Method for obtaining caudatin from White Polygonum Shou Bu extract

Abstract

The present invention relates to a method for producing caudatin, and more particularly to a method for obtaining caudatin from a White Polygonum multiflorum extract, and more specifically to a White Polygonum multiflorum extract containing a high concentration of caudatin produced thereby, or to caudatin isolated therefrom. By using the method for obtaining caudatin from *Polygonum multiflorum* extract according to the present invention, a *Polygonum multiflorum* extract containing a high concentration of caudatin can be produced through high-temperature hot water extraction or steaming steps, and a high yield of caudatin can be purified and separated from the *Polygonum multiflorum* extract.

Description

Technical Field

[0001] The present invention relates to a method for producing caudatin, particularly to a method for obtaining caudatin from a Cynanchum bungei extract, and specifically relates to a Cynanchum bungei extract containing high-concentration caudatin produced thereby, or caudatin isolated therefrom.

Background Art

[0002] Cynanchum bungei is a perennial vine plant belonging to the Apocynaceae family, and contains oxymethlanthraquinone derivatives, carbohydrates, an essential oil component called cynanchol, lecithin, rhapontin, a protein component having both hydrophilic and lipophilic groups called phosphatidylcholine, steroidal glycoside, and the like. In particular, previous research papers have reported that caudatin, which is a steroidal glycoside, has effects such as adipogenesis inhibition (Non-Patent Document 1), hepatitis B virus inhibition (Non-Patent Document 2), and breast cancer inhibition (Non-Patent Document 3).

[0003] Korean Registered Patent No. 10-2105661 (Patent Document 1), which is prior art, discloses a Cynanchum bungei composition that increases phenol content and exhibits antioxidant activity and NO inhibitory activity without exhibiting cytotoxicity by repeatedly performing steaming and drying using an infrared amplification device. In addition, Korean Registered Patent No. 10-1214347 (Patent Document 2) discloses a method for extracting a Chinese herbal medicine component that exhibits effects of reducing cytotoxicity, alleviating skin irritation, and skin whitening by steaming Chinese herbal medicine materials including Cynanchum bungei using a white rice steaming method. However, the above patent documents do not recognize caudatin as an active ingredient of the Cynanchum bungei extract, and therefore it is difficult to predict whether caudatin can be effectively obtained from the Cynanchum bungei extract produced by the method described in the documents.

[0004] Therefore, the present invention has confirmed that it is possible to obtain caudatin in a high content from among the multiple components of White Polygonum multiflorum after producing an extract of White Polygonum multiflorum including at least one of the steps of high-temperature extraction and steaming, and has completed a method for obtaining caudatin from a White Polygonum multiflorum extract containing a high content of caudatin. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Korean Registered Patent No. 10-2105661 Publication [Patent Document 2] Korean Registered Patent No. 10-1214347 Publication [Non-patent literature]

[0006] [Non-Patent Document 1] Cho Jae-seok, Effect of caudatin on adipogenesis and fat accumulation via hedgehog signaling (2020) [Non-Patent Document 2] Wang LJ et al. Design, Synthesis and Biological Evaluation of Caudatin Analogs as Potent Hepatitis B Virus Inhibitors. MedChem, 2015, 11(2), 165-79 [Non-Patent Document 3] Zhen X et al. Caudatin Isolated from Cynanchum auriculatum Inhibits Breast Cancer Stem Cell Formation via a GR / YAP Signaling. Biomolecules, 2020, 10, 925 [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention aims to provide a method for obtaining caudatin from Polygonum multiflorum extract.

[0008] Another object of the present invention is to provide a White Wheat Extract containing a high concentration of caudatin produced thereby, or caudatin isolated therefrom. [Means for solving the problem]

[0009] The present invention relates to a method for producing a compound represented by the following chemical formula 1 from an extract of Polygonum multiflorum.

[0010] In the present invention, the compound represented by the following chemical formula 1 is a compound with molecular formula C 28 H 42 It has O7, a molecular weight of 490.6, and CAS No.: 38395-02-7, and is named Caudatin.

[0011] [ka]

[0012] The following describes in more detail the method for obtaining caudatin from the extract of Polygonum multiflorum according to the present invention.

[0013] In the present invention, the method for obtaining caudatin from Polygonum multiflorum includes the step of adding a solvent to Polygonum multiflorum and heating to extract it.

[0014] Specifically, the heated extraction step includes adding a solvent 5 to 20 times (v / w) to Polygonum multiflorum and performing hot water extraction for 8 to 96 hours to obtain the extract.

