Cistanche deserticola polysaccharide as well as extraction method and application thereof

Abstract

The invention discloses cistanche deserticola polysaccharide and an extraction method and application thereof, and belongs to the technical field of cistanche deserticola polysaccharide extraction.The extraction method comprises the following steps that 1, coarse cistanche deserticola powder is subjected to ethyl acetate extraction three times and ethanol extraction three times in sequence and filtered, and degreased cistanche deserticola powder is obtained; (2) adding papain, cellulase and pectinase into the degreased cistanche powder for enzymolysis; and (3) after the step (2), carrying out microwave extraction, centrifuging, taking supernate, concentrating, then adding ethanol into the concentrated solution, standing, centrifuging to obtain a precipitate, and freeze-drying the precipitate. The extraction efficiency and the extraction quality of the cistanche polysaccharide can be improved by adopting an enzymolysis method in cooperation with a microwave method; and moreover, energy is saved, the safety and the stability are improved, and great popularization value is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of Cistanche polysaccharide extraction, and in particular relates to Cistanche polysaccharide and an extraction method and application thereof. Background Art

[0002] Cistanche deserticola Ma, also known as Xinjiang Yun, Dayun (Light Dayun), Cunyun, Cistanche (Sweet Cistanche, Light Cistanche), Dijing, Chagan Gaoya (Mongolian), belongs to the Orobanchaceae family, Cistanche genus. Cistanche is a parasitic plant that parasitizes on the roots of the desert tree Haloxylon ammodendron, absorbing nutrients and water from the Haloxylon ammodendron host. It is known as the "desert ginseng" and is also called "desert Cistanche". It has extremely high medicinal value and is a traditional precious Chinese medicinal material. In history, Cistanche was used as a treasure to pay tribute to the court by the Western Regions. It is also one of the most frequently used tonic medicines in the prescriptions of kidney-tonifying and aphrodisiac in the past dynasties. It is distributed in Inner Mongolia, Shaanxi, Gansu, Ningxia, Xinjiang and other places.

[0003] Cistanche plants have a variety of biological activities, such as kidney-tonifying and yang-strengthening, anti-aging, anti-oxidation, anti-fatigue, diuretic and laxative, sedative and analgesic, and memory-enhancing effects. Among them, polysaccharides are one of the important active ingredients of Cistanche plants, and therefore, the extraction technology of Cistanche polysaccharides is of vital importance. At present, the extraction methods of Cistanche polysaccharides are mainly traditional leaching, microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE) and enzymatic extraction (EE). The above 4 extraction methods of Cistanche polysaccharides, microwave-assisted extraction, ultrasound extraction and enzymatic extraction are all new extraction technologies, but the extraction rate of Cistanche polysaccharides, the loss of effective ingredients, the process steps and the extraction time, etc., need to be further optimized.

[0004] Therefore, there is an urgent need to provide a method for extracting Cistanche polysaccharide that can improve the extraction rate of Cistanche polysaccharide, shorten the extraction time and simplify the extraction process steps. Summary of the invention

[0005] In view of the above technical problems, the present invention provides a Cistanche deserticola polysaccharide and an extraction method and application thereof.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] One of the technical solutions of the present invention:

[0008] A method for extracting Cistanche deserticola polysaccharide comprises the following steps:

[0009] (1) extracting the crude powder of Cistanche deserticola with ethyl acetate and ethanol in sequence, collecting the residue to obtain defatted Cistanche deserticola powder;

[0010] (2) adding papain, cellulase, pectinase and water to the defatted Cistanche deserticola powder for enzymolysis;

[0011] (3) After step (2), microwave extraction is performed, the supernatant is centrifuged and concentrated, and then ethanol is added to the concentrate, the mixture is allowed to stand, and centrifuged to obtain a precipitate, which is then freeze-dried.

