阅读说明：本技术 一种抗衰老调控蛋白植物提取激活剂及其制备方法 (Anti-aging regulatory protein plant extraction activator and preparation method thereof ) 是由 刘玉亮 苏荣生 邓静 吴晓璇 于 2021-08-12 设计创作，主要内容包括：本发明属提供了一种抗衰老调控蛋白植物提取激活剂及其制备方法,抗衰老调控蛋白植物提取激活剂的原料主要包括丹参植物体和有机溶剂；制备方法至少包含以下几步：(1)一次回流提取；(2)二次回流提取；(3)回收干燥；(4)混合抽滤；(5)减压浓缩；(6)粉剂合并。本申请制备的Sirtuin蛋白植物提取激活剂具有良好的Sirtuin-3激发活性,细胞协助抗氧化衰老性,并且有效的进一步增强了激发后的Sirtuin-3活性,适宜在植物提取物领域推广,具有广阔的发展前景。(The invention provides an anti-aging regulatory protein plant extraction activator and a preparation method thereof, wherein the raw materials of the anti-aging regulatory protein plant extraction activator mainly comprise a salvia miltiorrhiza plant body and an organic solvent; the preparation method at least comprises the following steps: (1) carrying out primary reflux extraction; (2) secondary reflux extraction; (3) recovering and drying; (4) mixing and filtering; (5) concentrating under reduced pressure; (6) and (5) combining the powder. The Sirtuin protein plant extraction activator prepared by the application has good Sirtuin-3 excitation activity, helps the cells to resist oxidation and aging, effectively further enhances the Sirtuin-3 activity after excitation, is suitable for popularization in the field of plant extracts, and has wide development prospect.)

1. An anti-aging regulatory protein plant extraction activator, which is characterized in that: the raw materials at least comprise the following components: red sage root, organic solvent.

2. The anti-aging regulatory protein plant extract activator of claim 1, wherein: the Saviae Miltiorrhizae radix plant is at least one of root and leaf of Saviae Miltiorrhizae radix.

3. The anti-aging regulatory protein plant extract activator according to any one of claims 1 to 2, characterized in that: the organic solvent is at least one of methanol, ethanol, petroleum ether, ethyl acetate, chloroform, ethylene glycol, propylene glycol and diethylene glycol.

4. A method for preparing the anti-aging regulatory protein plant extract activator according to any one of claims 1 to 3, wherein the method comprises the steps of: the steps at least comprise the following steps: (1) carrying out primary reflux extraction; (2) secondary reflux extraction; (3) recovering and drying; (4) mixing and filtering; (5) concentrating under reduced pressure; (6) and (5) combining the powder.

5. The method of preparing the anti-aging regulatory protein plant extract activator of claim 4, wherein the method comprises the steps of: the specific operation of the primary reflux extraction is as follows: cutting and grinding the salvia miltiorrhiza plant body to prepare salvia miltiorrhiza coarse powder; and then placing the red sage root coarse powder into an extraction tank, adding an organic solvent, heating to 40-80 ℃, carrying out reflux reaction for 0.5-2 hours, and carrying out suction filtration to obtain a filtrate I and a residue I.

6. The method of preparing the anti-aging regulatory protein plant extract activator of claim 5, wherein the method comprises the steps of: the mass ratio of the organic solvent to the red sage root coarse powder is 7-10: 1.

7. The method for preparing the anti-aging regulatory protein plant extract activator according to any one of claims 5 to 6, wherein the method comprises the steps of: the specific operation of the secondary reflux extraction is as follows: and adding the residue I in the extraction tank into the organic solvent again, heating to 40-80 ℃, performing reflux reaction for 0.5-1 hour, and performing suction filtration to obtain a filtrate II and a residue II.

8. The method of preparing the anti-aging regulatory protein plant extract activator of claim 7, wherein: the mass ratio of the organic solvent to the residue I is 4-6: 1.

