A peony anti-aging extract and its preparation method and application

Abstract

The present application proposes a peony anti-aging extract and its preparation method and application. The peony plant is ground and filtered, and the filtrate is successively subjected to gradient centrifugation with increasing speed. After the precipitate is resuspended, it is centrifuged in a gradient sucrose solution to obtain the peony anti-aging extract. The entire extraction process is physical grinding extraction and centrifugal removal, and the extract is obtained by gradient separation in a sucrose solution. No organic reagents are used, and only physical methods are used. On the basis of taking into account the safety of the extract and the environmental protection of the preparation method, the activity of the active ingredients in the plant is retained to the greatest extent possible; therefore, it is a safe and efficient method for preparing a peony anti-aging extract.

Description

Technical Field

[0001] The present application relates to the technical field of natural product extraction, and in particular to a peony anti-aging extract and a preparation method and application thereof. Background Art

[0002] Since ancient times, peony has occupied a place in traditional Chinese medicine, and its root bark (i.e., moutan bark) has been widely used in various prescriptions to treat a variety of diseases.

[0003] In recent years, with the advancement of modern science and technology and people's pursuit of healthy life, the medicinal value of peony has been studied and developed more deeply. Modern scientific research has revealed that the active ingredients such as paeonol and paeoniflorin rich in moutan bark not only have significant antioxidant effects, but also effectively inhibit inflammatory responses. Inflammatory response is an important factor in skin aging, which can cause damage and death of skin cells and accelerate the aging process of the skin. Therefore, inhibiting inflammatory response has become a key link in the development of anti-aging products.

[0004] In the field of skin care products, peony extracts have shown great application potential due to their anti-inflammatory and antioxidant properties. As people age, their skin gradually shows signs of aging, such as wrinkles and spots. These aging phenomena are often closely related to oxidative damage and inflammatory responses of skin cells. The active ingredients in peony extracts can inhibit the production of free radicals, reduce oxidative damage to skin cells, and inhibit inflammatory responses, thereby delaying the aging process of the skin. This dual mechanism of action gives peony extracts a unique advantage in the development of anti-aging products.

[0005] Therefore, as a plant resource rich in bioactive substances, peony has great application potential in the field of skin care products. The anti-inflammatory and antioxidant components in its extracts can work synergistically to effectively inhibit the inflammatory response and oxidative damage during skin aging, thereby delaying skin aging. However, the existing preparation process still needs to be improved to improve the quality and stability of the extract. Summary of the invention

[0006] The present application provides a peony anti-aging extract and a preparation method and application thereof to solve the problems existing in the related technology. The technical solution is as follows:

[0007] In a first aspect, the present invention provides a method for preparing a peony anti-aging extract, comprising the following steps:

[0008] Grind and filter the peony plant, and collect the filtrate;

[0009] The filtrate was sequentially subjected to gradient centrifugation at increasing speeds, and the precipitate was collected;

[0010] The precipitate is resuspended and centrifuged in a gradient sucrose solution; the supernatant is the peony anti-aging extract.

[0011] In one embodiment, the gradient centrifugation process with increased speed is:

[0012] Centrifuge at 2500-4000 rpm for 5-20 min, collect the supernatant; centrifuge at 9000-13000 rpm for 25-40 min, collect the supernatant; centrifuge at 18000-20000 rpm for 45-70 min; collect the precipitate.

[0013] In one embodiment, the gradient centrifugation process with increased speed is:

[0014] Centrifuge at 4000 rpm for 15 min and collect the supernatant; centrifuge at 12000 rpm for 30 min and collect the supernatant; centrifuge at 20000 rpm for 60 min and collect the precipitate.

[0015] In one embodiment, the gradient sucrose solution is a gradient sucrose solution of 3-8%, 18-22%, 36-45%, and 65-75%.

[0016] In one embodiment, the conditions for centrifugation in a gradient sucrose solution are:

[0017] Centrifuge at 15000-20000 rpm for 1.5-2.5 h; preferably, centrifuge at 18000 rpm for 120 min.

[0018] In one embodiment, the pellet is resuspended in PBS buffer.