[0012] Preferably, the specific process of degreasing in step (1) is:

[0013] The Cistanche decoction pieces are dried and crushed to obtain Cistanche coarse powder, and 300-1000mL, 300-1000mL and 300-1000mL of ethyl acetate are sequentially added to the Cistanche coarse powder based on 100g of the Cistanche coarse powder, and the mixture is extracted for 3 times, each time for 2 hours; the mixture is filtered and the medicinal residue is collected; 300-1000mL, 300-1000mL and 300-1000mL of 95% ethanol are sequentially added to the medicinal residue for extraction for 3 times, each time for 2 hours, the mixture is filtered and the medicinal residue is collected to obtain defatted Cistanche powder.

[0014] Preferably, the material-liquid ratio in the enzymatic hydrolysis process in step (2) is 1:(20-25); wherein the amount of liquid in the material-liquid ratio refers to the amount of water added during the enzymatic hydrolysis and extraction of Cistanche polysaccharide; the material is the raw material defatted Cistanche powder; and the material-liquid ratio is a mass ratio.

[0015] Preferably, in step (2), the added amounts of papain, cellulase and pectinase are 1-2%, 1-2% and 0.5-0.8% respectively.

[0016] Preferably, the amount of papain added is 2%, the amount of cellulase added is 2%, and the amount of pectinase added is 0.8%, wherein the amount of each enzyme added is the mass percentage of the whole.

[0017] Preferably, the conditions for enzymatic hydrolysis in step (2) are:

[0018] Temperature 55℃, pH 5, enzymatic hydrolysis 1h.

[0019] Preferably, the parameters during the microwave treatment in step (3) are:

[0020] The microwave power is 500-800W and the microwave time is 3-4min.

[0021] Preferably, the amount of ethanol added is such that the ethanol concentration reaches 70%.

[0022] The second technical solution of the present invention:

[0023] The application of the Cistanche deserticola polysaccharide extracted by the above extraction method in anti-aging cosmetics.

[0024] Compared with the prior art, the present invention uses enzymatic hydrolysis combined with microwave method to extract Cistanche deserticola polysaccharide, which has the following advantages:

[0025] 1. Improve extraction efficiency: Enzymatic hydrolysis synergistic microwave extraction can accelerate the extraction process, improve extraction efficiency, shorten extraction time, and thus save production costs.

[0026] 2. Improve extraction quality: Enzymatic hydrolysis combined with microwave extraction can improve the quality of the extract, make the extract purer, and reduce impurity contamination.

[0027] 3. Save energy: Enzymatic hydrolysis combined with microwave extraction can reduce energy consumption and lower production costs.

[0028] 4. Improve safety: Enzymatic hydrolysis and microwave extraction can avoid the safety risks brought by high temperature and high pressure operations and improve production safety.

[0029] 5. Improve stability: Enzymatic hydrolysis combined with microwave extraction can make the extract more stable, extend the shelf life and improve product quality.

[0030] In summary, the present invention adopts the enzymatic hydrolysis method in coordination with the microwave method to improve the extraction rate and extraction quality of Cistanche deserticola polysaccharide; and further saves energy, improves safety and stability, and has great promotion value. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] The "room temperature" in the present invention refers to 20-30°C unless otherwise specified.

[0037] The "parts" described in the present invention, unless otherwise specified, refer to parts by mass.

[0038] The raw materials used in the present invention are all purchased from the market; the enzyme activities and manufacturers used in the following examples are:

[0039]

[0040] The technical solution of the present invention is further illustrated by the following embodiments.

