CN112656838A

CN112656838A - Pharmaceutical composition, preparation method and application thereof

Info

Publication number: CN112656838A
Application number: CN202011644495.3A
Authority: CN
Inventors: 孙云起; 熊盛; 赵辉; 陈伟; 聂艳峰; 郭朝万; 孙怀庆; 胡露
Original assignee: Guangdong Marubi Biological Technology Co Ltd
Current assignee: Guangdong Marubi Biological Technology Co Ltd
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2021-04-16
Anticipated expiration: 2040-12-31
Also published as: CN112656838B; CN114796310A; CN114796310B

Abstract

The application relates to the field of biological fermentation, in particular to a pharmaceutical composition, a preparation method and application thereof. A method of preparing a pharmaceutical composition comprising: culturing ATCC8014 in culture medium of ATCC8014 strain containing Japanese pepper, and collecting fermentation product; alternatively, ATCC9080 was cultured with ATCC9080 strain medium containing thyme, and then the fermentation product was collected. After the experiment of the inventor, the inventor finds that the addition of the thyme herb into the culture medium of the ATCC9080 strain and the ATCC8014 strain is beneficial to improving the performance of the fermentation product in whitening, antioxidation and anti-inflammatory aspects, and can be used for preparing skin care products, anti-inflammatory medicines, medicines for removing DPPH free radicals and medicines for inhibiting tyrosinase.

Description

Pharmaceutical composition, preparation method and application thereof

Technical Field

The application relates to the field of biological fermentation, in particular to a pharmaceutical composition, a preparation method and application thereof.

Background

The microbial conversion can use complete microbial cells or enzymes extracted from the microbial cells as biocatalysts, has strong regio-and stereoselectivity, mild reaction conditions, simple and convenient operation, lower cost and less public hazard, and can complete reactions which are difficult to carry out in organic chemical synthesis. The fermentation product contains rich active ingredients such as small molecular peptides, free amino acids, vitamins, nucleic acids, nucleotides and the like; the products after fermentation of different strains have different effects.

The present application aims to provide a process for the preparation of a pharmaceutical composition which aims to increase the performance of the fermentation product in skin care.

Disclosure of Invention

The embodiments of the present application aim to provide a pharmaceutical composition, a preparation method and applications thereof, which aim to provide a pharmaceutical composition beneficial to skin care.

The present application provides a method of preparing a pharmaceutical composition comprising:

ATCC8014 was cultured in a culture medium of ATCC8014 strain containing Japanese pepper, and the fermentation product was collected.

Alternatively, ATCC9080 was cultured with ATCC9080 strain medium containing thyme, and then the fermentation product was collected.

After the experiment of the inventor, the ground pepper is added into the ATCC9080 strain culture medium, so that the performance of the fermentation product in whitening, oxidation resistance and anti-inflammatory aspects is improved, and the fermentation product can be used for preparing skin care products, anti-inflammatory medicaments, medicaments for removing DPPH free radicals and medicaments for inhibiting tyrosinase.

After the experiment of the inventor, the ground pepper is added into the ATCC8014 strain culture medium, so that the performance of the fermentation product in whitening, oxidation resistance and anti-inflammatory aspects is improved, and the ground pepper can be used for preparing skin care products, anti-inflammatory medicaments, medicaments for removing DPPH free radicals and medicaments for inhibiting tyrosinase.

In some embodiments of the first aspect of the present application, the culture conditions for culturing ATCC8014 are: culturing at 200-240rpm and 35-40 ℃ for 20-35 hours.

Alternatively, the culture conditions for culturing ATCC9080 are: culturing at 200-240rpm and 35-40 ℃ for 20-35 hours.

In some embodiments of the first aspect of the present application, the step of collecting the fermentation product comprises: filtering with 0.2-0.3 μm filter membrane to obtain filtrate.

Optionally, the method further comprises removing the filter residue by centrifugation before filtration with a 0.2-0.3 μm filter membrane.

In some embodiments of the first aspect of the present application, the culture medium of ATCC8014 strain containing thymus serpyllum comprises about 0.5% to about 8% by weight of thymus serpyllum.

Optionally, the mass percent of the thymus serpentinatum in the culture medium of the ATCC8014 strain containing thymus serpentinatum is 4-8%.

In some embodiments of the first aspect of the present application, the culture medium of ATCC9080 species comprising thymus serpyllum comprises 0.5 to 9% by mass of thymus serpyllum.

Optionally, the mass percent of the sweet pepper in the culture medium of the ATCC9080 strain containing the sweet pepper is 7-9%.

The second aspect of the present application also provides a pharmaceutical composition, which is prepared by the above preparation method.

The application also provides an application of the pharmaceutical composition in preparing a skin care product.

The application also provides an application of the pharmaceutical composition in preparation of drugs for inhibiting the neuraminidase.

The application also provides an application of the pharmaceutical composition in preparing anti-inflammatory drugs.

The application also provides an application of the pharmaceutical composition in preparing a medicine for eliminating DPPH free radicals.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.

FIG. 1 shows the cell density of the mixture after 24-hour culture in examples 1-2 and comparative examples 1-2.

FIG. 2 shows the total ketone content in the pharmaceutical compositions of examples 1-2 and comparative examples 1-2.

