator, the liquid is changed. 2000 μl of complete medium was added per well of the blank control group; 2000. Mu.L of LPS solution with the concentration of 1. Mu.g/mL was added to each well of the inflammation model group, and the mixture was cultured for 24 hours. Adding dexamethasone solution with final concentration of 10 mu M into each hole of the positive control group; the extract prepared in example 4 was added to each well of the musk extract group at final concentrations of 4 and 12. Mu.g/mL, and LPS solution was added to each well to give final concentrations of 1. Mu.g/mL, and the mixture was cultured for 24 hours.

As shown in FIG. 10, the Meng Mao musk extract YSYZ was used for NF-κB p65 effects of nuclear translocation. LPS spikeNF in nucleus after excitationκThe red fluorescence intensity of B p65 protein is increased, which indicates NF-κB p65 protein undergoes a transfer from the cytoplasm to the nucleus. The Meng Cao musk extract obviously weakens NF-in cell nucleus at the concentration of 4 mu g/mL and 12 mu g/mLκB p65 protein fluorescence, which indicates that the Menghatti musk extract can inhibit NF-in cytoplasm after LPS inductionκNuclear translocation of B p65 protein.

Male Kunming mice (18-22 g) were purchased from Kunming medical university, yunnan province. The animal feed is fed according to standard animal feed conditions, and provides clean, comfortable and free living environment and sufficient and healthy food and drinking water. All animal experiment operations accord with animal welfare requirements, and animal ethics numbers: kib202303018. All mice were acclimatized for one week prior to the experiment.

The inhibition of the acute inflammation by the musk extract of Meng's hair was evaluated using the model of acute inflammation of the mouse foot swelling caused by carrageenan. Carrageenan was dissolved in physiological saline at a final concentration of 1% (w/v); the musk Meng's extract prepared in example 4 was dissolved in acetone solvent.

The experiment sets up an inflammation model group, a positive control group and a low, medium and high dose group of the Menghuak musk extract, each group comprising 6 mice. Pre-treatment was performed by priming the right rear foot of the positive control mice with 0.1mg dexamethasone cream 1h before inflammation induction; the right hind foot of the animals in the extract group was primed with 20 μl of the extract prepared in example 4 at concentrations of 4 μg/mL, 8 μg/mL and 12 μg/mL, respectively; and (3) coating 20 mu L of acetone on the right rear foot of the model group mice. After 1h, 50 μl carrageenan (1% w/v) was injected at the right hind foot sole of each mouse to cause inflammation.

The thickness of the right hind foot sole of each mouse was measured as a basal thickness before drug treatment, and 1h, 2h, 3h, 4h, 5h and 6h after carrageenan injection were measured as pathological thicknesses, respectively.

The rate of swelling of the feet of the mice was calculated according to the formula: swelling ratio (%) = [ (pathological thickness-basal thickness)/basal thickness ] ×100%.

As shown in fig. 11, the effect of the musk extract YSYZ on carrageenan-induced acute inflammation of swelling of the mouse foot. 4. The Musk extract with the concentration of mug/mL and 8 mug/mL can obviously inhibit the foot swelling degree of mice at the time of 2h after the induction of inflammation.

And evaluating whether the musk extract from the Meng's Musk has an inhibitory effect on acute inflammation by using an acute inflammation model of mouse ear swelling caused by the balm oil. The bara oil was dissolved in acetone at a final concentration of 2.5% (v/v); the musk Meng's extract prepared in example 4 was dissolved in acetone solvent.

The experiment sets up an inflammation model group, a positive control group and a low, medium and high dose group of the Menghuak musk extract, each group comprising 6 mice. Pre-treating the right ear of the mice in the positive control group by smearing 0.1mg dexamethasone cream 1h before inflammation induction; the right ear of the animals in the extract group was smeared with 20. Mu.L of the extract prepared in example 4 at concentrations of 4. Mu.g/mL, 8. Mu.g/mL and 12. Mu.g/mL, respectively, and the left ear was smeared with an acetone solution as a control. After 1h, the right ear of each mouse was smeared with a 2.5% solution of croton oil at 20 μl causing inflammation. After the ear swelling is induced for 6 hours, the cervical vertebra dislocation of the mice is killed, and round lugs at the same positions of left and right ears of the mice are removed by a 6 mm puncher and weighed.

The ear swelling rate of the mice was calculated according to the formula: swelling ratio (%) = [ (right ear weight-left ear weight)/left ear weight ] ×100%.