[0019] In one embodiment, the filtrate after centrifugation in the gradient sucrose solution is freeze-dried to obtain the peony anti-aging extract.

[0020] In one embodiment, freeze drying uses a lyoprotectant, which includes one or a combination of two or more of mannitol, sorbitol or lactose.

[0021] In a second aspect, an embodiment of the present application provides a peony anti-aging extract, which is prepared by any of the above-mentioned methods for preparing the peony anti-aging extract.

[0022] In a third aspect, the embodiments of the present application provide the application of the above-mentioned peony anti-aging extract in anti-aging products.

[0023] The advantages or beneficial effects of the above technical solution include at least:

[0024] The present application provides a method for preparing a peony anti-aging extract, comprising grinding and filtering peony plants, collecting filtrate; subjecting the filtrate to gradient centrifugation with increasing speed in turn, collecting precipitate; resuspending the precipitate; and centrifuging in a gradient sucrose solution; obtaining the peony anti-aging extract. The extract is obtained by physical grinding extraction and centrifugal removal, and gradient separation in a sucrose solution. The entire extraction process does not use organic reagents, but is only carried out by physical methods. On the basis of taking into account the safety of the extract and the environmental protection of the preparation method, the activity of the active ingredients in the plant is retained to the greatest extent possible; therefore, it is a safe and efficient method for preparing a peony anti-aging extract.

[0025] The peony anti-aging extract prepared in the present application has an outstanding anti-inflammatory effect, thereby achieving an anti-aging effect. DETAILED DESCRIPTION

[0026] In the following, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present application. Therefore, the description is considered to be exemplary rather than restrictive in nature.

[0027] The core objective of the present invention is to create a method for preparing a peony anti-aging extract, aiming to obtain a peony extract with anti-aging efficacy.

[0028] Therefore, the present application embodiment provides a method for preparing a peony anti-aging extract, comprising the following steps:

[0029] Grind and filter the peony plant, and collect the filtrate;

[0030] The filtrate was sequentially subjected to gradient centrifugation at increasing speeds, and the precipitate was collected;

[0031] The precipitate is resuspended and centrifuged in a gradient sucrose solution; the supernatant is the peony anti-aging extract.

[0032] By grinding, the tissues, cell walls, etc. in the peony plant can be broken, and the cell fluid, etc., containing active ingredients, can be released. In one embodiment, fresh peony plants are used in this embodiment, which have more active ingredients and liquids. And in this embodiment, after grinding, operations such as squeezing can be combined to promote the outflow of plant liquids. The filtrate can be impurities removed by gradient centrifugation; further centrifugation in a gradient sucrose solution can separate the unused active substances in layers. Therefore, the overall extraction process does not use organic reagents, but is only carried out by physical methods. On the basis of taking into account the safety of the extract and the environmental protection of the preparation method, the activity of the active ingredients in the plant is retained to the greatest extent possible.

[0033] In one embodiment, the gradient centrifugation process with increased speed is:

[0034] Centrifuge at 2500-4000 rpm for 5-20 min, collect the supernatant; centrifuge at 9000-13000 rpm for 25-40 min, collect the supernatant; centrifuge at 18000-20000 rpm for 45-70 min; collect the precipitate.

[0035] By performing gradient centrifugation with continuously increasing centrifugal speeds, at lower speeds of 2500-4000rpm and 9000-13000rpm, substances with higher density in the filtrate after peony grinding can be precipitated to the bottom of the centrifuge tube, while the active substances are retained in the filtrate; at high-speed centrifugation of 18000-20000rpm, relatively pure liquid is subjected to high-speed centrifugation, and the active substances therein are precipitated to the bottom of the centrifuge tube, thereby obtaining a precipitate containing the active substances.

[0036] In one embodiment, the gradient centrifugation process with increased speed is:

[0037] Centrifuge at 4000 rpm for 15 min and collect the supernatant; centrifuge at 12000 rpm for 30 min and collect the supernatant; centrifuge at 20000 rpm for 60 min and collect the precipitate.