[0015] In one embodiment, the heating extraction step can be performed by adding 5 to 20 times (v / w) of water for hot water extraction. For example, extraction can be performed by adding 5 to 7 times (v / w), 7 to 10 times (v / w), 10 to 12 times (v / w), 12 to 15 times (v / w), 15 to 17 times (v / w), or 17 to 20 times (v / w). Preferably, extraction can be performed by adding 5 to 15 times (v / w), more preferably 7 to 15 times (v / w), and even more preferably 7 to 12 times (v / w), which can contribute to an increase in caudatin content.

[0016] In one embodiment, the heated extraction step can be performed for 8 to 96 hours, for example, 8 to 12 hours, 12 to 16 hours, 16 to 20 hours, 20 to 24 hours, 24 to 28 hours, 28 to 32 hours, 32 to 36 hours, 36 to 40 hours, 40 to 44 hours, 44 to 48 hours, 48 ​​to 52 hours, 52 to 56 hours, 56 to 60 hours, 60 to 64 hours, 60 to 72 hours, 64 to 68 hours, 68 to 72 hours, 72 to 76 hours, 76 to 80 hours, 84 to 88 hours, 88 to 92 hours, or 92 to 96 hours. Preferably, the caudatin content can be increased by extracting for 16 to 92 hours, more preferably 24 to 82 hours, and even more preferably 44 to 78 hours.

[0017] The aforementioned heated extraction step, when the solvent is added 5 to 20 times (v / w) relative to Polygonum multiflorum and extracted for 8 to 96 hours, is advantageous in terms of efficiency in the extraction, concentration, or drying process for obtaining caudatin from Polygonum multiflorum.

[0018] Prior art, Korean Registered Patent No. 10-2105661 (Patent Document 1) and Korean Registered Patent No. 10-1214347 (Patent Document 2), do not recognize caudatin as an active ingredient in Polygonum multiflorum extract, and therefore do not present specific conditions for effectively obtaining caudatin from Polygonum multiflorum extract manufactured as described in the aforementioned documents.

[0019] On the other hand, in the present invention, by carrying out the hot water extraction step of *Polygonum multiflorum thunb* (Baiheshouwu) at a high temperature of 95°C or higher, preferably 100°C or higher, a Baiheshouwu extract with an increased content of caudatin can be obtained, and it has been confirmed that caudatin cannot be sufficiently extracted when the temperature is lower than 95°C.

[0020] Pressurization may be performed to reach said temperature, and more specifically, the extraction may be performed under 1 to 3 atmospheres.

[0021] In said heating extraction step, a mixture of water and an organic solvent can be used as the extraction solvent. For the extraction solvent, for example, water may account for 50% or more, 70% or more, 90% or more, 95% or more, 99% or more, 99.9% or more, or 100% of the solvent; using 100% water as the solvent is preferred, which is advantageous in terms of improving the yield of caudatin. In particular, when an alcohol is used as the organic solvent, for example, when a mixed extraction solvent containing 50% or more of ethanol is used, the hot water extraction temperature does not reach 95°C, so there is a problem that caudatin cannot be detected from the extract (Experimental Example 1).

[0022] The step of obtaining said primary extract may further comprise at least one of a step of steaming Baiheshouwu and a step of drying as pretreatment steps.

[0023] Said steaming step softens the cell walls and tissues of Baiheshouwu using steam, thereby promoting the elution of caudatin and increasing the content of active ingredients in the final extract.

[0024] Said steaming step according to the present invention can be performed at a temperature of 90 to 130°C. The steaming temperature may be, for example, 90 to 95°C, 95 to 100°C, 100 to 105°C, 105 to 110°C, 110 to 115°C, 115 to 120°C, 120 to 125°C, or 125 to 130°C; performing the steaming at 95 to 125°C is preferred, whereby a Baiheshouwu extract with an increased content of caudatin can be obtained (Experimental Example 2).

[0025] In the aforementioned steaming stage, the steaming time may be 1 to 80 hours. The steaming time may be, for example, 1 to 80 hours, over 1 to 80 hours, over 2 to 80 hours, over 4 to 80 hours, 1 to 5 hours, 5 to 10 hours, 10 to 15 hours, 15 to 20 hours, 20 to 25 hours, 25 to 30 hours, 30 to 35 hours, 35 to 40 hours, 40 to 45 hours, 45 to 50 hours, 50 to 55 hours, 55 to 60 hours, 60 to 65 hours, 65 to 70 hours, 70 to 75 hours, and 75 to 80 hours, preferably at 100°C with over 4 hours By performing the procedure for 80 hours, 5-80 hours, 6-80 hours, 7-80 hours, 8-80 hours, 16-80 hours, 24-80 hours, or 72-80 hours, or by performing the procedure at 110°C for more than 2 hours to 80 hours, 3 hours to 80 hours, or 4 hours to 80 hours, or by performing the procedure at 120°C for 1-80 hours, more than 1 hour to 80 hours, 2 hours to 80 hours, 3 hours to 80 hours, 4 hours to 80 hours, or 5 hours to 80 hours, an extract of Polygonum multiflorum with increased caudatin content can be obtained (Experimental Example 2).

