CN113384592A

CN113384592A - Application of jasminum grandiflorum extract or effective components thereof in preparation of elastase inhibitor

Info

Publication number: CN113384592A
Application number: CN202110554643.0A
Authority: CN
Inventors: 李冬利; 李慧芳; 马航; 周致言; 彭兴家; 张奕孜; 谢寅正; 吴盼盼; 刘文锋; 徐学涛; 张焜
Original assignee: International Healthcare Innovation Institute (jiangmen); Wuyi University
Current assignee: Guangzhou Dayu Chuangfu Technology Co ltd
Priority date: 2021-05-20
Filing date: 2021-05-20
Publication date: 2021-09-14
Anticipated expiration: 2041-05-20
Also published as: CN113384592B

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an application of jasminum grandiflorum extract or an active ingredient thereof in preparation of an elastase inhibitor, wherein the jasminum grandiflorum extract is an ethyl acetate layer extract of jasminum grandiflorum; the effective components of the jasminum grandiflorum extract comprise oleuropein and hydroxytyrosol. The oleuropein, hydroxytyrosol or jasminum extract disclosed by the invention has the effect of inhibiting the activity of elastase, so that diseases such as skin aging and emphysema caused by over-expression of elastase are avoided, and when the oleuropein and the hydroxytyrosol with equal molar concentrations are mixed in equal volumes, the inhibition effect on elastase is stronger. Therefore, the oleuropein, hydroxytyrosol or jasminum grandiflorum extract provides a new choice for improving skin aging or treating emphysema chronic complications.

Description

Application of jasminum grandiflorum extract or effective components thereof in preparation of elastase inhibitor

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an application of jasminum grandiflorum extract or an active ingredient thereof in preparation of an elastase inhibitor.

Background

Elastase is an endopeptidase which can hydrolyze elastin and collagen, and the hydrolysis of elastin and collagen can cause skin to lose elasticity, relax and wrinkle, and other aging manifestations, although the process is a normal process under healthy skin aging conditions, environmental factors and oxidative stress of oxidation level (ROS) in internal cells such as fibroblasts in the dermis layer can over-express elastase, promote the hydrolysis of elastin and collagen and further exacerbate the loss of skin mechanical structure, and cause skin to lose elasticity, relax and wrinkle, reduce the number of fibroblasts in the dermis layer of skin, change the morphology of fibroblasts, and further accelerate skin aging; in addition, under normal physiological conditions, elastase can help phagocytes to eliminate harmful bacteria, promote wound healing and tissue regeneration, but its overexpression can cause inflammatory response of the body, promote cancer cell proliferation and metastasis, and induce various diseases including emphysema. Therefore, the development of elastase inhibitors, which can inhibit the activity of elastase, avoid the over-expression of elastase, effectively avoid the induction of inflammatory reaction of the body, slow down the hydrolysis of elastin and collagen, enhance the elasticity of skin and hair, reduce wrinkles and pigmentation, and have important significance for delaying skin aging and avoiding the induction of related inflammatory reaction.

The elastase inhibitor can inhibit the activity of elastase, thereby reducing the inflammatory reaction of organisms and delaying skin aging, the natural product-derived elastase inhibitor can effectively slow down the degradation speed of elastin, plays roles in delaying aging and reducing wrinkles, has the advantages of rich sources, greenness and safety compared with artificially synthesized inhibitors, and has wider development prospect.

Disclosure of Invention

The invention aims to develop an elastase inhibitor derived from a natural product, and particularly relates to an application of jasminum grandiflorum extract or an active ingredient thereof in preparation of the elastase inhibitor.

Based on the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides an application of jasminum grandiflorum extract or an active ingredient thereof in the preparation of an elastase inhibitor, wherein the jasminum grandiflorum extract is an ethyl acetate layer extract of jasminum grandiflorum; the effective components of the Jasminum grandiflorum extract include Oleuropein (OLE) and Hydroxytyrosol (HT).