[0038] In one embodiment, the gradient sucrose solution is a gradient sucrose solution of 3-8%, 18-22%, 36-45%, 65-75%. Preferably, the gradient sucrose solution is a gradient sucrose solution of 5%, 20%, 40%, 70%.

[0039] In one embodiment, the conditions for centrifugation in a gradient sucrose solution are:

[0040] Centrifuge at 15000-20000 rpm for 1.5-2.5 h; preferably, centrifuge at 18000 rpm for 120 min.

[0041] In one embodiment, the pellet is resuspended and then centrifuged in a gradient sucrose solution:

[0042] Add sucrose solutions of different concentrations into the centrifuge tube according to the above ratio to form a density gradient with gradually increasing density;

[0043] Add the mixture to be separated to the surface of the density gradient in the centrifuge tube;

[0044] Use high-speed centrifugation to sediment the sample in the density gradient, and biomolecules of different densities will gather in different locations according to their density;

[0045] According to the sedimentation positions of molecules with different densities, the purified layer of the target molecule can be collected. This method has the advantages of strong selectivity, good separation effect, and simple operation. It can remove bacteria, apoptotic bodies, and aggregated exosomes; and the exosomes will settle in sucrose solutions of different concentrations, thereby achieving the purpose of impurity removal.

[0046] In one embodiment, the pellet is resuspended in PBS buffer.

[0047] In one embodiment, the filtrate after centrifugation in the gradient sucrose solution is freeze-dried to obtain the peony anti-aging extract.

[0048] In one embodiment, freeze drying uses a lyoprotectant, which includes one or a combination of two or more of mannitol, sorbitol or lactose.

[0049] The embodiment of the present application provides a peony anti-aging extract, which is prepared by any of the above-mentioned methods for preparing the peony anti-aging extract.

[0050] The examples of the present application provide the application of the above-mentioned peony anti-aging extract in anti-aging products.

[0051] The following is a further description with reference to a specific implementation method.

[0052] Example 1

[0053] Fresh peony plants are ground and filtered to collect the filtrate; the filtrate is centrifuged at 4000rpm for 15 minutes, and the supernatant is collected; the supernatant is further centrifuged at 12000rpm for 30 minutes, and the supernatant is collected again; the supernatant is centrifuged at 20000rpm for 60 minutes, and the precipitate is collected to remove impurities; the precipitate is resuspended in PBS and transferred to a centrifuge tube with a gradient sucrose solution of 5%, 20%, 40% and 70% respectively, and centrifuged at 18000rpm for 120 minutes to prepare the filtrate, and different layers are freeze-dried to obtain freeze-dried powder, wherein the 5% layer is the peony anti-aging extract A1, the 20% layer is the peony anti-aging extract A2, the 40% layer is the peony anti-aging extract A3, and the 70% layer is the peony anti-aging extract A4.

[0054] Example 2

[0055] Fresh peony plants are ground and filtered to collect the filtrate; the filtrate is centrifuged at 2500rpm for 20 minutes and the supernatant is collected; the supernatant is further centrifuged at 13000rpm for 25 minutes and the supernatant is collected again; the supernatant is centrifuged at 18000rpm for 70 minutes to collect the precipitate and remove impurities; the precipitate is resuspended in PBS and transferred to a centrifuge tube containing a gradient sucrose solution of 3%, 22%, 45% and 65% respectively, and centrifuged at 15000rpm for 2.5h to prepare the filtrate, and different layers are freeze-dried to obtain freeze-dried powder, wherein the 5% layer is the peony anti-aging extract B1, the 20% layer is the peony anti-aging extract B2, the 40% layer is the peony anti-aging extract B3, and the 70% layer is the peony anti-aging extract B4.

[0056] Example 3

[0057] Fresh peony plants are ground and filtered to collect the filtrate; the filtrate is centrifuged at 3500rpm for 5 minutes, and the supernatant is collected; the supernatant is further centrifuged at 9000rpm for 40 minutes, and the supernatant is collected again; the supernatant is centrifuged at 19000rpm for 45 minutes, and the precipitate is collected to remove impurities; the precipitate is resuspended in PBS and transferred to a centrifuge tube with a gradient sucrose solution of 8%, 18%, 36%, and 75% respectively, and centrifuged at 20000rpm for 90 minutes to prepare the filtrate, and different layers are freeze-dried to obtain freeze-dried powder, wherein the 5% layer is the peony anti-aging extract C1, the 20% layer is the peony anti-aging extract C2, the 40% layer is the peony anti-aging extract C3, and the 70% layer is the peony anti-aging extract C4.