[0026] The aforementioned steaming stage can be carried out by applying pressure to reach a high temperature, and the pressure at this time may be, for example, 1 atmosphere or more, 2 atmospheres or more, or 3 atmospheres or more.

[0027] In this case, if the conditions are not met, caudatin may not be sufficiently extracted, or the caudatin may be destroyed, making it impossible to improve the extraction yield.

[0028] The manufacturing method of the present invention may further include a drying step of concentrating the extract obtained from the heating extraction step to obtain a concentrated liquid.

[0029] The step of obtaining the concentrated liquid may include a filtration step to remove solids and foreign matter, and a filtration membrane of 0.01 to 1.5 μm can be used for the filtration step.

[0030] In the step of obtaining the concentrated solution, the solvent is evaporated to prepare the concentrated solution to a concentration of 10-20 Brix.

[0031] The drying step may further include a step of pulverizing the concentrate, and the pulverizing step may be carried out using a conventional method commonly used in the industry, such as a spray dryer, but is not limited thereto.

[0032] The drying step may further include a purification step.

[0033] The purification step is the process of removing other substances from the Polygonum bracteatum extract to obtain caudatin. This purification step is carried out by conventional purification methods commonly used in the industry, such as separation using differences in solubility, separation using filtration membranes, and separation using various chromatography methods (designed for separation based on size, charge, hydrophobicity, or affinity).

[0034] In this invention, it was confirmed in a specific example that the caudatin content of the White Polygonum multiflorum extract increases through the high-temperature hot water extraction and / or steaming stage (Tables 1 to 3). These results show that a high content of caudatin can be obtained from the White Polygonum multiflorum extract under the high-temperature hot water extraction and / or steaming conditions of this invention.

[0035] The present invention provides a high-concentration extract of Polygonum multiflorum containing caudatin, produced by the above-described manufacturing method, or caudatin isolated therefrom.

[0036] In one embodiment, the extract of Polygonum multiflorum is provided in the form of a concentrated liquid or a powder.

[0037] Specifically, the caudatin content may be between 0.001 and 10 mg / g based on the total weight of the concentrated extract of Polygonum bracteatum.

[0038] Specifically, the caudatin content may be between 0.01 and 10 mg / g based on the total weight of the powdered White Polygonum multiflorum extract.

[0039] Furthermore, the present invention provides a composition containing *Polygonum multiflorum* extract, produced by the above-described manufacturing method, as an active ingredient.

[0040] The composition according to the present invention may be a food composition for alleviating menopausal symptoms, comprising Polygonum multiflorum extract as an active ingredient.

[0041] In this invention, "menopausal symptoms" may refer to "menopausal osteoporosis," which occurs when hormone production decreases due to menopause in women. Menopausal osteoporosis refers to the symptoms of osteoporosis that occur in menopausal women due to an imbalance between osteoblasts, which are involved in bone formation, and osteoclasts, which are involved in tissue destruction and resorption, caused by a decrease in hormone production.

[0042] In this invention, "alleviation" means any action that improves or beneficially alters the target symptoms by administering the composition of this invention. A person with ordinary skill in the art to which this application belongs should be able to understand the precise criteria for the disease and determine the degree of improvement, enhancement, and treatment by referring to materials presented by the Korean Medical Association and other organizations.

[0043] The term "food" as used herein means a natural product or processed product containing one or more nutrients, for example, something that has been processed through a certain process to become ready for direct consumption, and in its usual sense, includes all health functional foods, functional foods, beverages, food additives, and beverage additives. The term "food additive" or "beverage additive" contains or consists of a compound represented by chemical formula 1 or a pharmaceutically acceptable salt thereof, and is in a form in which the compound represented by chemical formula 1 or a pharmaceutically acceptable salt thereof is included in an effective amount in the food containing the additive. The term "effective amount" as used herein means the amount of extract that has the effect of improving or beneficially altering menopausal symptoms or menopausal osteoporosis, or promoting the differentiation and proliferation of osteoblasts.

[0044] Specifically, in the present invention, the White Wheat Extract may be included in the total weight of the food composition in amounts of, for example, 0.001 to 30% by weight, 0.001 to 28% by weight, or 0.001 to 26% by weight.