The jasminum grandiflorum extract and OLE and HT obtained by further extracting and purifying the jasminum grandiflorum extract both have the effect of inhibiting the activity of elastase, and are concentration-dependent on the inhibition of the activity of elastase, and the inhibition effect of the jasminum grandiflorum extract or OLE and HT on elastase is gradually enhanced along with the increase of the concentration of the jasminum grandiflorum extract or OLE and HT, so that the jasminum grandiflorum extract, OLE and HT can be used for preparing elastase inhibitors. In addition, the jasminum grandiflorum extract, OLE and HT used for preparing the elastase inhibitor have the advantages of rich sources, greenness and safety, and have a relatively high application prospect.

Further, the molar ratio of oleuropein to hydroxytyrosol used for preparing the elastase inhibitor is 1:1, and the volume ratio is 1: 1.

Both OLE and HT of the present invention have anti-elastase effect, wherein OLE induces reversible inhibition of enzyme activity by competing with elastase substrate for binding to the enzyme active center. HT causes inhibition of a decrease in enzyme activity by binding to the enzyme-substrate complex, but not to the free enzyme. The test results show that when the OLE and HT are mixed in a volume ratio of 1:1, the compound has a better synergistic inhibition effect on the activity of the elastase.

Further, the preparation method of the jasminum grandiflorum extract comprises the following steps:

drying and crushing the jasminum grandiflorum, extracting and concentrating the dried and crushed jasminum grandiflorum in 75-95% ethanol water solution to obtain an ethanol extract, adding water into the ethanol extract for redissolution, extracting by ethyl acetate, and concentrating to obtain an ethyl acetate layer extract of the jasminum grandiflorum.

Further, the preparation method of the active ingredient OLE in the jasminum grandiflorum extract comprises the following steps:

taking dichloromethane and methanol mixed solution as eluent, carrying out gradient elution on the ethyl acetate layer extract of the jasminum grandiflorum, collecting the eluent, eluting the eluent by chloroform and methanol mixed solution, and carrying out reverse phase column chromatography on methanol aqueous solution to collect the eluent, thus obtaining the active ingredient OLE of the jasminum grandiflorum extract.

Further, in the process of gradient elution by using a mixed solution of dichloromethane and methanol, the volume ratio of dichloromethane to methanol is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 and 1:1 in sequence, and fractions eluted by the mixed solution of dichloromethane and methanol with the volume ratio of 6:1 are collected for next step elution; the volume ratio of chloroform to methanol in the chloroform-methanol mixed solution is 1: 1; the volume fraction of methanol in the aqueous methanol solution was 40%.

Further, the preparation method of the active ingredient HT in the jasminum grandiflorum extract comprises the following steps:

gradient elution is carried out on the ethyl acetate layer extract of the jasminum grandiflorum by taking a mixed solution of petroleum ether and ethyl acetate as an eluent, the eluent is collected, and the collected eluent is eluted by a mixed solution of chloroform and methanol to obtain the active ingredient HT of the jasminum grandiflorum extract.

Further, in the process of gradient elution by using a mixed solution of petroleum ether and ethyl acetate, the volume ratio of the petroleum ether to the ethyl acetate is 50:1, 40:1, 30:1, 20:1, 10:1, 1:1 and 0:1 in sequence, and fractions eluted by the mixed solution of the petroleum ether and the ethyl acetate in the volume ratio of 1:1 are collected for next step elution; the volume ratio of chloroform to methanol in the chloroform-methanol mixture is 1: 1.

In a second aspect, the present invention provides an elastase inhibitor comprising an extract of jasminum grandiflorum or an active ingredient thereof; the effective components of the extract of Jasminum grandiflorum comprise OLE and HT.

Further, the molar ratio of OLE to HT was 1:1 and the volume ratio was 1: 1.

In a third aspect, the invention provides the use of an elastase inhibitor in the preparation of an anti-aging cosmetic.

The elastase inhibitor comprises jasminum grandiflorum extract or active ingredients OLE and HT thereof, and experiments prove that the jasminum grandiflorum extract and the active ingredients thereof can obviously inhibit the activity of elastase and can reduce the content of H₂O₂The invention is directed to the oxidative induction of ROS levels in fibroblasts in the dermis of the skin, reducing the oxidative stress of environmental factors and oxidative levels (ROS) in internal cells, such as fibroblasts in the dermis, and preventing the overexpression of elastase, thereby delaying skin agingThe elastase inhibitor can be used for preparing antiaging cosmetic.