FIG. 3 shows the total protein content in the pharmaceutical compositions of examples 1-2 and comparative examples 1-2.

FIG. 4 is a graph showing the in vitro inhibition rate of tyrosinase by the pharmaceutical compositions of examples 1-2 and comparative examples 1-2.

FIG. 5 is a graph showing the DPPH clearance rate of the pharmaceutical compositions of examples 1-2 and comparative examples 1-2.

Fig. 6 is a toxicity analysis of the pharmaceutical compositions of example 1 and example 2 on B16 cells.

FIG. 7 is a toxicity analysis of the pharmaceutical compositions of examples 1 and 2 on Hacat cells.

Fig. 8 is a toxicity analysis of the pharmaceutical compositions of example 1 and example 2 on 3T3 cells.

FIG. 9 is a graph of the effect of the pharmaceutical compositions of examples 1 and 2 on Collagen1 and HO-1 in 3T3 cells.

Fig. 10 shows an analysis of the inhibition of the neuraminidase by the pharmaceutical compositions of example 1 and example 2 on B16 cells.

FIG. 11 shows anti-inflammatory analysis of IL-8 on HaCat cells by the pharmaceutical compositions of example 1 and example 2.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The pharmaceutical compositions, methods of preparation, and uses of the embodiments of the present application are described in detail below.

The present application generally provides two pharmaceutical compositions; a first pharmaceutical composition and a second pharmaceutical composition.

The preparation method of the first pharmaceutical composition comprises the following steps:

The ground pepper is added into the basic culture medium of the ATCC8014 strain and then used for culturing the ATCC8014, and a fermentation product is collected after the culture is carried out for a period of time.

Thymus aquicostatus cak is a plant of the genus Thymus of the family Labiatae. The main active ingredients of the thyme herb are volatile oil and flavonoid ingredients. The herba Thymi Japonici extract has effects of resisting tumor, resisting bacteria, resisting oxidation, reducing blood sugar and improving cardiovascular and cerebrovascular ischemia. The Mongolian thyme herb has the functions of dispelling wind, relieving exterior syndrome, promoting qi circulation, relieving pain, clearing heat and removing toxicity, and is clinically used for treating various diseases. The thyme herb is used as a natural medicine and food, has strong antioxidation, and can effectively prevent and treat cardiovascular and cerebrovascular diseases and has potential black eye removing effect.

In the present application, the culture medium of ATCC8014 strain containing thyme comprises a basal medium and thyme, wherein the percentage by mass of thyme is 0.5-8%, for example, 0.5%, 0.6%, 1%, 1.2%, 1.6%, 2.0%, 2.5%, 3%, 3.6%, 4%, 4.5%, 5%, 6%, 7%, 8%, etc. Basal media other than sweet pepper can be purchased commercially or can be configured on-site, and can be configured according to the growth and metabolic properties of the ATCC8014 species.

In the examples of the present application, the sweet pepper may adopt at least one part of the root, stem and leaf of sweet pepper. In the present application, the ground pepper powder is added to the culture medium, and it is understood that in other embodiments of the present application, an aqueous extract, an alcohol extract, etc. of the ground pepper may be added to the culture medium. The ground pepper of this application chooses for use Ningxia ground pepper, in other embodiments of this application, can choose for use the ground pepper of other producing areas.

By way of illustration, in some embodiments, the culture conditions for culturing ATCC8014 are: culturing at 200-240rpm and 35-40 ℃ for 20-35 hours.

In some embodiments, the ATCC8014 strain is inoculated with a culture medium of ATCC8014 strain containing thymus serpyllum, at a density OD of 1.5 to 1.9 (e.g., 1.5, 1.6, 1.7, 1.8, 1.9); then culturing at 200-240rpm (such as 200rpm, 210rpm, 220rpm, 230rpm, 240rpm) at 35-40 deg.C (such as 35 deg.C, 36 deg.C, 37 deg.C, 39 deg.C, 40 deg.C) for 20-35 hr; the incubation time may be, for example, 20 hours, 22 hours, 24 hours, 28 hours, 30 hours, or 35 hours, and the like.

In other examples of the present application, if the temperature, time and rotation speed of the culture conditions are out of the above ranges, the ratio of Japanese pepper in the culture medium of ATCC8014 species containing Japanese pepper is out of the above ranges, regardless of the yield and the culture efficiency.

After the completion of the culture, the fermentation product was collected.

In some embodiments of the present application, the means for collecting the fermentation product comprises filtering the product after cultivation to remove filter residues which cannot be emptied by 0.2-0.3 μm filtration. For example, the filtrate is collected by filtration with a filter of 0.2 to 0.3 μm (for example, 0.2 μm, 0.22 μm, 0.25 μm, 0.28 μm, 0.3 μm, etc.).

In some embodiments of the present application, in the process of collecting the fermentation product, in order to improve the filtration efficiency and save the filtration time, the filtration may further include centrifugal separation to remove the filter residue. For example, centrifugation is carried out at 4000rpm for 10 min.

The application also provides a pharmaceutical composition which is mainly prepared by the preparation method of the first pharmaceutical composition.