[0058] Performance Testing

[0059] The peony anti-aging extract prepared in the example was subjected to an experimental study on the anti-inflammatory effect, comprising the following steps: an experiment on the peony anti-aging extract on rat skin swelling induced by 2,4-dinitrofluorobenzene (DNFB), and immunohistochemical detection of inflammatory factors TNF-α and IL-6, as follows:

[0060] 2,4-Dinitrofluorobenzene (DNFB) induced dermatitis in rats: 90 rats were randomly divided into 9 groups, 10 rats in each group, as normal group, model group, dexamethasone ointment group, peony anti-aging extract group. The peony anti-aging extract group includes peony anti-aging extract A1, peony anti-aging extract A2, peony anti-aging extract A3, peony anti-aging extract A4 in Example 1, peony anti-aging extract B3 in Example 2, and peony anti-aging extract C3 in Example 3. The rats' abdominal hair was removed with electric clippers and electric shavers, with an area of ​​about 2cm×3cm, and 20μl of 5% 2,4-dinitrofluorobenzene (DNFB) was applied to the skin for the first sensitization. On the third day, 50μl of 0.2% DNFB was applied to the shaved area on the left back for secondary sensitization, and the symmetrical shaved area on the right back was not treated with drugs as a control site. After that, the stimulation was performed once every 3 days for 3 consecutive times. On the 9th day, the left back of the rats showed more obvious erythema, edema, papules, erosion or exudation than the right back, indicating that the model was successfully established.

[0061] Starting from the 10th day, the model group, dexamethasone ointment group, and peony anti-aging extract group were given 1ml of sterile distilled water, 1ml of 50mg / Kg of dexamethasone ointment, and 1ml of 50mg / Kg of peony anti-aging extract, respectively, twice a day, once in the morning and once in the afternoon, for 9 consecutive days. On the 21st day after modeling, the animals were killed by cervical dislocation, and two pieces of skin from the left modeling area of ​​the rat's back and one piece of symmetrical healthy skin on the right were cut off with sterile surgical scissors. The first piece of modeling area skin and the right healthy skin were punched with a 6mm diameter circular puncher to punch the same area of ​​skin and weighed; the skin swelling and inhibition rate were calculated, and the results are shown in Table 1. The calculation formula is as follows:

[0062] Skin swelling degree = inflamed skin weight - healthy skin weight;

[0063] Swelling rate % = (inflamed skin weight - healthy skin weight) / healthy skin weight × 100%.

[0064] Table 1 Effects of different peony anti-aging extracts on skin swelling in rats with acute eczema

[0065]

[0066] The second piece of skin in the modeling area was placed in an EP tube treated with DEPC water and fixed in a 10% neutral formaldehyde solution for 48 hours for immunohistochemical examination. Immunohistochemical detection of TNF-α and IL-6 content The above fixed skin tissue was taken, dehydrated with alcohol step by step, immersed in wax, embedded in paraffin, and sliced ​​into 2 pieces with a thickness of 4μm. Immunohistochemical SP method was used for TNF-α and IL-6 staining respectively. Positive substance judgment standard Positive substances are brown, yellow, light yellow, granular, filamentous, and located in the cytoplasm or interstitium. TNF-α and IL-6 immunohistochemical staining sections were observed under a microscope. The distribution, range and position of positive substances were first observed at low magnification (×100), and 5 fields of view where positive substances were most obvious were selected. Then, at medium magnification (×200), the Image-ProPlus image analysis system (Media Cybernetics, Inc.) was used to semi-quantitatively detect the integrated optical density (IOD) of the positive substances in the above 5 fields of view. The sum of the integrated optical density of the positive substances in the 5 fields of view was calculated to represent the TNF-α and IL-6 content. The results are shown in Table 2.