Further, the cosmetic comprises a toner, essence, cream, mask or body lotion.

In a fourth aspect, the invention provides the use of an elastase inhibitor for the manufacture of a medicament for the prevention or treatment of emphysema.

The elastase inhibitor can reduce the free radical of H₂O₂Oxidation-induced ROS levels in fibroblasts of the dermis layer of the skin, reducing oxidative stress of environmental factors and oxidative levels (ROS) in interior cells, such as fibroblasts in the dermis layer, inhibiting elastase overexpression, thereby avoiding emphysema induced by elastase overexpression.

Compared with the prior art, the invention has the following beneficial effects:

the oleuropein, hydroxytyrosol or jasminum jasminoides extract disclosed by the invention has the effect of inhibiting the activity of elastase, so that diseases such as skin aging and emphysema caused by over-expression of elastase are avoided, and when the molar concentration ratio of the oleuropein to the hydroxytyrosol is 1:1, the inhibition effect on the elastase is stronger. Therefore, the oleuropein, hydroxytyrosol or jasminum grandiflorum extract provides a new choice for improving skin aging or treating emphysema chronic complications.

Drawings

FIG. 1 is a graph of the inhibitory effect of jasminum grandiflorum Extract (Extract), OLE, HT and OLE and HT on elastase at different concentrations and ratios;

FIG. 2 is a graph showing the synergistic inhibitory effect of OLE and HT on elastase;

FIG. 3 is a double reciprocal plot of OLE and HT;

FIG. 4 is a schematic diagram of the binding sites of OLE, HT and elastase;

FIG. 5 shows OLE, HT reduction H₂O₂Graphs of ROS levels in induced human dermal fibroblasts.

FIG. 6 is a graph showing the synergistic reduction of H by OLE and HT₂O₂Graphs of ROS levels in induced human dermal fibroblasts.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. It will be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.

Example 1 extraction and purification of Jasminum lanceolatum extract and its effective ingredients

Sun drying and oven drying radix Jasmini Lanceolatae, pulverizing into powder, extracting 8kg of radix Jasmini Lanceolatae powder with 10L of 80% ethanol water solution at normal temperature and pressure for three times (each for 3 days) to obtain ethanol extractive solution, and concentrating under reduced pressure to obtain ethanol extract; adding 2L of distilled water into the ethanol extract to dissolve the ethanol extract, sequentially extracting with 1L of petroleum ether for three times (1L × 3), sequentially extracting the residue with 1L of ethyl acetate for three times (1L × 3) and 1L of n-butanol for three times (1L × 3), mixing the ethyl acetate extracts, and concentrating to obtain the ethyl acetate layer extract of jasminum grandiflorum.

The effective components of the ethyl acetate layer extract of the jasminum grandiflorum comprise OLE and HT, and the HT is prepared by separating and purifying the ethyl acetate layer extract of the jasminum grandiflorum as follows:

performing medium-pressure preparative chromatography on the ethyl acetate layer extract of jasminum grandiflorum, performing gradient elution on the ethyl acetate layer extract of jasminum grandiflorum by taking a mixed solution of petroleum ether and ethyl acetate as an eluent, collecting fractions eluted from the mixed solution of petroleum ether and ethyl acetate in a volume ratio of 1:1, 40:1, 30:1, 20:1, 10:1, 1:1 and 0:1 in sequence in the elution process, performing LH-20 gel column chromatography on the collected fractions, eluting by taking chloroform-methane in a volume ratio of 1:1 as the eluent, collecting the eluent and concentrating to obtain HT.

The method for preparing OLE by separating and purifying ethyl acetate layer extract of jasminum grandiflorum comprises the following steps:

separating ethyl acetate layer extract of jasminum grandiflorum by medium pressure preparative chromatography, performing gradient elution on the ethyl acetate layer extract of jasminum grandiflorum respectively by using dichloromethane and methanol according to the volume ratio of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 and 1:1, collecting the fraction eluted from a mixed solution of dichloromethane and methanol with the volume ratio of 6:1, performing LH-20 gel column chromatography, eluting by using chloroform-methanol with the volume ratio of 1:1 as a mobile phase, performing reverse phase silica gel column chromatography, eluting by using 40% methanol aqueous solution as the mobile phase, collecting the fraction eluted from the fraction, and concentrating to obtain the compound OLEE.