The pharmaceutical composition provided by the embodiment of the application has better effects on whitening, antioxidation, anti-inflammation and inhibition of the neuraminidase. After the experiment of the inventor, the ground pepper is added into the ATCC8014 strain culture medium, so that the performance of the fermentation product in whitening, oxidation resistance and anti-inflammatory aspects is improved, and the ground pepper can be used for preparing skin care products, anti-inflammatory medicaments, medicaments for removing DPPH free radicals and medicaments for inhibiting tyrosinase.

The present application also provides a second pharmaceutical composition, the second pharmaceutical composition prepared by a method comprising:

ATCC9080 was cultured with ATCC9080 strain medium containing Japanese pepper, and then the fermentation product was collected.

In the present application, the ATCC9080 culture medium containing thyme comprises a basic culture medium and thyme, wherein the thyme is 0.5-9% by mass, for example, 0.5%, 0.7%, 1%, 1.3%, 1.5%, 2.0%, 2.6%, 3%, 3.5%, 4%, 4.7%, 5%, 7%, 9%, etc. The basic culture medium except the thyme herb can be purchased in the market or can be configured on site, and can be configured according to the growth and metabolic properties of ATCC9080 strains.

Accordingly, at least one part of the root, stem and leaf of Japanese pepper can be added to the basal medium. The Capsicum annuum can be added in the form of powder, water extract, alcohol extract, etc.

Illustratively, in some embodiments, the culture conditions for culturing ATCC9080 species are: culturing at 200-240rpm at 25-30 ℃ for 20-35 hours.

In some embodiments, ATCC9080 strain is inoculated in a culture medium of ATCC9080 strain containing thymus serpyllum at a thalli density OD of 1.5-1.9 (e.g., 1.5, 1.6, 1.7, 1.8, 1.9); then culturing at 200-240rpm (such as 200rpm, 210rpm, 220rpm, 230rpm, 240rpm) at 35-40 deg.C (such as 35 deg.C, 36 deg.C, 37 deg.C, 39 deg.C, 40 deg.C) for 20-35 hr; the incubation time may be, for example, 20 hours, 22 hours, 24 hours, 28 hours, 30 hours, or 35 hours, and the like.

In other examples of the present application, if the yield, the culture efficiency and other factors are not considered, the temperature, time and rotation speed of the culture conditions may not be in the above-mentioned ranges, and accordingly, the proportion of sweet pepper in the culture medium of ATCC9080 species containing sweet pepper may not be in the above-mentioned ranges.

After the completion of the culture, the fermentation product was collected.

In some embodiments of the present application, in the process of collecting the fermentation product, in order to improve the filtration efficiency and save the filtration time, the filtration may further include centrifugal separation to remove the filter residue. For example, the residue is removed by centrifugation at 4000rpm for 10 min.

The pharmaceutical composition (second pharmaceutical composition) provided by the embodiment of the application has better effects in whitening, antioxidation, anti-inflammation and inhibition of the neuraminidase. After the experiment of the inventor, the ground pepper is added into the ATCC9080 strain culture medium, so that the performance of the fermentation product in whitening, oxidation resistance and anti-inflammatory aspects is improved, and the fermentation product can be used for preparing skin care products, anti-inflammatory medicaments, medicaments for removing DPPH free radicals and medicaments for inhibiting tyrosinase.

The application also provides an application of the first pharmaceutical composition in preparing a skin care product. Or the second medicinal composition is applied to preparing skin care products.

The application also provides an application of the first pharmaceutical composition in preparing a medicament for inhibiting the neuraminidase. Or the second medicine composition is applied to preparing medicines for inhibiting the tyrosinase.

The application also provides an application of the first pharmaceutical composition in preparing an anti-inflammatory drug. Or the application of the second medicinal composition in preparing anti-inflammatory medicaments.

The application also provides an application of the first medicine composition in preparing a medicine for eliminating DPPH free radicals. Alternatively, the use of the second pharmaceutical composition as described above for the manufacture of a medicament for scavenging DPPH radicals.

The first medicinal composition and the second medicinal composition provided by the application have better effects in resisting inflammation, eliminating DPPH free radicals and inhibiting neuraminidase.

The features and properties of the present application are described in further detail below with reference to examples.

The strains and sources of culture medium in the various examples are listed below:

ATCC1.91 strain: purchased from north nai and originally abbreviated as LJ03 in the text and drawings.

ATCC204508 strain: it is purchased from Beinai Chuang Union, and is abbreviated as ZL03 in the text and the drawings.

ATCC8014 strain: purchased from north na institute of technology, hereinafter abbreviated as MRS.

ATCC9080 strain: purchased from Beinanjian union of Industrial chemistry, hereinafter referred to as YPD.

The basic culture medium of the ATCC1.91 strain and the ATCC204508 strain is LB culture medium; a507002 from Sangon Biotech.

The basic culture medium of the ATCC8014 strain is an MRS culture medium; m8540-250g from Solarbio.

The basic culture medium of the ATCC9080 strain is a YPD culture medium; LA0250-250g from Solarbio.