9. The method for preparing the anti-aging regulatory protein plant extract activator according to any one of claims 7 to 8, wherein the method comprises the steps of: the concrete operation of mixing and suction filtration is as follows: taking out the residue II, volatilizing residual organic solvent, placing the residue II into a new extraction tank, adding hydrochloric acid buffer solution with the pH value of 4-5, heating at 140 ℃ for 3-6 hours, and finally performing suction filtration to obtain filtrate III.

10. The method of preparing an anti-aging regulatory protein plant extract activator of claim 9, wherein: the mass ratio of the hydrochloric acid buffer solution to the residue II is 10-15: 1.

Technical Field

The invention relates to the field of A23L33/105, in particular to an anti-aging regulatory protein plant extraction activator and a preparation method thereof.

Background

Since the discovery of the Sirtuin family of proteins, it has gradually become an important class of Sirtuin proteins that are important in anti-aging, stress resistance and regulation of human metabolism. Among them, the Sirtuin proteins which have been widely studied and used include three types, Sirtuin-3, Sirtuin-4 and Sirtuin-5, which are mainly present in mammalian mitochondria and participate in the regulation of metabolic processes in the human body. In particular, endogenous Sirtuin-3 is a soluble protein located in the mitochondrial matrix of cells and has been well documented in recent years to have excellent skin antiaging and disease treatment assisting effects. In particular, Sirtuin-3 has more excellent repairing and inhibiting effects on skin aging caused by oxidative stress. In actual use, the Sirtuin protein can show more effect under the activation of an activator.

The prior art (CN201510268673.X) provides a Sirt-1 activator and an application thereof, wherein the main components of the Sirt-1 activator are resveratrol and the like, and the resveratrol serving as a common activator in the field of Sirtuin proteins has poor performance in promoting the expression level and oxidation resistance of the Sirtuin proteins.

Therefore, there is a need for a plant extract activator and a product thereof that can effectively increase the expression level of Sirtuin proteins and the antioxidant activity.

Disclosure of Invention

In order to solve the above problems, the present invention provides, in a first aspect, an anti-aging regulatory protein plant extract activator, characterized in that: the raw materials at least comprise the following components: red sage root, organic solvent.

In a preferred embodiment, the salvia miltiorrhiza plant is at least one of roots and leaves of salvia miltiorrhiza.

In a preferred embodiment, the organic solvent is at least one of methanol, ethanol, petroleum ether, ethyl acetate, chloroform, ethylene glycol, propylene glycol, and diethylene glycol.

As a more preferred embodiment, the organic solvent is ethanol.

As a most preferred embodiment, the organic solvent is an ethanol solution with a mass concentration of 90%.

The second aspect of the present invention provides a method for preparing the anti-aging regulatory protein plant extraction activator, which at least comprises the following steps: (1) carrying out primary reflux extraction; (2) secondary reflux extraction; (3) recovering and drying; (4) mixing and filtering; (5) concentrating under reduced pressure; (6) and (5) combining the powder.

As a preferred scheme, the specific operation of the primary reflux extraction is as follows: cutting and grinding the salvia miltiorrhiza plant body to prepare salvia miltiorrhiza coarse powder; and then placing the red sage root coarse powder into an extraction tank, adding an organic solvent, heating to 40-80 ℃, carrying out reflux reaction for 0.5-2 hours, and carrying out suction filtration to obtain a filtrate I and a residue I.

As a preferable scheme, the mass ratio of the organic solvent to the red sage root coarse powder is 7-10: 1.

As a more preferable scheme, the mass ratio of the organic solvent to the red sage root coarse powder is 8: 1.

As a preferred scheme, the secondary reflux extraction specifically comprises the following operations: and adding the residue I in the extraction tank into the organic solvent again, heating to 40-80 ℃, performing reflux reaction for 0.5-1 hour, and performing suction filtration to obtain a filtrate II and a residue II.

Preferably, the mass ratio of the organic solvent to the residue I is 4-6: 1.

In a more preferred embodiment, the mass ratio of the organic solvent to the residue I is 5: 1.