Example 2 Jasminum leptostachyum extract and its effective component inhibition effect test on elastase

In this example, the inhibition effect of OLE and HT on elastase is examined, and the specific test method is as follows:

1. preparation of test solution:

the ethyl acetate layer extract of jasminum grandiflorum prepared in example 1 was prepared into a stock solution with a concentration of 100mg/mL using Dimethyl sulfoxide (DMSO), and sequentially diluted to final concentrations of 1000. mu.g/mL, 500. mu.g/mL, 250. mu.g/mL, 125. mu.g/mL, 62.5. mu.g/mL.

HT obtained in example 1 was prepared in stock solution at a concentration of 100mM in DMSO, and diluted sequentially to final concentrations of 1000. mu.M, 500. mu.M, 250. mu.M, 125. mu.M, and 62.5. mu.M. .

OLE from example 1 was prepared in DMSO as a stock solution at a concentration of 100mM, and diluted sequentially to final concentrations of 1000. mu.M, 500. mu.M, 250. mu.M, 125. mu.M, 62.5. mu.M. .

2. The test method is as follows:

mu.L of 20mmol/L Tris-HCl buffer solution (pH 8.0) was used as a test system, 20. mu.L elastase solution dissolved in Tris-HCl buffer solution was added to the test system, 10. mu.L of test solutions with different concentrations were added to the test system, and 50. mu.L of substrate N-Succinyl-Ala-Ala-Ala-p-nitroanilide (N-Succinyl-Ala-Ala-Ala-p-nitroanilide available from Sigma-Aldrich) solution was added to the test system, and the mixture was mixed and measured for absorbance at 410nm using a microplate reader. In addition, 10. mu.L of Tris-HCl buffer solution was added as a blank instead of the test solution as a control.

3. Test results

The inhibition of elastase by different test samples is shown in fig. 1, where fig. 1A is the effect of different concentrations of jasminum grandiflorum extract on elastase activity. The activity of elastase treated in the blank control group was set to 100%, and there was no inhibition of elastase activity. As can be seen from fig. 1A, the elastase activity of the blank control group is highest, and the elastase activities after adding the jasminum grandiflorum extracts with different concentrations are all lower than that of the blank control group, which indicates that the jasminum grandiflorum extracts have an inhibitory effect on elastase; the activity of the elastase is lower along with the increase of the concentration of the jasminum grandiflorum extract, which shows that the concentration of the jasminum grandiflorum extract on the inhibition of the elastase is dependent, and the higher the concentration of the jasminum grandiflorum extract is, the stronger the inhibition of the elastase is.

FIG. 1C is a graph showing the inhibitory effect of OLE at various concentrations on elastase; as can be seen from fig. 1C, the elastase activities after treatment with different concentrations of OLE were significantly lower than the control group, indicating that OLE has an inhibitory effect on the elastase activities; furthermore, the lower the elastase activity with increasing OLE concentration indicates that OLE has concentration dependency on the elastase inhibitory effect, and the higher the concentration, the stronger the elastase inhibitory effect.

FIG. 1D is a graph showing the inhibitory effect of different concentrations of HT on elastase; as can be seen from the figure, the elastase activities after different concentrations of HT treatment were all lower than the control group, indicating that HT has inhibitory effect on elastase activity; furthermore, the lower the elastase activity with increasing concentrations of HT, the more concentration-dependent the inhibitory effect of HT on elastase, and the higher the concentration, the stronger the inhibitory effect on elastase.

Next, the inhibitory effect of OLE and HT on elastase was tested at the same concentration (250 μ M) in different volume ratios, and the results are shown in fig. 1B, where it can be seen that when the volume ratio of OLE and HT is 1:1, the elastase inhibitory activity is stronger.

