Salvia miltiorrhiza and earthworm composition as well as preparation method and application thereof

Abstract

The invention belongs to the field of natural pharmaceutical compositions, and particularly relates to a salvia miltiorrhiza and earthworm composition as well as a preparation method and application thereof. According to the specific technical scheme, the skin care product comprises a radix salviae miltiorrhizae extract, and the radix salviae miltiorrhizae extract is an alcohol extract of roots and stems of radix salviae miltiorrhizae; the components of the skin care product also comprise earthworm polypeptide. The invention finds that the salvia miltiorrhiza extract and the earthworm polypeptide provided by the invention respectively have the effects of reducing the activity of tyrosinase in cells and reducing the content of melanin, and the effect of the composition is better. Therefore, the radix salviae miltiorrhizae extract, the earthworm polypeptide and the composition thereof have the potential of preparing whitening and anti-blackening products.

Description

Technical Field

[0001] The present invention belongs to the field of natural medicine compositions, and particularly relates to a danshen and earthworm composition, a preparation method thereof, and an application thereof. Background Art

[0002] Consumers' demand for "natural, safe, and highly effective" skin care ingredients continues to rise. Although traditional chemically synthesized ingredients (such as hydroquinone, retinol derivatives, etc.) have significant effects, they generally have risks such as irritation, photosensitivity, and environmental accumulation. Therefore, the research on natural medicine extracts as skin care ingredients is increasing.

[0003] Danshen, as a traditional Chinese medicine, has multiple effects such as antioxidant, anti-inflammatory, antibacterial, improving microcirculation, and regulating tissue repair and regeneration. Earthworms have the effects of promoting blood circulation to remove blood stasis, scavenging free radicals, and delaying aging, and the low-molecular-weight peptides therein have excellent transdermal permeability, so they have certain development potential in functional skin care.

[0004] In summary, if a new composition of natural ingredients with whitening and anti-blackening effects can be provided, it will have great application potential. Summary of the Invention

[0005] The purpose of the present invention is to provide a danshen and earthworm composition, a preparation method thereof, and an application thereof.

[0006] To achieve the above invention purpose, the technical solution adopted by the present invention is: a skin care product, the ingredients of the skin care product include danshen extract, and the danshen extract is an ethanol extract of danshen rhizome. The ingredients of the skin care product also include earthworm polypeptide, and the amino acid sequence of the earthworm polypeptide is: AVFPSIVGR.

[0007] Preferably, the danshen extract is obtained after ethanol extraction and elution. The leaching solution obtained after ethanol extraction is concentrated under reduced pressure to obtain an extract, and the extract is dispersed in water for elution. The eluate is concentrated under reduced pressure to obtain the danshen extract. Preferably, elution is carried out with an 80% ethanol aqueous solution.

[0008] Correspondingly, a skin care product, the ingredients of the skin care product include earthworm polypeptide, and the amino acid sequence of the earthworm polypeptide is: AVFPSIVGR. Preferably, the ingredients of the skin care product also include danshen extract, and the danshen extract is an ethanol extract of danshen rhizome.

[0009] The skin care product has whitening and anti-blackening functions.

[0010] Correspondingly, a composition, the composition includes danshen extract and earthworm polypeptide, the danshen extract is an ethanol extract of danshen rhizome, and the amino acid sequence of the earthworm polypeptide is: AVFPSIVGR.

[0011] Correspondingly, an external medicine prepared by using the composition; or; an external medicine containing the composition. The medicine has the functions of whitening and anti-blackening.