[0067] Table 2 Effects of different peony anti-aging extracts on TNF-α and IL-6 in the skin of rats with acute eczema

[0068]

[0069]

[0070] As can be seen from Table 1, the peony anti-aging extracts prepared in the examples of the present application have a certain effect on inhibiting skin swelling. The degree of skin swelling in the model group is 44.3%, and the degree of swelling using the peony anti-aging extracts in Example 1 has decreased, especially the peony anti-aging extract A3 in Example 1, the peony anti-aging extract B3 in Example 2, and the peony anti-aging extract C3 in Example 3, the degree of inhibition of swelling is almost close to that of the dexamethasone ointment group, showing excellent anti-inflammatory effects. The peony anti-aging extracts A2 and A4 in Example 1 can also significantly inhibit swelling.

[0071] As can be seen from Table 2, compared with the model group, the TNF-α and IL-6 contents of the peony anti-aging extracts of the examples of the present application were greatly reduced. After treatment with the peony anti-aging extract A3 of Example 1, the peony anti-aging extract B3 of Example 2, and the peony anti-aging extract C3 of Example 3, the TNF-α and IL-6 contents were close to those of the dexamethasone ointment group and the normal group, while the peony anti-aging extract A1 of Example 1, the peony anti-aging extract A2, and the peony anti-aging extract A4, compared with the model group, the TNF-α content decreased by nearly half, while the IL-6 content decreased by nearly 60%.

[0072] In summary, the peony anti-aging extract prepared from the 40% centrifugal layer in the example has a significant therapeutic effect on eczema, and its mechanism may be through down-regulating the content of TNF-α and moderately regulating the content of IL-6.

[0073] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples, unless they are contradictory.

[0074] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0075] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of various changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A method for preparing a peony anti-aging extract, characterized in that: The following steps are involved: Grind and filter the peony plant, and collect the filtrate; The filtrate was sequentially subjected to gradient centrifugation at increasing speeds, and the precipitate was collected; The precipitate is resuspended; The mixture is centrifuged in a gradient sucrose solution; the supernatant is the peony anti-aging extract.

2. The method for preparing the peony anti-aging extract according to claim 1, characterized in that: The process of gradient centrifugation with increasing speed is: Centrifuge at 2500-4000 rpm for 5-20 min, collect the supernatant; centrifuge at 9000-13000 rpm for 25-40 min, collect the supernatant; centrifuge at 18000-20000 rpm for 45-70 min; collect the precipitate.

3. The method for preparing a peony anti-aging extract according to claim 1, characterized in that: The process of gradient centrifugation with increasing speed is: Centrifuge at 4000 rpm for 15 min and collect the supernatant; centrifuge at 12000 rpm for 30 min and collect the supernatant; centrifuge at 20000 rpm for 60 min and collect the precipitate.

4. The method for preparing the peony anti-aging extract according to claim 1, characterized in that: The gradient sucrose solution is a gradient sucrose solution of 3-8%, 18-22%, 36-45%, and 65-75%.

5. The method for preparing the peony anti-aging extract according to claim 1, characterized in that: The conditions for centrifugation in a gradient sucrose solution are: Centrifuge at 15000-20000 rpm for 1.5-2.5 h; preferably, centrifuge at 18000 rpm for 120 min.

6. The method for preparing the peony anti-aging extract according to claim 1, characterized in that: The pellet was resuspended in PBS buffer.

7. The method for preparing the peony anti-aging extract according to claim 1, characterized in that: The filtrate after centrifugation in the gradient sucrose solution is freeze-dried to obtain the peony anti-aging extract.

8. The method for preparing the peony anti-aging extract according to claim 7, characterized in that: The freeze drying uses a lyoprotectant, which includes one or a combination of two or more of mannitol, sorbitol or lactose.

9. A peony anti-aging extract, characterized in that: The peony anti-aging extract is prepared by the preparation method of the peony anti-aging extract according to any one of claims 1-8.

10. Use of the peony anti-aging extract according to claim 9 in anti-aging products.

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