EXAMPLE 3 synergistic inhibitory Effect of Oleuropein and Hydroxytyrosol on Elastase

In this example, to investigate whether Oleuropein (OLE) and Hydroxytyrosol (HT) have synergistic inhibitory effect on elastase, the specific test method is as follows:

1. elastase activity inhibition assay

(1) Sample preparation:

sequentially diluting samples with the concentration of 100mM to enable the final concentrations of OLE and HT to be 62.5 muM, 125 muM, 250 muM, 500 muM and 1000 muM to serve as samples to be detected; meanwhile, the mixture of OLE and HT (OLE + HT) with equal volume and equal molarity is used as the sample to be tested.

(2) Test method and test result

mu.L of each sample to be tested was added to a 96-well plate, and then 120. mu.L of Tris-HCl buffer (2 mM, pH 8) was added to the 96-well plate, and 20. mu.L of an elastase solution having a concentration of 0.5U/mL was further added to the 96-well plate.

The 96-well plate is placed in a constant temperature incubator at 37 ℃ for incubation for 15min, then 50 mu L of substrate (N-Succinyl-Ala-Ala-Ala-p-nitroanilide) with the concentration of 1mg/mL is added, the absorbance of the sample at 410nm is measured by using an enzyme label reader, and the inhibition rate of the sample to be tested on the elastase activity is calculated. The elastase activity inhibition rate was calculated by the following formula:

the elastase activity inhibition ratio (%) is [ (. DELTA.A-. DELTA.B)/. DELTA.A ]. times.100%,. DELTA.A represents the absorbance value of the blank control group containing no sample to be tested, and. DELTA.B represents the absorbance value of the test solution at different concentrations.

The test results are shown in fig. 2A, and it can be seen that, with the increase of the concentration of the sample to be tested, the inhibition rates of OLE, HT, OLE + HT on the elastase activities all show an increasing trend, and under the condition of equal concentration, the inhibition rate of OLE + HT on the elastase activities is higher than the inhibition rate of OLE and HT on elastase alone, which indicates that OLE and HT may have a synergistic inhibition effect on the elastase activities.

To further determine the synergistic inhibitory effect of the same amount of OLE and HT on elastase, different concentration gradients of HT + OLE (equal molar concentration mixed solution) were used as test solutions, the inhibition rate of the test solutions on elastase was determined by the above method, and the synergy index (CI) of the test solutions was measured by CompuSyn software to evaluate the synergy of the test solutions, wherein CI <1 is defined as synergistic effect, CI ═ 1 is defined as additive effect, and CI >1 is defined as antagonistic effect.

By testing the inhibition effect of the combination of OLE and HT on elastase, the results are shown in FIG. 2B, when OLE and HT are mixed in equal molar concentrations and equal volumes, the CI values after different concentrations of OLE + HT treatment are less than 1, which indicates that OLE and HT have synergistic inhibition effect on elastase when being mixed in equal molar concentrations and equal volumes, and the results provide theoretical basis for the combination of OLE and HT.

2. Analysis of Elastase inhibition types by OLE and HT

Testing the inhibition type of OLE and HT on elastase by enzymatic reaction kinetics, and setting the concentration gradient of OLE and HT at 0 μ M, 250 μ M, 500 μ M and 1000 μ M; the substrate concentrations were 4mg/mL, 2mg/mL, 1.32mg/mL, 1mg/mL, 0.8mg/mL, and 0.4mg/mL in this order, and the absorbance at 410nm was measured in the kinetic mode according to the above method, and the inhibitors were subjected to kinetic analysis using a Lineweaver-Burk plot to determine the inhibition pattern of OLE and HT on elastase.

The Lineweaver-Burk plot is shown in fig. 3, where the lines of OLE at different concentrations intersect at the Y-axis (fig. 3A), and as the OLE concentration increases, the Michaelis-Menton constant (Km) of OLE increases while its maximum reaction rate (Vmax) remains unchanged (fig. 3C), indicating that OLE is a competitive inhibitor of elastase activity. While the lines for different concentrations of HT were parallel (fig. 3B) and as the concentration of HT increased, its Km and Vmax decreased (fig. 3C), indicating that HT inhibited elastase activity in an anti-competitive manner.