As shown in FIG. 12, the effect of the extract YSYZ of Meng Mao musk on acute inflammation of ear swelling in mice induced by croton oil is shown. The musk extract can obviously reduce the auricle swelling of the mice caused by the croton oil at the concentration of 4 mug/mL, and the weight difference between the right ear and the left ear of the mice is 2.620 +/-3.114 mg.

Male C57BL/6 mice (6-8 weeks) were purchased from Kunming medical university, yunnan province. The animal feed is fed according to standard animal feed conditions, and provides clean, comfortable and free living environment and sufficient and healthy food and drinking water. All animal experiment operations accord with animal welfare requirements, and animal ethics numbers: kib202303018. All mice were acclimatized for one week prior to the experiment.

A histamine dependent acute pruritus model was established using histamine as an inducer. Dissolving histamine in physiological saline with the concentration of 500 mug/50 mu L; the musk Meng's extract prepared in example 4 was dissolved in DMSO (4.8%) solvent.

Two days before molding, the hair on the back neck of the mouse was shaved by an electric shaver, and the area was about 2X 2cm. Mice were placed in a behavioural observation box for 30 minutes prior to the start of the experiment.

The experiment sets a blank control group, a pruritus model group and a low, medium and high dose group of the musk extract of the Menghatti, and each group comprises 6 mice. The rear part of the neck of the mice in the blank control group is smeared with DMSO (4.8%) solution, 50 mu L of physiological saline solution is injected at the administration position under the transient anesthesia of isoflurane after 1h, and then the scratching behavior of the mice within 1h is immediately recorded. The posterior cervical part of the mice of the pruritus model group was smeared with DMSO (4.8%) solution, and after 1h, 50 μl (500 μg) of histamine physiological saline solution was injected at the administration site under brief anesthesia of isoflurane, followed by immediately recording the scratching behavior of the mice within 1 h. Coating the musk extract of Mentha glabra prepared in example 4 on the shaved area of each mouse for 1h before itch induction, wherein the concentration is respectively 4 mug/mL, 8 mug/mL and 12 mug/mL, and recording the scratching behavior of the mice within 1h after coating; after 1h, 50 μl (500 μg) of histamine physiological saline solution was injected at the mice administration site under brief anesthesia with isoflurane, followed immediately by recording the scratching behavior of the mice over 1 h. After the recording is finished, counting the scratching behavior of the mice, and counting the scratching of the modeling skin area by the rear paw after the mice are lifted, or counting the scratching of the modeling skin area by the rear paw after the rear paw is scratched.

As shown in table 9, mice were scratched for 60min after application of DMSO (4.8%) solvent and the musk extract alone. The number of spontaneous scratching times of the mice coated with DMSO (4.8%) solution alone in 1h is 8.0+/-1.342 times, and after the mice are coated with the musk extract of the Meng hair (4 mug/mL, 8 mug/mL and 12 mug/mL) with different concentrations, the scratching times of the mice in 1h are respectively 4.0+/-1.673 times, 4.8+/-0.583 times and 7.0+/-0.707 times, compared with the mice coated with the DMSO (4.8%) solution alone, the scratching times are not significantly different, which indicates that the coating of the musk extract of the Meng hair does not cause itching of the skin of the mice.

As shown in table 10 and fig. 13, the number of scratches in the mice 1h of the pruritus model group was 111.4± 7.298 times, which is significantly higher than that of the blank group of 3±0.894 times. The number of scratches of mice at 1h after pretreatment with 4 μg/mL and 12 μg/mL of the Musk extract was significantly reduced to 56.2.+ -. 19.438 and 51.4.+ -. 9.288. The animal experiment results show that the Menghai musk extract can relieve acute itching induced by histamine.

TABLE 9 scratching times of mice over 60min after separate application of Meng Caoshan musk extract

TABLE 10 Effect of Meng Cao musk extracts on histamine-induced acute itching scratching behavior in mice

Example 11 use of chloroquine as an inducer to model acute itch that is not histamine dependent

And (3) utilizing chloroquine as an inducer to establish a histamine-independent acute pruritus model. Chloroquine is dissolved in normal saline, and the concentration is 200 mug/50 mu L; the musk Meng's extract prepared in example 4 was dissolved in DMSO (4.8%) solvent.

Two days before molding, the hair on the back neck of the mouse was shaved by an electric shaver, and the area was about 2X 2cm. Mice were placed in advance in the behavioural observation box for 30 minutes before the start of the experiment.