As a preferred scheme, the specific operations of recovering and drying are as follows: mixing filtrate I and filtrate II, recovering organic matter under reduced pressure, and vacuum drying to obtain powder extract I.

As a preferred scheme, the specific operations of the mixing and suction filtration are as follows: taking out the residue II, volatilizing residual organic solvent, placing the residue II into a new extraction tank, adding hydrochloric acid buffer solution with the pH value of 4-5, heating at 140 ℃ for 3-6 hours, and finally performing suction filtration to obtain filtrate III.

Preferably, the mass ratio of the hydrochloric acid buffer solution to the residue II is 10-15: 1.

More preferably, the mass ratio of the hydrochloric acid buffer solution to the residue II is 12: 1.

According to the method, the hydrochloric acid buffer solution with the pH value of 4-5 is added, and the mass ratio of the hydrochloric acid buffer solution to the residue is controlled to be 12:1, so that the extraction purity of the active substances of the salvia miltiorrhiza bunge is effectively improved, and the activation activity of Sirtuin-3 of the extraction activator is finally improved. The applicant speculates that: the hydrochloric acid buffer solution in a weakly acidic environment can ensure that the hydrochloric acid buffer solution can dissolve strong insoluble organic substances, and simultaneously, the required active substances are released in the filter residue in an unlocking manner, so that the impurity content in the filtrate is reduced. And on the other hand, the existence of a proper amount of hydrochloric acid buffer solution can also form a positive ion band in the filtrate, so that colloidal molecular chains adsorbing series negative ion impurities are agglomerated in series, the filtration and purification are easier, and the extraction efficiency is improved.

As a preferable scheme, the vacuum concentration is specifically performed by: concentrating the filtrate III under reduced pressure, and vacuum drying to obtain powder extract II.

As a preferred scheme, the powder combining specifically comprises the following operations: mixing the powdery extract I and the powdery extract II, stirring and combining.

The third aspect of the application also provides a skin external preparation containing the anti-aging regulatory protein plant extract activator, and the raw materials at least comprise the anti-aging regulatory protein plant extract activator, dihydric alcohol, plant extract, humectant, essence and deionized water for supplementing the balance.

As a preferable scheme, the anti-aging regulatory protein plant extraction activator has a mass content of 0.001-20%.

As a more preferable scheme, the anti-aging regulatory protein plant extraction activator has a mass content of 1-20%.

As a most preferred scheme, the skin external preparation containing the anti-aging regulatory protein plant extraction activator comprises the following raw materials in percentage by mass: 5% of anti-aging regulatory protein plant extraction activator, 5% of butanediol, 2% of tremella extract, 1% of glycerol, 0.1% of dipotassium glycyrrhizinate, 0.5% of aloe barbadensis leaf juice, 0.5% of decursia decursiva root extract, 0.3% of caprylyl glycol, 0.3% of phenoxyethanol, 0.02% of ethylhexyl glycerol, 0.1-2% of triethanolamine, 0.1-1% of essence and the balance of deionized water.

Has the advantages that:

1. the anti-aging regulatory protein plant extraction activator prepared in the application has better Sirtuin protein activation capability than the traditional activator, particularly has the Sirtuin-3 activation capability compared with the activator in the prior art, effectively stimulates the relative expression capability of Sirtuin-3, and has excellent anti-aging activity.

2. The anti-aging regulatory protein plant extraction activator prepared by the application has the advantages of simple operation method, mild and pollution-free extraction reaction conditions, high utilization rate and extraction rate of plant components, and excellent extraction effect of active ingredients of salvia miltiorrhiza.

3. The anti-aging regulatory protein plant extraction activator prepared by the application can also be effectively applied to an external preparation for inhibiting skin aging caused by ultraviolet-induced oxidative stress, and can effectively stimulate Sirtuin-3 activity of skin cells, so that the anti-aging capability of the external skin preparation is obviously improved.

Drawings

FIG. 1 is a graph of relative expression level test data of Sirtuin-3 protein in Salvia miltiorrhiza extract activator, resveratrol activator and blank control groups prepared in example 1 of the present invention.