3. Molecular docking test

Molecular docking was then used to predict the binding force of OLE and HT to elastase, where the chemical structures of OLE and HT and elastase are available from the national center for biotechnology information. Molecular docking analysis was performed using Autodock 4.2 under saturation conditions of the computational model provided by the university of rhode island (RI-INBRE). The crystal structure of elastase protein (PDB ID: 1LVY) was retrieved in PDB format from the RCSB protein database (www.rcsb.org), and partial charges were assigned using AutoDock Tools with water molecules removed and hydrogen atoms added. The rigid structure of the elastase protein was used as a receptor for docking calculations and Discovery Studio software was used to obtain a reasonable active binding site. Docking simulation is carried out by using a Lamarkian genetic algorithm, and an optimized protein structure, construction of side chains and rings, a binding site and a binding fraction of a ligand are obtained on the basis of free band energy and hydrogen bonds of the Lamarkian genetic algorithm.

By computational molecular docking studies to predict the interaction between elastase and its inhibitors, as shown in figure 4, OLE and HT were able to bind to elastase at different binding sites with free binding energies of-7.21 kcal/mol and-4.72 kcal/mol, respectively. The OLE binding sites located at the catalytic site of the elastase proteins include the amino acid residues Val85, Asp60, Leu63, Val90, Glu62, which contribute to the formation of 4 hydrogen bonds and one carbon-hydrogen bond to stabilize the enzyme-ligand complex. HT interacts with elastase proteins and forms 4 hydrogen bonds and one pi-cationic bond with amino acid residues Leu73, Glu70, Gln34, Gln38 and Arg65A (fig. 4). This result theoretically supports that OLE and HT inhibit elastase activity.

Example 4 Oleuropein and Hydroxytyrosol synergistically reduce H₂O₂Induced ROS level assay in fibroblasts

Since the most important cellular component in the dermis of the skin is fibroblasts, the reduction in the number, the change in the form, the decrease or the decline of the secretory synthesis function of which is closely related to skin aging, Reactive Oxygen Species (ROS) play an important role in natural aging and photoaging, and induce skin aging by the damaging effect on the cellular component. Endogenous aging and exogenous aging of skin cells have a common molecular mechanism, namely, the skin cells are subjected to oxidative stress caused by ROS, and a series of subsequent effects occur, so that the aging of the skin cells and even the skin is influenced. Therefore, this example is to explore the effect of OLE, HT on ROS levels in human dermal fibroblasts, and the specific test method is as follows:

the human dermal fibroblasts were seeded in a 96-well plate at a density of about 5000 to 6000 cells per well, allowed to attach for 12 hours, and then treated with assay samples OLE, HT, OLE + HT at different concentrations for 24 hours, wherein the assay samples had concentrations of 12.5 μ M, 25 μ M, 50 μ M, and 100 μ M in this order. The medium was then removed and 100. mu.L of fresh medium containing 20. mu.M of the fluorescent reagent 2', 7' -dichlorofluorescein diacetate (DCF-DA) was added to each well and incubated with the cells for 20 minutes. The cells were then incubated with 100. mu.L of 100. mu.M H₂O₂After 1 hour of treatment, fluorescence intensity of each well was measured using a microplate reader at excitation and emission wavelengths of 485nm and 525nm, respectively.

Evaluating the antioxidant effect of OLE and HT by measuring the improvement of oxidative stress-induced reactive oxygen species in human dermal fibroblasts by OLE and HT in non-toxic concentrations (6.25-100 μ M), hydrogen peroxide being used as an oxidative stress inducer, wherein a sample without hydrogen peroxide added and OLE or HT added is used as a blank control, and a sample without hydrogen peroxide added but OLE or HT added is used as H₂O₂The results of the stimulated group are shown in FIG. 5, H is compared with the blank control group₂O₂ROS levels in stimulated cells increased 6.8-fold. Although the treatment of OLE (12.5, 25, 50 and 100. mu.M) slightly reduced H₂O₂Trend of induced ROS levels, but do not show significant antioxidant effects in fibroblasts. Higher concentrations (50 and 100. mu.M) of HT respectively convert H₂O₂Induced ROS levels were reduced by 14.5% and 15.2%, respectively.