Example 1

The embodiment provides a pharmaceutical composition, which is mainly prepared by the following steps:

inoculating the MRS strain to 10ml of MRS culture medium to be cultured for one day to obtain MRS strain;

mixing the stem and leaf powder of the thyme herb and the MRS culture medium according to the mass ratio of 1:25, 1:50 and 1:100 respectively to prepare three culture media; then inoculating MRS strains cultured on the first day into the three mixed culture media at a thallus density of OD 1.7; culturing at 220rpm and 37 deg.C for 24 hr; the fermented mixture was collected, and the cell density was measured to evaluate the growth. After centrifugation at 4000rpm for 10min, the supernatant was collected and filtered through a 0.22 μm filter (commercially available from Merck Millipore, SLGP033RB) and collected for use.

Example 2

the YPD strain was inoculated into 10ml of YPD medium and cultured for one day to obtain YPD strain.

Mixing the stem and leaf powder of the Japanese pepper and the YPD culture medium according to the mass ratio of 1:25, 1:50 and 1:100 respectively to prepare three culture media; then, the YPD strains cultured on the first day are respectively inoculated to the three mixed culture media at a cell density of OD 1.7; culturing at 220rpm and 37 deg.C for 24 hr; the fermented mixture was collected, and the cell density was measured to evaluate the growth state. And centrifuged at 4000rpm for 10 min. The supernatant was collected and filtered through a 0.22 μm filter (commercially available from Merck Millipore, SLGP033RB) and collected for use.

Comparative example 1

The present comparative example provides a pharmaceutical composition, prepared by the steps of:

inoculating the LJ03 strain to 10ml LB culture medium to obtain LJ03 strain;

mixing the stem and leaf powder of the Japanese pepper and the LB culture medium according to the mass ratio of 1:25, 1:50 and 1:100 respectively to prepare three culture media; then respectively inoculating the LJ03 strain cultured on the first day to the three mixed culture media at the density of the strain with OD being 1.7; culturing at 220rpm and 37 deg.C for 24 hr; the fermented mixture was collected, and the cell density was measured to evaluate the growth state. And centrifuged at 4000rpm for 10 min. The supernatant was collected and filtered through a 0.22 μm filter (commercially available from Merck Millipore, SLGP033RB) and collected for use.

Comparative example 2

inoculating ZL03 strain into 10ml LB culture medium to culture for one day to obtain ZL03 strain;

mixing the stem and leaf powder of the Japanese pepper and the LB culture medium according to the mass ratio of 1:25, 1:50 and 1:100 respectively to prepare three culture media; then respectively inoculating the strain ZL03 cultured on the first day into the three mixed culture media at the density of the strain with OD (OD) of 1.7; culturing at 220rpm and 37 deg.C for 24 hr; the fermented mixture was collected, and the cell density was measured to evaluate the growth state. And centrifuged at 4000rpm for 10 min. The supernatant was collected and filtered through a 0.22 μm filter (commercially available from Merck Millipore, SLGP033RB) and collected for use.

Comparative example 3

The present comparative example, which differs from example 1 in that thymus serpyllum is not mixed in the medium, provides a pharmaceutical composition.

Comparative example 4

The present comparative example, which differs from example 2 in that thymus herb is not mixed in the medium, provides a pharmaceutical composition.

In fig. 1-9: LJ03 represents the pharmaceutical composition of comparative example 1; ZL03 represents the pharmaceutical composition of comparative example 2; YPD represents the pharmaceutical composition of example 2; MRS represents the pharmaceutical composition of example 1.

Test example 1

Example 1-2 the results of measuring the cell density of the mixture after 24 hours of culture and fermentation are shown in FIG. 1.

As can be seen from FIG. 1, the growth of the cells was better in examples 1-2, which indicates that the growth of the corresponding bacterial species was not affected by the Thymus mongolicus.

Test example 2

Measurement of Total Ketone content in the pharmaceutical compositions of examples 1-2

Rutin (available in Solarbio, SR8250-100mg) is used as standard substance, 60% ethanol (available in Tianjin Damao, 3852) is used for preparing rutin standard solution of 1000 mug/mL, 800 mug/mL, 600 mug/mL, 400 mug/mL, 200 mug/mL, 100 mug/mL, 50 mug/mL and 0 mug/mL, and the rutin standard solution is divided into partsRespectively taking 300 mu L of rutin standard solution and fermentation sample to 2mL of EP (eppendorf) tube, and respectively adding 100 mu L of 5% NaNO₂(purchased from Macklin, S818033), mixing, standing for 6min, and adding 100 μ L of 10% Al (NO)₃)₃(purchased from Tianjin Damao) solution, mixing, standing for 6min, adding 500 μ L of 4% NaOH (purchased from Macklin, S835850) solution, mixing by vortex, standing for 15min, absorbing 200 μ L of the mixed solution in a 96-well enzyme label plate, and measuring the OD value at the wavelength of 510 nm. And (4) calculating the total ketone content in each fermentation sample according to the standard curve. FIG. 2 shows the total ketone content in the pharmaceutical compositions of examples 1-2 and comparative examples 1-2.