The experiment sets a blank control group, a pruritus model group and a low, medium and high dose group of the musk extract of the Menghatti, and each group comprises 6 mice. The rear part of the neck of the mice in the blank control group is smeared with DMSO (4.8%) solution, 50 mu L of physiological saline solution is injected at the administration position under the transient anesthesia of isoflurane after 1h, and then the scratching behavior of the mice within 1h is immediately recorded. The posterior part of the neck of the mice in the pruritus model group was smeared with DMSO (4.8%) solution, and after 1h, 50. Mu.L (200. Mu.g) chloroquine physiological saline solution was injected at the administration site under brief anesthesia of isoflurane, followed by immediately recording the scratching behavior of the mice within 1 h. Coating the musk extract of Mentha glabra prepared in example 4 on the shaved area of each mouse for 1h before itch induction, wherein the concentration is respectively 4 mug/mL, 8 mug/mL and 12 mug/mL, and recording the scratching behavior of the mice within 1h after coating; after 1h, 50 μl (200 μg) of chloroquine physiological saline solution was injected at the mice administration site under brief anesthesia with isoflurane, followed immediately by recording the scratching behavior of the mice over 1 h. After the recording is finished, counting the scratching behavior of the mice, and counting the scratching of the modeling skin area by the rear paw after the mice are lifted, or counting the scratching of the modeling skin area by the rear paw after the rear paw is scratched.

As shown in table 11 and fig. 14, the number of scratches in the mice 1h of the pruritus model group was 52.6± 7.380 times, which is significantly higher than that of the blank group by 3.0±0.894 times. The number of scratches of mice at 1h after pretreatment with 4. Mu.g/mL, 8. Mu.g/mL and 12. Mu.g/mL of the Meng/mL musk extract was significantly reduced to 14.4.+ -. 5.085, 21.+ -. 6.107 and 22.8.+ -. 9.831. The animal experiment results show that the Meng's musk extract can relieve acute itch induced by chloroquine.

TABLE 11 Effect of Meng Cao musk extract on chloroquine-induced acute itching scratching behavior in mice

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims

1. A preparation method of a Menghua musk extract is characterized by comprising the following steps: the method comprises the following steps:

1) Pulverizing dried Meng Caoshan musk inflorescence into coarse powder;

2. The method for preparing the musk extract from the Menghuamao as claimed in claim 1, wherein the method comprises the following steps: in the step 2), coarse powder is prepared according to the weight/volume ratio: 95% methanol=1:5; the ultrasonic time was 30min.

3. The method for preparing the musk extract from the Menghuamao as claimed in claim 1, wherein the method comprises the following steps: in the step 3), the suspension is homogenized according to the volume ratio: petroleum ether = 1:1, the standing time of the separating funnel is 5-10min.

4. The method for preparing the musk extract from the Menghatti as claimed in claim 2, wherein: in the step 4), according to the volume ratio, the solution I: ethyl acetate = 1:1, the standing time of the separating funnel is 5-10min.

5. Use of the musk extract of the horseradish tree prepared by the method according to any one of claims 1 to 4 for preparing anti-inflammatory drugs.

6. Use of a musk extract from a human being as defined in any one of claims 1 to 4 for the preparation of a medicament for inhibiting acute inflammation caused by carrageenan or croton oil.

7. The use according to claim 5, wherein: the Meng Cao musk extract can inhibit 5-LOX enzyme or COX-2 enzyme activity, inhibit RAW264.7 cell NO generation caused by LPS induction, inhibit IL-6, TNF-alpha and IL-10 generation, inhibit TLR4, myD88, NF-kappa B P-P65/P65 and iNOS protein expression, and achieve antiinflammatory effect.

8. The use according to claim 6, wherein: the Meng Cao musk extract can inhibit 5-LOX enzyme or COX-2 enzyme activity, inhibit RAW264.7 cell NO generation caused by LPS induction, inhibit IL-6, TNF-alpha and IL-10 generation, inhibit TLR4, myD88, NF-kappa B P-P65/P65 and iNOS protein expression, and achieve antiinflammatory effect.

9. Use of the musk extract of the horseradish tree as defined in any one of claims 1 to 4 for preparing anti-itch drugs.

10. Use of a musk extract of the group consisting of the musk and the musk, as prepared by the method according to any one of claims 1 to 4, for preparing a medicament for alleviating acute itch caused by histamine or chloroquine.