FIG. 2 is a data diagram of ROS content testing of the activators of Salvia miltiorrhiza Bunge, resveratrol activators and placebo prepared in example 1 of the present invention.

Fig. 3 is a data diagram of relative expression level test data of SOD2 protein in the salvia miltiorrhiza extract activator, resveratrol activator and blank control group prepared in example 1 of the invention.

Detailed Description

The contents of the present invention can be more easily understood by referring to the following preferred embodiments of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

Examples

The technical solution of the present invention is described in detail by the following examples, but the scope of the present invention is not limited to all of the examples. The starting materials of the present invention are all commercially available unless otherwise specified.

Example 1

In a first aspect, embodiment 1 provides an anti-aging regulatory protein plant extract activator, the raw materials include root of salvia miltiorrhiza and 90% ethanol solvent by mass concentration.

In a second aspect of this embodiment, a method for preparing the anti-aging regulatory protein plant extract activator comprises the steps of: (1) primary reflux extraction: cutting root of Saviae Miltiorrhizae radix, and grinding to obtain Saviae Miltiorrhizae radix coarse powder; then placing the red sage root coarse powder into an extraction tank, adding an ethanol solvent, heating to 70 ℃, carrying out reflux reaction for 1 hour, and carrying out suction filtration to obtain filtrate I and residue I; (2) secondary reflux extraction: adding the residue I in the extraction tank into the ethanol solvent again, heating to 60 ℃, performing reflux reaction for 40 minutes, and performing suction filtration to obtain filtrate II and residue II; (3) and (3) recovering and drying: uniformly mixing the filtrate I and the filtrate II, recovering organic matters under reduced pressure, and drying in vacuum to obtain a powder extract I; (4) mixing and suction filtering: taking out the residue II, volatilizing residual ethanol solvent, placing into a new extraction tank, adding hydrochloric acid buffer solution with pH of 5, heating at 140 deg.C for 4 hr, and vacuum filtering to obtain filtrate III; (5) and (3) concentrating under reduced pressure: concentrating the filtrate III under reduced pressure, and vacuum drying to obtain powder extract II; (6) powder merging: mixing the powdery extract I and the powdery extract II, stirring and combining.

In this example, the mass ratio of the ethanol solvent to the salvia miltiorrhiza raw powder material is 8: 1.

In this example, the mass ratio of the ethanol solvent to the residue I was 5: 1.

In this example, the mass ratio of the hydrochloric acid buffer solution to the residue II was 12: 1.

In this embodiment, the third aspect further provides a skin external preparation containing the anti-aging regulatory protein plant extract activator, which comprises the following raw materials by mass: 5% of anti-aging regulatory protein plant extraction activator, 5% of butanediol, 2% of tremella extract, 1% of glycerol, 0.1% of dipotassium glycyrrhizinate, 0.5% of aloe barbadensis leaf juice, 0.5% of decursia decursiva root extract, 0.3% of caprylyl glycol, 0.3% of phenoxyethanol, 0.02% of ethylhexyl glycerol, 0.5% of triethanolamine, 0.2% of lavender essence and the balance of deionized water.

In this embodiment, the tremella extract is a tremella extract product sold by wales biotechnology limited, lanzhou.

In this embodiment, the decursia violacea extract is sold by schannsinsett biotechnology limited in shanxi.

In this embodiment, the lavender essence is a lavender essence product sold by shanxi morning-ming biotechnology limited company.

In this example, the aloe vera leaf juice is an aloe vera leaf juice product sold by Shandong cosmetics Biotech limited.

Example 2

The embodiment of the present invention is different from embodiment 1 in that: adding hydrochloric acid buffer solution with pH of 6.5 into the step (4)

Example 3

The embodiment of the present invention is different from embodiment 1 in that: the mass ratio of the ethanol solvent to the red sage root coarse powder is 7: 1.

Comparative example 1

The embodiment of this comparative example is the same as example 1 except that: adding hydrochloric acid buffer solution with pH of 2 in the step (4).