[0041] Example 1

[0042] A method for extracting Cistanche deserticola polysaccharide comprises the following steps:

[0043] (1) Drying and crushing the Cistanche deserticola slices to obtain Cistanche deserticola powder, adding 300 mL of ethyl acetate to the Cistanche deserticola powder for extraction based on 100 g of Cistanche deserticola powder, and repeating the extraction three times, each time for 2 hours; filtering and collecting the medicinal residue; adding 300 mL of 95% ethanol to the medicinal residue for extraction, and repeating the extraction three times, each time for 2 hours, filtering and collecting the medicinal residue to obtain defatted Cistanche deserticola powder;

[0044] (2) adding complex enzyme and water to the defatted Cistanche deserticola powder, and performing enzymolysis at 55°C and pH 5 for 1 h;

[0045] The material-liquid ratio during the enzymatic hydrolysis process was 1:20;

[0046] The addition amounts of papain, cellulase, and pectinase were 2%, 2%, and 0.8%, respectively;

[0047] (3) After step (2), microwave treatment was performed at a microwave power of 800 W for 4 min of microwave extraction. The supernatant was centrifuged and concentrated. Then, ethanol was added to the concentrate until the ethanol concentration was 70%. The concentrate was allowed to stand and centrifuged to obtain a precipitate, which was freeze-dried.

[0048] Example 2

[0049] The difference from Example 1 is that

[0050] In step (2), the added amounts of papain, cellulase and pectinase are 1.5%, 1.5% and 0.6% respectively.

[0051] Example 3

[0052] The difference from Example 1 is that

[0053] In step (2), the added amounts of papain, cellulase and pectinase are 1%, 1% and 0.5% respectively.

[0054] Example 4

[0055] The difference from Example 1 is that

[0056] The material-liquid ratio in the enzymatic hydrolysis process in step (2) is 1:22.

[0057] Example 5

[0058] The difference from Example 1 is that

[0059] The material-liquid ratio in the enzymatic hydrolysis process in step (2) is 1:25.

[0060] Example 6

[0061] The difference from Example 1 is that

[0062] In step (3), the microwave power is 600 W, and microwave extraction is performed for 4 minutes.

[0063] Example 7

[0064] The difference from Example 1 is that

[0065] In step (3), the microwave power is 500 W, and microwave extraction is performed for 4 minutes.

[0066] Comparative Example 1

[0067] The difference from Example 1 is that

[0068] Only enzymatic hydrolysis was used, i.e., the microwave treatment in step (3) of Example 1 was not performed.

[0069] Comparative Example 2

[0070] The difference from Example 1 is that

[0071] Only the microwave method was used, i.e. step (2) of Example 1 was not performed.

[0072] The extraction rates of Cistanche deserticola polysaccharides obtained by the extraction methods of Examples 1-7 and Comparative Examples 1-2 are shown in Table 1:

[0073] Table 1

[0074] Extraction rate / % Example 1 68 Example 2 65 Example 3 66 Example 4 63 Example 5 65 Example 6 63 Example 7 66 Comparative Example 1 35 Comparative Example 2 38

[0075] It can be seen from the extraction rates in Table 1 that, compared with comparative examples 1 and 2 in which enzymatic hydrolysis and microwave methods are used alone to extract Cistanche polysaccharide, the present invention uses enzymatic hydrolysis in conjunction with microwave method to significantly improve the extraction rate of Cistanche polysaccharide.

[0076] Effect verification

[0077] The Cistanche deserticola polysaccharide extracted in Example 1 was used to verify its anti-aging effect.

[0078] XTT assay for keratinocyte activity

[0079] 1) Experimental principle

[0080] The thickness of the epidermis of aged skin becomes significantly thinner, the division rate of the main component cells, keratinocytes, slows down, and the metabolism level also decreases significantly, causing the skin to lose vitality, become dull, have more fine lines, and lose moisture quickly. Therefore, increasing the metabolism and proliferation capacity of epidermal cells has become one of the important means to delay aging.

[0081] Mitochondria are the site of cellular oxidative metabolism, and their activity can directly reflect the metabolic capacity of cells. The present invention detects the mitochondrial activity of keratinocytes by the XTT method, thereby reflecting the metabolic level of cells. XTT is a tetrazole nitrogen derivative that can be degraded by mitochondrial dehydrogenase in living cells to produce brown-yellow water-soluble formazan, which has an absorbance at 450nm, and the amount of formazan generated corresponding to its reading is positively correlated with the mitochondrial activity of cells. In the in vitro cell platform of the laboratory, the increase in the number of mitochondria caused by cell proliferation can also cause an increase in the XTT result. That is, the XTT experimental results involved in the present invention reflect the comprehensive results of the cell metabolism level and proliferation capacity.