[0045] If the content of each active ingredient is below the lower limit, the effect of alleviating menopausal symptoms in women may not be achieved. If it is included above the upper limit, the physical properties, color, and unique flavor and characteristics of the active ingredient itself may adversely affect the product.

[0046] The aforementioned foods include all foods in the ordinary sense, such as various food products, beverages, gum, tea, vitamin complexes, and health functional foods. Furthermore, in the present invention, foods include, but are not limited to, special nutritional foods (e.g., powdered milk, infant food, etc.), processed meat products, fish products, tofu products, jellies, noodles (e.g., ramen, soba, etc.), health supplements, seasoned foods (e.g., soy sauce, miso, gochujang, mixed seasonings, etc.), sauces, confectionery (e.g., snacks), dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various types of kimchi, pickles, etc.), beverages (e.g., fruit and vegetable beverages, soy milk products, fermented beverages, etc.), and natural seasonings (e.g., instant noodle soup, etc.).

[0047] The aforementioned foods, functional foods, health functional foods, beverages, food additives, and beverage additives can be manufactured by conventional manufacturing methods. The term "functional food" is synonymous with "food for special health use (FoSHU)" and refers to foods with high medical and therapeutic effects that are processed to effectively express biological regulatory functions in addition to providing nutrients. Here, "functionality" means regulating nutrients in relation to the structure and function of the human body, or obtaining effects useful for health purposes such as physiological effects. The functional foods of the present invention can be manufactured by methods commonly used in the industry, and during the manufacturing process, raw materials and components commonly added in the industry can be added. Furthermore, there are no restrictions on the dosage form of the functional foods, as long as it is a form recognized as a functional food. The food composition of the present invention can be manufactured in various dosage forms, and unlike general pharmaceuticals, it has the advantage of not having side effects that can occur from long-term use of pharmaceuticals because it is made from food as a raw material, and it is also highly portable. Therefore, the functional foods of the present invention can be taken as an adjunct to enhance the alleviation effect of menopausal symptoms or menopausal osteoporosis.

[0048] The food may contain food-grade food additives, and may further contain suitable carriers, excipients, and diluents commonly used in the manufacture of functional foods.

[0049] The composition may contain additional components commonly used in food compositions that can improve odor, taste, visual appeal, etc. For example, it may contain vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. It may also contain minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), and copper (Cu). Furthermore, it may contain amino acids such as lysine, tryptophan, cysteine, and valine.

[0050] The composition may contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder, high-grade bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytriene (BHT), etc.), colorants (tar dyes, etc.), color fixatives (sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (monosodium glutamate (MSG), etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, potassium bitartrate, etc.), fortifiers, emulsifiers, thickeners, coating agents, gum bases, defoamers, solvents, and improvers. The additives may be selected and used in appropriate amounts depending on the type of food.

[0051] The components contained in the food composition according to the present invention may preferably be included in the food composition according to the present invention in a manner that does not exceed the maximum amount of use specified in the relevant regulations concerning "food use permits" stipulated by each national government. [Effects of the Invention]

[0052] By using the method for obtaining caudatin from *Polygonum multiflorum* extract according to the present invention, a *Polygonum multiflorum* extract containing a high concentration of caudatin can be produced through the steps of high-temperature hot water extraction and steaming, and a high yield of caudatin can be purified and separated from the *Polygonum multiflorum* extract. [Modes for carrying out the invention]

[0053] The figures described herein should be interpreted as including their equivalent range unless otherwise specified.

[0054] The present invention will be described in detail below with reference to examples and other details to aid in understanding the present invention. However, the examples of the present invention can be modified in various forms, and the scope of the present invention should not be construed as being limited to the following examples. The examples of the present invention are provided to give a more complete explanation of the present invention to a person of average knowledge in the art.

[0055] Experimental Example 1. Acquisition of caudatin by extraction solvent, extraction temperature, and extraction time. [extraction] Tests were conducted to determine whether the yield of caudatin was affected by the extraction solvent, extraction temperature, or extraction time. 1 g of dried White Wheat Root was placed in 10 mL of each solvent as shown in Table 1, and then extracted under specified temperature and time conditions.

[0056] [Quantitative analysis of caudatin] First, 1 g of the extracted sample was taken and transferred to a 10 mL volumetric flask. Methanol was added up to 20 mL, and the sample was sonicated at room temperature for 1 minute. Next, a portion of the sample was dispensed into vials and quantitatively analyzed by high-performance liquid chromatography (HPLC). For HPLC, a C18 column was used as the stationary phase, and 0.1% phosphate-containing water and acetonitrile were used as the mobile phase. Detection was performed at 222 nm using an ultraviolet detector. The quantitative results were calculated by comparing the results with calibration curves obtained from 1 ppm, 10 ppm, 100 ppm, and 500 ppm samples prepared using caudatin standards. The results are shown in Table 1.