[0012] The present invention has the following beneficial effects: The present invention discovers that the salvia miltiorrhiza extract and earthworm polypeptide provided by the present invention respectively have the effects of down-regulating the activity of tyrosinase in cells and reducing the content of melanin, and the effect of the composition is better. Therefore, the salvia miltiorrhiza extract, earthworm polypeptide and their composition all have the potential to prepare whitening and anti-blackening products. Description of the Drawings

[0013] Figure 1 It is the LC-MS / MS spectrum of earthworm polypeptide;

[0014] Figure 2 It is the control chart of the influence of salvia miltiorrhiza extract and earthworm polypeptide on the viability of A375 cells;

[0015] Figure 3 It is the control chart of the influence of salvia miltiorrhiza extract and earthworm polypeptide on the activity of tyrosinase;

[0016] Figure 4 It is the control chart of the influence of salvia miltiorrhiza extract and earthworm polypeptide on the content of melanin. Detailed Embodiments

[0017] The present invention provides a salvia miltiorrhiza and earthworm composition, and the composition includes a salvia miltiorrhiza extract and an earthworm polypeptide. The salvia miltiorrhiza extract is preferably obtained by extracting the salvia miltiorrhiza rhizome with ethanol, and then eluting with an 80% ethanol aqueous solution. The amino acid sequence (single letter abbreviation) of the earthworm polypeptide is: AVFPSIVGR. A preferred scheme is: in the composition, by mass ratio, salvia miltiorrhiza extract: earthworm polypeptide = (1-25):(1-25).

[0018] The present invention also provides a preparation method of the composition, which specifically includes the following steps:

[0019] 1. Obtain the salvia miltiorrhiza extract: Crush the dried salvia miltiorrhiza rhizome and soak it in ethanol or an ethanol aqueous solution or pure water (preferably an 80% ethanol aqueous solution) at room temperature for 5-10 days, extract 3-5 times in total, and combine the extraction solutions. After filtration, carry out vacuum concentration to obtain a salvia miltiorrhiza extract. Disperse the extract in water, adsorb it with D101 macroporous resin for 1-3 h, and then elute it with an ethanol aqueous solution (preferably an 80% ethanol aqueous solution), collect the eluate, and carry out vacuum concentration to obtain the salvia miltiorrhiza extract.

[0020] 2. Obtain earthworm polypeptides. Crush the dried earthworms, reflux and extract them with sodium chloride solution at 100 °C, concentrate the filtrate to obtain a crude extract. Dissolve the crude extract in 50% methanol, centrifuge to obtain the supernatant. Load the supernatant onto a Sephadex LH-20 gel filtration column, elute with 50% methanol solution, and monitor by thin layer chromatography (TLC). Combine the fractions containing the same components, and then elute with water (A) and methanol (B) as the mobile phases, and collect the eluate.

[0021] Analyze the components of the eluate by liquid chromatography-tandem mass spectrometry (LC-MS / MS). After reduction with dithiothreitol in a 55 °C water bath, add iodoacetamide, and react in the dark at room temperature for 0.5 h for LC-MS / MS analysis. The mobile phases are solvent A (0.1% (v / v) formic acid (FA) in ultrapure water) and solvent B (0.1% (v / v) FA in acetonitrile). A total of 4 components are separated and obtained, and the polypeptide with the amino acid sequence AVFPSIVGR is the earthworm polypeptide used in the present invention.

[0022] Alternatively, according to needs, the earthworm polypeptide can be artificially synthesized by means known in the art.

[0023] 3. Mix the salvia miltiorrhiza extract and the earthworm polypeptide in the required ratio to obtain the composition. Alternatively, according to needs, the salvia miltiorrhiza extract and the earthworm polypeptide can be dissolved / dispersed separately or together in other solvents / substrates, such as being mixed with sodium acetylated hyaluronate, tranexamic acid silanol, etc., to prepare the required drugs / skin care products, etc.

[0024] The salvia miltiorrhiza extract and the earthworm polypeptide respectively have the effects of down-regulating the intracellular tyrosinase activity and reducing the melanin content, and the effect of the composition is better.

[0025] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. If not specifically specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the data obtained are the averages obtained after at least 3 repetitions, and all the repetitions obtained are valid data.