[0082] 2) Experimental methods

[0083] Add 50 μL of cell culture medium containing 0.2% growth factor to each well of a 96-well plate, and then add 150 μL of fibroblast suspension with a spray gun. The cell inoculation amount is 8500 cells / well, and the cells are placed in a 37°C, 5% CO2 incubator for 24 hours. Aspirate 150 μL of culture medium, and then add 150 μL / well of culture medium containing Cistanche polysaccharide to make the final concentrations 0.1 mg / mL and 1 mg / mL, respectively. All drug concentrations have been tested for cytotoxicity and have no toxic effect on fibroblasts at this cell density. After continuing to culture for 48 hours, replace the culture medium again according to the method described above, aspirate the culture medium after 24 hours, wash with PBS, add 125 μL of 0.2 mg / mL XTT reaction solution (containing 0.1% PMS, Sigma), and then culture for another 2 hours before measuring the absorbance at 450nm on an enzyme reader.

[0084] 3) Experimental results

[0085] The experiment showed that the corresponding readings (cell activity) of the experimental group cultured with 1mg / mL Cistanche polysaccharide and the experimental group cultured with 0.1mg / mL Cistanche polysaccharide were significantly higher than those of the blank control group. In other words, the amount of XTT degraded by mitochondria in cells treated with Cistanche polysaccharide was significantly greater, proving that its cell activity (comprehensive result of metabolic level and proliferation ability) was strong, and Cistanche polysaccharide could effectively delay aging.

[0086] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application 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 extracting Cistanche deserticola polysaccharide, characterized in that: The following steps are involved: (1) extracting the Cistanche deserticola powder three times with ethyl acetate and three times with ethanol, filtering, and obtaining defatted Cistanche deserticola powder; (2) adding papain, cellulase, pectinase and water to the defatted Cistanche deserticola powder for enzymolysis; (3) After step (2), microwave extraction is performed, the supernatant is centrifuged and concentrated, and then ethanol is added to the concentrate, allowed to stand, centrifuged, and freeze-dried.

2. The method for extracting Cistanche deserticola polysaccharide according to claim 1, characterized in that: In step (1), the ratio of the crude powder of Cistanche deserticola to ethyl acetate or ethanol is 100 g: 300-1000 mL; Each extraction lasted 2 h.

3. The method for extracting Cistanche deserticola polysaccharide according to claim 1, characterized in that: The material-liquid ratio in the enzymatic hydrolysis process in step (2) is 1:(20-25).

4. The method for extracting Cistanche deserticola polysaccharide according to claim 1, characterized in that: In step (2), the added amounts of papain, cellulase and pectinase are 1-2%, 1-2% and 0.5-0.8% respectively.

5. The method for extracting Cistanche deserticola polysaccharide according to claim 4, characterized in that: In step (2), the amount of papain added is 2%; the amount of cellulase added is 2%; and the amount of pectinase added is 0.8%.

6. The method for extracting Cistanche deserticola polysaccharide according to claim 1, characterized in that: The conditions for enzymolysis in step (2) are: Temperature 55℃, pH 5, enzymatic hydrolysis 1h.

7. The method for extracting Cistanche deserticola polysaccharide according to claim 1, characterized in that: The parameters in the microwave treatment process in step (3) are: The microwave power is 500-800W and the microwave time is 3-4min.

8. The method for extracting Cistanche deserticola polysaccharide according to claim 1, characterized in that: The amount of ethanol added is such that the ethanol concentration reaches 70%.

9. Application of Cistanche deserticola polysaccharide in anti-aging cosmetics, characterized in that: The Cistanche deserticola polysaccharide is extracted by the extraction method according to any one of claims 1 to 8.

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