Test example 3

Measurement of Total protein content in the pharmaceutical compositions of examples 1-2

Using BSA as a standard, 2000. mu.g/mL, 1500. mu.g/mL, 1000. mu.g/mL, 750. mu.g/mL, 500. mu.g/mL, 250. mu.g/mL, 125. mu.g/mL, 25. mu.g/mL, 0. mu.g/mL BSA standard solutions were prepared using 1 XPBS (purchased from Gibco,10010023) according to the protein quantification kit (purchased from Thermo, 23225). According to the kit specification, working solution is prepared in a dark condition according to the proportion of 50:1, and meanwhile, standard solution and corresponding pharmaceutical composition are added into each 10 mu L of 96-hole enzyme label plate; then 200. mu.L of the working solution was added thereto, and the mixture was incubated at 37 ℃ for 30 min. And measuring the OD value of the 96-hole enzyme label plate at the wavelength of 562 nm. And calculating the total protein content of each fermentation sample according to the standard curve. FIG. 3 shows the total protein content in the pharmaceutical compositions of examples 1-2 and comparative examples 1-2.

Test example 4

The efficiency of the pharmaceutical compositions of examples 1-2 and comparative examples 1-2 in inhibiting tyrosinase in vitro was measured

Multiple parallel experimental groups, standard control group, sample background group and blank control group are set up.

Adding 80. mu.L, 120. mu.L, 160. mu.L and 200. mu.L of PBS buffer solution with the pH value of 6.80.2M into the experimental group, the standard control group, the sample background group and the blank control group respectively;

adding 40. mu.L of each of the pharmaceutical compositions of examples 1-2 and comparative examples 1-2 to a plurality of wells of the experimental group; then 80. mu.L of 1.5mmol/L L-tyrosine (ex Solebao, T0010-25g) was added to each well of the experimental group;

to a plurality of wells of the sample background group, 40. mu.L of each of the pharmaceutical compositions of examples 1-2 and comparative examples 1-2 was added.

To each well of the standard control group was added 80. mu.L of 1.5mmol/L L-tyrosine (ex Solebao, T0010-25 g).

Finally, 40. mu.L of 500U/mL tyrosinase (available from Solambio, T8830-25KU) was added to each well.

The incubation was carried out at 37 ℃ for 20min, and the OD was measured at a wavelength of 475 nm. Tyrosinase in vitro inhibition rate ═ (experimental OD value-sample background OD value)/(standard control OD value-blank control OD value).

As can be seen from FIG. 4, the addition of Zanthoxylum piperitum to the culture medium can significantly improve the tyrosinase inhibition rate of the final fermentation product.

Test example 5

The DPPH radical scavenging efficiency of the pharmaceutical compositions of examples 1-2 and comparative examples 1-2 was measured

The 96-well ELISA plates are divided into 3 groups, D1 group, D2 group and D3 group, each group comprises a plurality of wells.

Part of the wells of group D1 were filled with the pharmaceutical composition of example 1, part of the wells were filled with the pharmaceutical composition of example 2, part of the wells were filled with the pharmaceutical composition of comparative example 1, and part of the wells were filled with the pharmaceutical composition of comparative example 2; the addition amount is 100 mu L; then 100. mu.L of 2X 10 were added to each well of the D1 group^-4A solution of DPPH (from TCI, D0909) in mol/L absolute ethanol.

Part of the wells of group D2 were filled with the pharmaceutical composition of example 1, part of the wells were filled with the pharmaceutical composition of example 2, part of the wells were filled with the pharmaceutical composition of comparative example 1, and part of the wells were filled with the pharmaceutical composition of comparative example 2; the addition amount is 100 mu L; then 100. mu.L of absolute ethanol solution was added to each well of the D2 group.

Each well of D3 group was filled with 100. mu.L of 2X 10^-4A solution of DPPH (from TCI, D0909) in mol/L in absolute ethanol and100 μ L of absolute ethanol solution.

Then, the 96-well microplate was placed at 37 ℃ for 30 min. OD values were measured at a wavelength of 517nm (D1, D2, D3). The radical clearance was calculated from the radical clearance (%) (D2+ D3-D1)/D3X 100%.

From test example 1 to test example 5, it can be seen that: the content of total flavone in the supernatant and the content of total protein of the pharmaceutical compositions provided in example 1 and example 2 are far greater than those of the pharmaceutical compositions of comparative example 1 and comparative example 2; the pharmaceutical compositions provided in examples 1 and 2 have higher tyrosinase inhibition rates in vitro and DPPH clearance rates than the pharmaceutical compositions of comparative examples 1 and 2.

The effect of the thyme herb on the fermentation products of the ATCC8014 strain and the ATCC9080 strain is illustrated, and the positive effect on the antioxidant performance, the free radical scavenging capacity and the tyrosinase external inhibition performance in the fermentation products is realized.

Test example 6

Toxicity analysis of the pharmaceutical compositions of example 1 and example 2 on B16, Hacat, 3T3 cells

Two experiments were performed, one for studying the pharmaceutical composition provided in example 1, and the pharmaceutical composition obtained in example 1 by mixing the stem and leaf powder of thyme herb and the culture medium prepared from MRS medium at a mass ratio of 1:25 was used for the experiments. One group was used to study the pharmaceutical compositions provided in example 2. An experiment was performed on a pharmaceutical composition obtained from a culture medium prepared by mixing the stem and leaf powder of Japanese pepper and the YPD medium at a mass ratio of 1:50 in example 2.