[0026] Example 1: Obtain the salvia miltiorrhiza extract, the earthworm polypeptide and the salvia miltiorrhiza-earthworm composition

[0027] 1. Obtaining Salvia miltiorrhiza extract: The dried rhizomes of Salvia miltiorrhiza were crushed and soaked in 80% edible ethanol at room temperature for 7 days, with a total of 4 extraction times. The extraction solutions were combined, filtered, and then concentrated under reduced pressure to obtain a Salvia miltiorrhiza extract paste. The paste was dispersed in distilled water, adsorbed with D101 macroporous resin for 1 h, and then eluted with distilled water, 20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol, and 100% ethanol respectively. The eluates of each part were collected and concentrated under reduced pressure to obtain the extracts (Salvia miltiorrhiza extracts) of each part, which were named as: DS-H2O, DS-20% EtOH, DS-40% EtOH, DS-60% EtOH, DS-80% EtOH, and DS-100% EtOH.

[0028] 2. Obtaining earthworm polypeptides: The dried earthworms were crushed and reflux-extracted with 0.9% sodium chloride solution (1×10 L) at 100 °C for 1 h. After filtration, 0.9% sodium chloride solution (1×8 L) was added again, and secondary extraction and reflux were carried out at 100 °C for 30 min. The filtrates were concentrated together to obtain a crude extract.

[0029] The crude extract was dissolved in 50% methanol, and the supernatant was obtained by centrifugation. The supernatant was loaded onto a Sephadex LH-20 gel filtration column (200 cm × 2.5 cm) and eluted with 50% methanol solution, monitored by thin-layer chromatography (TLC). The components containing the same ingredients were combined, and their cell activities were evaluated using the CCK-8 assay. Then, elution was carried out with ultrapure water (A) and methanol (B) as the mobile phase, and the fraction with higher neuroprotective activity was further purified using an RP-C18 column. The eluate was collected.

[0030] Using Q Exactive TM Hybrid quadrupole-orbitrap TM Mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used for liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis of the eluate components. After reduction with 10 mmol / L dithiothreitol in a 55 °C water bath for 30 min, 15 mmol / L iodoacetamide was added, and the reaction was carried out in the dark at room temperature for 0.5 h for LC-MS / MS analysis. The mobile phase was solvent A (0.1% (v / v) formic acid (FA) in ultrapure water) and solvent B (0.1% (v / v) FA in acetonitrile). The required earthworm polypeptides were separated, and their LC-MS / MS spectra were as shown in Figure 1 shown, and the amino acid sequence was: AVFPSIVGR.

[0031] 3. Obtaining the Salvia miltiorrhiza-earthworm composition: The required Salvia miltiorrhiza extract and earthworm polypeptides were mixed in the required proportions to obtain the composition.

[0032] Example 2: Safety Assessment of Salvia miltiorrhiza Extract and Earthworm Polypeptide

[0033] Human melanoma A375 cells were added to fresh DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and placed in an incubator at 37°C and 5% CO2. The cytotoxic effects of each Salvia miltiorrhiza extract and earthworm polypeptide on A375 cells were evaluated by the CCK-8 method. When the cells reached 80-90% confluence, 2 mL of trypsin was added and digested for 3-5 min. Subsequently, on a 96-well plate, plating was carried out at 1.0×10 4 cells / well and incubated in an incubator at 37°C and 5% CO2 for 24 h.

[0034] Subsequently, 100 μL of DMEM medium containing different concentrations (1, 5, 25, 50, 100, 200 μg / mL) of each Salvia miltiorrhiza extract and earthworm polypeptide was added, and incubation was continued in an incubator at 37°C and 5% CO2 for 24 h. At the same time, a positive control group was set up: 100 μL of DMEM medium containing the same concentration of α-arbutin was added; and a blank control group (control) was set up: 100 μL of DMEM medium was added; each group had 5 replicates and the results were averaged. After 24 h, 10 μL of CCK8 reagent was added to each well, reacted at 37°C for 1 h, and the absorbance value at 450 nm was measured.

[0035] Cell viability = (OD 450给药组 / OD 450空白对照组 )×100%.