Comparative example 2

The embodiment of this comparative example is the same as example 1 except that: the mass ratio of the hydrochloric acid buffer solution to the residue II was 6: 1.

Evaluation of Performance

1. Relative protein expression: human umbilical vein endothelial cells (Wuhan Punuo Sai) were seeded on collagen-coated 6-well microplate at a density of 1X 10⁶Individual cells/well, cultured until cells fuse to 80%. Then, the cells were incubated in the activator for plant extraction prepared in examples or comparative examples for 48 hours, proteins were recovered using PhosphoSafeExtraction reagent (derived from Novagen), protein concentrations were measured using BCA protein quantification kit, and the samples were adjusted so that the protein concentrations were equivalent, 1-antibody using anti-Sirtuin-3 (Baeyer), anti-beta actin (Boshide biosciences), and 2-antibody using anti-r, respectivelyThe abbit, anti-mouse were evaluated by western blotting (western blot), 5 specimens were tested for each example comparative example, and the average value of the measured values is shown in Table 1.

ROS content: and detecting the intracellular ROS level by adopting a DCFH-DA method of an active oxygen detection kit. Human umbilical vein endothelial cells (wuhan kino) were seeded in 6-well plates and incubated for 48 hours in the presence of the plant extract activator prepared in the examples or comparative examples, following the ROS detection kit (from merck) instructions. The cells were resuspended in DCFH-DA at a final concentration of 10. mu. mol/L, incubated at 37 ℃ for 20 minutes in a cell culture chamber, washed 3 times with serum-free cell culture medium, and transferred to a flow cytometer for measuring the mean fluorescence intensity, 5 samples were tested for each example comparative example, and the mean of the values measured is reported in Table 1.

SOD2 activity: the activity of SOD2 in cells was detected by WST-8 method. Human umbilical vein endothelial cells (wuhan ponuosai) were seeded in 6-well plates, and after incubation in medium containing salvia miltiorrhiza extract a, salvia miltiorrhiza extract B, resveratrol (EBM2), pre-cooled PBS washed 2 times. Collecting cells, adding precooled PBS for homogenizing, taking homogenate, centrifuging at 4 ℃, and collecting supernatant. Protein quantification is carried out by a BCA method for later use. WST-8/enzyme solution and reaction starting solution are prepared according to the instruction of a CuZn/MnSOD activity detection kit (WST-8 method, from Biyuntian). mu.L of the sample was added with 160. mu.L of the enzyme solution and 20. mu.L of the reaction starting solution in this order, and incubated at 37 ℃ for 30 minutes. The absorbance was measured at a wavelength of 450nm, 5 samples were tested for each example comparative example, and the average of the measured values is shown in Table 1.

As shown in fig. 1 to 3, the processing method comprises: human umbilical vein endothelial cells (wuhan kino sai) were seeded on collagen-coated 6-well microplates, cultured to sub-confluency, grouped according to different treatment conditions: control group (30 mJ/cm)²Ultraviolet irradiation), Saviae Miltiorrhizae radix extract group A (pretreated with 10 μmol/L Saviae Miltiorrhizae radix extract for 30 min, and 30mJ/cm²Ultraviolet irradiation), Saviae Miltiorrhizae radix extract B group (30mJ/cm after pretreatment of 20 μmol/L Saviae Miltiorrhizae radix extract for 30 min²Ultraviolet irradiation), resveratrol (5. mu.M after 30 minutes of pretreatment, 30mJ/cm²Ultraviolet irradiation).

TABLE 1

Through the examples 1-3, the comparative examples 1-2 and the table 1, it can be known that the anti-aging regulatory protein plant extraction activator and the preparation method thereof provided by the invention have good Sirtuin-3 stimulating activity, assist the anti-oxidation and anti-aging performance of cells, effectively and further enhance the stimulated Sirtuin-3 activity, are suitable for popularization in the field of plant extracts, and have wide development prospects. Wherein, the example 1 obtains the best performance index under the factors of the best preparation raw material proportion, the best preparation process and the like.