(1) Culturing at 100cm²B16, Hacat, and 3T3 cells in the cell culture dish were digested with 1mL of 0.25% trypsin solution (purchased from Gibco, 25200-072), respectively, and when the cells were to be detached from the dish, the trypsin solution was aspirated, and then digestion of B16 cells was terminated with 6mL of RP1640 MI (purchased from Gibco, C11875500CP) containing 10% serum (purchased from Gibco, 10270106) and 1% double antibody; digestion of Hacat, 3 was stopped with 6mL of DMEM medium containing 10% serum (purchased from Gibco, 10270106) and 1% double antibody (purchased from Gibco, C11995500BT)T3 cells. The cells were then dispersed by gentle pipetting, collected in a centrifuge tube and centrifuged at 800rpm for 5 min. The medium was removed by centrifugation, the cells were resuspended in 2mL of medium containing 0.02% serum, 10. mu.g/mL human transferrin (from simga, T2252) and 200. mu.g/mL BSA (from Macklin, B824162) DMEM/F12(1:1) (from Gibco, C11330500BT), 20uL of the cell suspension was aspirated, mixed with 4% trypan blue solution (from Solibao, C0040) at a ratio of 1:1 and counted using a countstar counter. Each well was plated with 5000 cells in 80. mu.L to a 96-well plate, wherein 100. mu.L of DMEM/F12(1:1) medium was added to the outermost periphery of the 96-well plate.

(2) At 37 deg.C, 5% CO₂Culturing for 24 hours in the incubator; then, one group was diluted by mixing the pharmaceutical composition of example 1 with DMEM/F12(1:1) medium at a mass ratio of 1:4, and 20. mu.L of the sample was added to each well at 37 ℃ with 5% CO₂Culturing for 48h in an incubator; then 10. mu.L of CCK-8 solution (from selelck, B34304) was added per well. Another group was diluted by mixing the pharmaceutical composition of example 2 with DMEM/F12(1:1) medium at a mass ratio of 1:4, and 20. mu.L of the sample was added to each well at 37 ℃ with 5% CO₂Culturing for 48h in an incubator; then 10. mu.L of CCK-8 solution (from selelck, B34304) was added per well.

(3) At 37 deg.C, 5% CO₂The incubator was incubated for 2 hours in the absence of light, and then the OD value at a wavelength of 450nm and the OD value at a wavelength of 630nm were measured, and the difference was determined. Then according to the formula: relative proliferation rate (% relative proliferation rate) (sample OD-blank)/(blank OD-medium OD) was obtained.

Fig. 6 is a toxicity analysis of the pharmaceutical compositions of example 1 and example 2 on B16 cells. FIG. 7 is a toxicity analysis of the pharmaceutical compositions of examples 1 and 2 on Hacat cells. Fig. 8 is a toxicity analysis of the pharmaceutical compositions of example 1 and example 2 on 3T3 cells. As can be seen from fig. 6-8, the pharmaceutical compositions of example 1 and example 2 are less toxic to B16, Hacat, 3T3 drugs. According to the results of FIGS. 6 to 8, three concentrations of less toxic substances, 0.3125% (H), 0.078125(M), and 0.019532(L), were selected for test example 6 and test example 7.

Test example 7

Antioxidant analysis of 3T3 cells treated with the pharmaceutical compositions of examples 1 and 2

(1) Culturing at 100cm²3T3 cells in the cell culture dish were digested with 1mL of 0.25% trypsin solution, and when the cells were to be detached from the dish, the trypsin solution was aspirated, the digestion was stopped with 6mL of 10% serum, 1% double anti DMEM medium, and gently flicked to disperse the cells, and the cells were collected in a centrifuge tube and centrifuged at 800rpm for 5 min. The medium was removed by centrifugation, the cells were resuspended in 2mL DMEM/F12(1:1) medium, 20uL of the cell suspension was aspirated, mixed with 4% trypan blue solution at a 1:1 ratio and counted using a countstar counter. At 1.5X 10⁶Each cell is planted at 60cm per well²Cell culture dishes were maintained and supplemented with DMEM/F12(1:1) medium to 3 mL. Placing at 37 ℃ with 5% CO₂Culturing in a cell culture box for 24 h.

(2) Respectively preparing the pharmaceutical compositions of the example 1 and the example 2 with the concentration of 0.3125%, 0.078125% and 0.019531% in volume fraction; then added to different dishes separately so that the final volume was 4 mL. Placing at 37 ℃ with 5% CO₂Culturing in a cell culture box for 48 h. The medium was then removed, washed twice with PBS, and the cells were lysed with 300uL of RIPA (ex Bilun, P0013B) lysate containing 1mM PMSF (ex Bilun, ST506), the cell lysate was collected and centrifuged at 13000rpm for 30min at 4 ℃. The centrifuged supernatant was transferred to a new EP (eppendorf) tube. Protein quantification method reference test example 2 protein quantification kit (purchased from Thermo, 23225).

(3) The samples with quantified protein were sampled in equal mass, made up to equal volume with PBS solution, added with 1/5 volume of 5 × Loading buffer (from Solebao, P1040), vortexed, mixed well, and heated in a 100 deg.C water bath for 10 min. And (5) spotting after cooling.

(4) Electrophoresis was performed using a pre-made gel (available from invitrogen, EC6025BOX), 20 μ L of the prepared sample was added to the pre-made gel, the sample was electrophoresed to the separation gel at 80V for 30min, and then electrophoresed at 120V until the indicator reached the bottom.