[0036] The results are as Figure 2 shown. In the figure, compared with the blank control group, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001; where not otherwise specified, the meaning of * is the same as here. The results show that DS-80% EtOH, DS-100% EtOH and earthworm polypeptide (peptide) did not significantly inhibit cell viability at concentrations of 1-25 μg / mL. DS-80% EtOH and earthworm polypeptide also only showed weak cytotoxicity at 100 μg / mL. It shows that under the condition of controlling the concentration, Salvia miltiorrhiza extract and earthworm polypeptide have biological safety.

[0037] It can be seen from the results that after elution with different concentrations of ethanol, the effects of the obtained Salvia miltiorrhiza extracts on cell viability do not strictly show a dose-dependent linear relationship. Since each group was repeated 5 times, technical errors or operation mistakes and other interferences can be excluded; generally speaking, at concentrations of 25 μg / mL and below, each Salvia miltiorrhiza extract has a certain biological safety.

[0038] In addition, analysis of the components of each Danshen extract revealed that: the components of DS-80% EtOH and DS-100% EtOH were highly similar, both being tanshinone compounds; the main component in DS-20% EtOH was phenolic acid compounds. Therefore, considering factors such as cost, safety, and component composition, in subsequent examples, DS-20% EtOH and DS-80% EtOH were mainly selected as the Danshen extracts.

[0039] Example 3: Effects of Danshen extract and earthworm polypeptide on tyrosinase activity and melanin content

[0040] 1. Effects of individual substances on tyrosinase activity

[0041] A375 cells were cultured in a DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) in an incubator at 37°C and 5% CO2. They were seeded in a 6-well plate at a density of 2×10 5 cells / well. After adherent growth for 24 h, 2 mL of DMEM medium containing 1 μM α-MSH (α-melanocyte-stimulating hormone) was added. DS-20% EtOH, DS-80% EtOH, and earthworm polypeptide with final concentrations of 1, 5, and 25 μg / mL were added to each DMEM medium to treat the cells. At the same time, a model group (model), a blank control group (control), and a positive control group were set up; the model group was: adding 2 mL of DMEM medium containing 1 μM α-MSH; the blank control group was adding 2 mL of DMEM medium; the positive control group was: adding 2 mL of DMEM medium containing 1 μM α-MSH, and α-arbutin was added to the medium with final concentrations of 1, 5, and 25 μg / mL. Each group had 5 replicates, and the results were averaged.

[0042] After incubation in an incubator at 37°C and 5% CO2 for 48 h, the cells were lysed with 1% Triton X-100. After collecting the cells, 5 mM L-dopa was added and incubated at 37°C for 30 minutes, and the absorbance value at 475 nm was measured.

[0043] Relative tyrosinase activity = (OD 475给药组 / OD 475空白对照组 )×100%.

[0044] The results were as Figure 3 shown. The results showed that: compared with the blank control group, the tyrosinase activity in the model group increased extremely significantly, indicating that the model was successfully established. DS-80% EtOH and earthworm polypeptide could significantly inhibit the tyrosinase activity in A375 cells at 1 μg / mL, and showed a dose-dependent effect.

[0045] 2. Effects of individual substances on melanin content

[0046] A375 cells were cultured in DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) in an incubator at 37 °C and 5% CO2. They were incubated at a density of 1.25×10 5 cells / well in a 6-well plate for 24 h until the cells adhered. Then, 2 mL of DMEM medium containing 1 μM α-MSH was added, and DS-20% EtOH, DS-80% EtOH, and earthworm polypeptide with final concentrations of 1, 5, and 25 μg / mL were added to each DMEM medium to treat the cells.

[0047] Meanwhile, a model group (model), a blank control group (control), and a positive control group were set up. The model group was: adding 2 mL of DMEM medium containing 1 μM α-MSH; the blank control group was adding 2 mL of DMEM medium; the positive control group was: adding 2 mL of DMEM medium containing 1 μM α-MSH, and arbutin was added to the medium with final concentrations of 1, 5, and 25 μg / mL of arbutin. Each group had 5 replicates, and the results were averaged.