Furthermore, the synergistic effect of the combination of OLE and HT on oxidative stress in fibroblasts was assessed by calculating CI values. The combination of OLE and HT at a ratio of 1:1 (from 6.25/6.25. mu.M to 100/100. mu.M) showed a synergistic protective effect in fibroblasts due to the combination with H₂O₂OLE + HT decreased ROS levels in fibroblasts by 7.6, 18.4, 21.0, 27.6, and 37.3% respectively (FIG. 6A), all higher than ROS levels in fibroblasts when acted on alone, compared to the stimulated groupThe extent of the reduction in level. Furthermore, the CI values for the combination of OLE and HT (from 6.25/6.25 μ M to 100/100 μ M) were each less than 1 (0.44, 0.17, 0.26, 0.28 and 0.27, respectively), indicating that OLE and HT exert a synergistic cytoprotective effect in fibroblasts (fig. 6B).

The above results indicate that OLE and HT inhibit elastase activity and that the combination exerts a synergistic effect in the elastase inhibition assay, which may contribute to the overall anti-skin aging effect of the plant extracts containing OLE and HT. In addition, the combination of OLE and HT synergistically reduces reactive oxygen species levels in human dermal fibroblasts. Thus, OLE and HT can be used as promising bioactive cosmeceutical products for antioxidants and skin care products.

In conclusion, the jasminum grandiflorum extract, OLE and HT all have an anti-elastase effect, wherein the OLE and HT can enhance the inhibition rate of elastase and significantly reduce the activity of elastase when mixed at equal molar concentrations and equal volumes, and therefore, the jasminum grandiflorum ethyl acetate layer extract, OLE and HT can be used for preparing anti-aging cosmetics and medicines for preventing or treating emphysema complications.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. The application of the jasminum grandiflorum extract or the effective components thereof in preparing the elastase inhibitor is characterized in that the jasminum grandiflorum extract is an ethyl acetate layer extract of the jasminum grandiflorum; the effective components of the jasminum grandiflorum extract comprise oleuropein and hydroxytyrosol.

2. The use as claimed in claim 1, wherein the molar ratio of oleuropein to hydroxytyrosol for the preparation of elastase inhibitors is 1:1 and the volume ratio is 1: 1.

3. The use of claim 1, wherein the preparation method of the jasminum grandiflorum extract comprises the following steps:

4. The use as claimed in claim 1, wherein the preparation method of oleuropein as the effective component of the jasminum grandiflorum extract comprises the following steps:

taking mixed solution of dichloromethane and methanol as eluent, performing gradient elution on the ethyl acetate layer extract of the jasminum grandiflorum, collecting the eluent, eluting the eluent with mixed solution of chloroform and methanol, and performing reverse phase column chromatography with aqueous solution of methanol to collect the eluent, thereby obtaining the oleuropein serving as the effective component of the jasminum grandiflorum extract.

5. The use of claim 1, wherein the preparation method of hydroxytyrosol as an effective component in the jasminum jasminoides extract comprises the following steps:

gradient elution is carried out on the ethyl acetate layer extract of the jasminum grandiflorum by taking a mixed solution of petroleum ether and ethyl acetate as an eluent, the eluent is collected, and the collected eluent is eluted by a mixed solution of chloroform and methanol to obtain the hydroxytyrosol as the effective component of the jasminum grandiflorum extract.

6. An elastase inhibitor, wherein the protease inhibitor comprises an extract of jasminum grandiflorum or an active ingredient thereof; the effective components of the jasminum grandiflorum extract comprise oleuropein and hydroxytyrosol.

7. The protease inhibitor according to claim 6, wherein said oleuropein and hydroxytyrosol are present in a molar ratio of 1:1 and in a volume ratio of 1: 1.

8. Use of the elastase inhibitor as defined in claim 6 or 7 for the preparation of an anti-aging cosmetic.

9. The use according to claim 8, wherein the cosmetic comprises a toner, essence, cream, mask or body lotion.

10. Use of an elastase inhibitor in the manufacture of a medicament for the prevention or treatment of emphysema.