(5) The gel was attached to an appropriate size NC membrane (purchased from Millipore, HATF00010) in a sandwich configuration, and membrane transfer was performed for 90min with the addition of membrane transfer solution at 300mA constant current. Then 5% skimmed milk powder (purchased from BD, 232100) was added and blocked for 1h at room temperature. The membrane was washed with PBS solution (available from Sangon Biotech, E607008) containing Tween-20 (available from VETEC, V900548) at a concentration of 0.1 vol% PBST for 5min at 40 rpm. And (4) cutting NC according to the molecular weight of the protein to be detected. The volume ratio of the components is 1: 1000 series Mouse anti-GAPDH (available from Proteintech, 60004-1-Ig) was diluted in 0.1 vol% PBST solution and overlaid on NC membrane; or, the volume ratio is 1: 1000 series HO-1Rabbit mAb (from CST,86806S) was diluted in 0.1 vol% PBST solution and coated on NC membrane; or, the volume ratio is 1: 1000 columns Rabbit Anti-Collagen I antibodies (from bios, bs-10423R) were diluted in 0.1 vol% PBST solution and overlaid on NC membrane. Incubate at 4 ℃ for 12 h. The membrane was then washed with 0.1% PBST at 40rpm for 5min, repeated three times, and excess antibody was washed away. Then, the ratio of 1: Anti-Rabbit IgG (L + H) HRP (from Proteintech, SA00001-2) diluted in 5000 columns was incubated in 0.1 vol% PBST solution onto the corresponding NC membrane; the above membrane washing step was repeated to add ECL chemiluminescence solution to the NC membrane surface, and photographic exposure was performed using a gel imager (Bio-Rad). Mixing the raw materials in a ratio of 1: 5000 dilution of Anti-Mouse IgG (L + H) HRP (from Proteintech, SA00001-1) in 0.1 vol% PBST solution, incubated on the corresponding NC membrane; the above membrane washing step was repeated to add ECL chemiluminescence solution to the NC membrane surface, and photographic exposure was performed using a gel imager (Bio-Rad).

FIG. 9 is a graph of the effect of the pharmaceutical compositions of examples 1 and 2 on Collagen1 and HO-1 in 3T3 cells. In fig. 9, L, M, H represent the different concentrations of the pharmaceutical compositions obtained from the culture medium prepared by mixing the stem and leaf powder of sweet pepper and the MRS medium in a mass ratio of 1:25 in example 1, 10.3125% (H), 0.078125(M), and 0.019532(L), respectively. L, M, H represent the concentrations of the pharmaceutical compositions obtained by mixing the stem and leaf powder of Japanese pepper and YPD medium at a mass ratio of 1:50 in example 2, 10.3125% (H), 0.078125(M), and 0.019532(L), respectively.

As can be seen in fig. 9: under the premise of consistent GAPDH (internal reference), the higher the grayscale of the bands of collagen1 (type 1 collagen) and HO-1 (heme oxygenase-1) indicates that more proteins correspond to the collegen, while collagen1 (type 1 collagen) and HO-1 (heme oxygenase-1) are important detection indexes in the anti-oxidation or anti-aging detection, and the higher the contents of collagen1 and HO-1 proteins indicate that the anti-oxidation performance of the medicine is better, and as can be seen from FIG. 9, the three pharmaceutical compositions provided in example 1 and example 2 have better anti-oxidation or anti-aging effects.

Test example 8

Analysis of the pharmaceutical compositions of examples 1 and 2 for the inhibition of the neuraminidase on B16 cells

(1) Culturing at 100cm²The B16 cells in the cell culture dish were digested with 1mL of 0.25% trypsin solution, and when the cells were to be detached from the dish, the trypsin solution was aspirated, the digestion was stopped with 6mL of RPMI1640 medium, and gently flicked to disperse the cells, and the cells were collected in a centrifuge tube and centrifuged at 800rpm for 5 min.

After centrifugation the medium was removed, the cells were resuspended in 2mL DMEM/F12(1:1) medium, 20uL of the cell suspension was aspirated, mixed with 4% trypan blue solution at a 1:1 ratio and counted using a countstar counter. At 1.5X 10⁶Each cell is planted at 60cm per well²Cell culture dishes were maintained and supplemented with DMEM/F12(1:1) medium to 3 mL.

(2) Placing at 37 ℃ with 5% CO₂Culturing in a cell culture box for 24 h. After 24h of incubation, the test samples were prepared and according to the cck-8 toxicity test results, concentrations consistent with those of test example 5 were selected and the drug was diluted with 1mL of DMEM/F12(1:1) medium. A final concentration of 0.5mM alpha-MSH (from MCE, HY-P0252) was added, with no blank added. And (5) after the dosing is finished. Placing at 37 ℃ with 5% CO₂Culturing in a cell culture box for 48 h.

(3) The medium was aspirated off, washed once with PBS solution, digested with 300uL of 0.25% trypsin solution, digested with 1mL of RPMI1640 medium, and the cells were blown up evenly, collected and centrifuged at 800rpm for 5min and washed three times with PBS solution, leaving the cell pellet. 500uL of a RIPA-rich lysate was used as in (2) and (3) of test example 6.