[0048] After incubation in the incubator for 72 h, the cells were then washed 2 - 3 times with ice-cold PBS, immediately 2 mL of trypsin was added to lyse for 3 - 5 min. After collecting the cells, the supernatant was discarded by centrifugation. 1 M NaOH and 10% DMSO (dimethyl sulfoxide) were added and heated at 80 °C for 60 min to release the intracellular melanin, and then the absorbance value of each sample was measured at 405 nm.

[0049] Melanin content = (OD 405给药组 / OD 405空白对照组 )×100%.

[0050] The results are as Figure 4 shown. The results showed that the melanin content in the model group was extremely significantly higher than that in the blank control group, indicating that the model was successfully established. Each component could significantly reduce the melanin content in A375 cells at concentrations of 5 - 25 μg / mL, and at low concentrations of 1 and 5 μg / mL, the effects were better than those of the positive control group of arbutin. It should be noted that: at a concentration of 25 μg / mL, earthworm polypeptide could almost restore the melanin content induced by α-MSH to the normal level.

[0051] 3. Effect of salvia miltiorrhiza and earthworm composition on melanin content

[0052] Based on the above results, we selected a combination of DS-80% EtOH and earthworm polypeptide. Following the methods and procedures of Steps 1 and 2, we tested the effects of this combination on α-MSH-induced tyrosinase activity and melanin content in A375 cells. Blank and model groups were also established. The results are shown in Table 1. The data from the first replicate of the blank control group is considered 100%, with slight fluctuations in the data between replicates.

[0053] Table 1 Comparison of the effects of various tested drugs on tyrosinase activity and melanin content

[0054]

[0055]

[0056] The results showed that compared with the use of Salvia miltiorrhiza extract or earthworm polypeptide alone, the mixed composition had a relatively more obvious trend of reducing tyrosinase activity and melanin content, indicating that there was a certain synergistic effect between the two, and earthworm polypeptide was dominant.

[0057] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various deformations, modifications, and substitutions made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A skin care product, characterized in that: The components of the skin care product include Salvia miltiorrhiza extract, and the Salvia miltiorrhiza extract is an ethanol extract of the rhizome of Salvia miltiorrhiza.

2. The skin care product according to claim 1, wherein: The components of the skin care product also include earthworm polypeptide, and the amino acid sequence of the earthworm polypeptide is: AVFPSIVGR.

3. The skin care product according to claim 1 or 2, characterized in that: The Salvia miltiorrhiza extract is obtained after ethanol extraction and elution. The extract obtained after ethanol extraction is concentrated under reduced pressure to obtain an extract paste, and the extract paste is dispersed in water for elution. The eluate is concentrated under reduced pressure to obtain the Salvia miltiorrhiza extract.

4. The skin care product according to claim 3, characterized in that: Elution is carried out with an 80% aqueous ethanol solution.

5. A skin care product, characterized in that: The components of the skin care product include earthworm polypeptide, and the amino acid sequence of the earthworm polypeptide is: AVFPSIVGR.

6. The skin care product according to claim 5, characterized in that: The components of the skin care product also include Salvia miltiorrhiza extract, and the Salvia miltiorrhiza extract is an ethanol extract of the rhizome of Salvia miltiorrhiza.

7. The skin care product according to claim 5, characterized in that: The Salvia miltiorrhiza extract is obtained after ethanol extraction and elution. The extract obtained after ethanol extraction is concentrated under reduced pressure to obtain an extract paste, and the extract paste is dispersed in water for elution. The eluate is concentrated under reduced pressure to obtain the Salvia miltiorrhiza extract.

8. A composition, characterized in that: The composition includes Salvia miltiorrhiza extract and earthworm polypeptide. The Salvia miltiorrhiza extract is an ethanol extract of the rhizome of Salvia miltiorrhiza, and the amino acid sequence of the earthworm polypeptide is: AVFPSIVGR.

9. An external medicine for whitening and anti-blackening prepared by using the composition according to claim 8.

10. An external medicine for whitening and anti-blackening containing the composition according to claim 8.

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