(4) 60uL of lysis supernatant was added to each well and a blank control was prepared. 140uL of a 2mM L-Dopa (available from Macklin, L807435) solution was then added. Incubation was carried out at 37 ℃ for 30min, and the OD of the sample was measured at a wavelength of 475nm and the data was processed. At equal protein amounts, a higher OD at 475nm of the sample indicates a higher activity of the neuraminidase. And vice versa. Therefore, the relative activity of the neuraminidase is equal to the activity of the neuraminidase in each unit of protein in the experimental group/the activity of the neuraminidase in each unit of protein in the alpha-MSH group, namely the relative activity of the neuraminidase in the 0.5 mu M alpha-MSH model group is 100%.

Fig. 10 shows an analysis of the inhibition of the neuraminidase by the pharmaceutical compositions of example 1 and example 2 on B16 cells. In FIG. 10, Ctrl represents a blank control group, and L, M, H represents the same meaning as in FIG. 9.

As can be seen in FIG. 10, the relative activity of the neuraminidase in the experimental group to which the pharmaceutical compositions of example 1 and example 2 were added was less than 0.5mM α -MSH model group and less than the blank control group, indicating that the above pharmaceutical compositions have better inhibitory effects on the inhibition of the neuraminidase.

Test example 9

Anti-inflammatory assay of IL-8 on HaCat cells with the pharmaceutical compositions of examples 1 and 2

(1) Referring to (1) and (2) in test example 5, supernatants of HaCat cells treated with drugs at different concentrations were collected.

(2) Detection was performed using the IL-8Elisa Kit (from Union, EK 108). Add 300uL of Wash Buffer to each well, let stand for 30s, pour away Wash Buffer, and pat dry the Elisa plate. 50uL of the pharmaceutical composition provided in the different examples and IL-8 standards were added separately, and 50uL of detection antibody was added to each well. Sealing films are pasted on the mixture and incubated for 1.5h at 37 ℃.

(3) Removing supernatant, adding 300uL of Wash Buffer into each hole, standing for 30s each time, and repeatedly washing for 6 times. 100uL of anti-streptomycin HRP antibody is added into each hole, a sealing membrane is attached, and the mixture is incubated at 37 ℃ for 1 h.

(4) Removing supernatant, adding 300uL of Wash Buffer into each hole, standing for 30s each time, and repeatedly washing for 6 times. Adding 100uL of TMB color development solution into each well in a dark place, sticking a sealing film, and incubating at 37 ℃ for 10 min. The reaction was then terminated by adding 100uL of NaOH solution per well.

(5) Then, the OD value at a wavelength of 450nm and the OD value at a wavelength of 630nm were measured, and the difference was determined. And obtaining a standard curve and a formula according to the standard substance, and calculating the concentration or the quality of the IL-8 sample according to the OD of the sample.

FIG. 11 shows anti-inflammatory analysis of IL-8 on HaCat cells by the pharmaceutical compositions of example 1 and example 2. As can be seen in fig. 11: the excessive secretion of IL-8 in the skin can accelerate the aging of the skin and the skin relaxation, and compared with a blank control group, the two medicines of the application can well inhibit the secretion of IL-8 under different concentrations.

From test example 1 to test example 8, it can be seen that: the red pepper is added into the culture medium of ATCC8014 strain and ATCC9080 strain, which is beneficial to positively enhancing the performance of the fermentation product in anti-inflammatory drugs, scavenging DPPH free radicals and inhibiting tyrosinase; accordingly, the culture medium of ATCC1.91 and ATCC204508 to which Japanese pepper has been added have poor anti-inflammatory activity, DPPH radical scavenging activity and tyrosinase inhibiting activity.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method of preparing a pharmaceutical composition, comprising:

culturing ATCC8014 in culture medium of ATCC8014 strain containing Japanese pepper, and collecting fermentation product;

2. The method of preparing a pharmaceutical composition according to claim 1,

the culture conditions for culturing ATCC8014 were as follows: culturing at 200-240rpm and 35-40 deg.C for 20-35 hr;

3. The method of preparing a pharmaceutical composition according to claim 1,

the step of collecting the fermentation product comprises: filtering with 0.2-0.3 μm filter membrane to obtain filtrate;

4. The method for preparing the pharmaceutical composition according to any one of claims 1 to 3, wherein the culture medium of the Mongolian thyme herb-containing ATCC8014 strain contains 0.5 to 8% by weight of Mongolian thyme herb;

5. The method for preparing a pharmaceutical composition according to any one of claims 1 to 3, wherein the mass percentage of the thymus serpyllum in the culture medium of ATCC9080 species containing thymus serpyllum is 0.5 to 9%;

6. A pharmaceutical composition prepared by the process of any one of claims 1 to 5.

7. Use of the pharmaceutical composition of claim 6 for the preparation of a skin care product.

8. Use of the pharmaceutical composition of claim 6 for the manufacture of a medicament for inhibiting a neuraminidase.

9. Use of a pharmaceutical composition according to claim 6 for the preparation of an anti-inflammatory medicament.

10. Use of a pharmaceutical composition according to claim 6 for the preparation of a medicament for scavenging DPPH radicals.