[0057] [Table 1]

[0058] The results above confirm that caudatin can only be obtained by performing the extraction at a temperature exceeding 90°C. Furthermore, since the boiling point of ethanol is 78°C, it is difficult to raise the extraction temperature above 90°C when ethanol is used as a solvent, and therefore it was confirmed that it is undesirable to contain more than 50% ethanol. Finally, it was confirmed that it is preferable to use water with a boiling point of 100°C as a solvent when obtaining caudatin from Polygonum multiflorum.

[0059] Therefore, it is preferable to perform heated extraction using water as the solvent at a temperature of 95°C or higher for 8 to 96 hours.

[0060] Experimental Example 2. Acquisition of caudatin by steaming. Experiments were conducted to confirm the change in caudatin yield depending on whether or not the white hollyhock is steamed. First, 1 kg of dried white hollyhock was placed in a steaming apparatus and steamed while varying the temperature and time. After steaming was completed, the steamed white hollyhock was dried in a dryer at 40°C for 24 hours. Next, the white hollyhock from each example was used as a sample, and caudatin was quantified using the same method as the quantitative analysis of caudatin described in Experimental Example 1, and the results are shown in Table 2.

[0061] [Table 2]

[0062] The results above confirm that the caudatin content obtained from Polygonum multiflorum changes depending on the steaming conditions. In particular, it was confirmed that the yield of caudatin increases with increasing steaming time, and that the yield of caudatin can be improved in a shorter time by increasing the steaming temperature.

[0063] Therefore, it is efficient and preferable to perform the steaming time within the range of 4 to 80 hours at 100°C, 2 to 80 hours at 110°C, or 1 to 80 hours at 120°C.

[0064] Experimental Example 3. Acquisition of caudatin by a combination of hot water extraction and steaming. In Experimental Examples 1 and 2 described above, it was confirmed that pretreatment by high-temperature hot water extraction or steaming is necessary to obtain caudatin from Polygonum multiflorum. Furthermore, it was investigated whether combining these methods improves the effectiveness of caudatin acquisition.

[0065] White He Shou Wu, which had been steamed under the conditions of Example 5 in Experimental Example 2, was extracted with water for 24 hours at room temperature (25°C) or 100°C, respectively. After the extraction was complete, caudatin was quantified using the same method as the quantitative analysis of caudatin described in Experimental Example 1, and the results are shown in Table 3.

[0066] [Table 3]

[0067] From the results above, it was confirmed that even if the White He Shou Wu plant becomes rich in caudatin through steaming, an extract rich in caudatin cannot be produced unless the extraction is carried out at a temperature exceeding 90°C. Furthermore, it was confirmed that steaming the White He Shou Wu before hot water extraction increases the amount of caudatin obtained compared to using hot water extraction alone.

Claims

1. A method for producing caudatin from Polygonum multiflorum, comprising the step of adding a solvent to Polygonum multiflorum and performing a heating and extraction.

2. The method for obtaining caudatin from Polygonum multiflorum according to claim 1, wherein the heating extraction step is performed at a temperature of 95 to 110°C.

3. The method for obtaining caudatin from Polygonum multiflorum according to claim 1, wherein the heating extraction step involves adding a solvent to Polygonum multiflorum at a ratio of 5 to 20 times (v / w) relative to Polygonum multiflorum and extracting for 8 to 96 hours.

4. The method for obtaining caudatin from Polygonum multiflorum according to claim 1, wherein the heating extraction step is performed by extracting with less than 50% ethanol as the solvent.

5. The method for obtaining caudatin from Polygonum multiflorum according to claim 1, wherein the heating extraction step is performed by extracting with water as the solvent.

6. A method for producing caudatin from Polygonum multiflorum, including a step of steaming the plant.

7. The method for producing caudatin from Polygonum multiflorum according to claim 6, wherein the aforementioned steaming step is carried out at a temperature of 90 to 130°C for 2 to 72 hours.

8. The stage of steaming and ripening White He Shou Wu; and A method for obtaining caudatin from Polygonum multiflorum, comprising the step of adding a solvent to steamed Polygonum multiflorum and heating to extract the mixture.

9. A method for obtaining caudatin from Polygonum multiflorum according to claim 8, further comprising the step of concentrating the Polygonum multiflorum extract obtained by the aforementioned heating extraction step.

10. A method for obtaining caudatin from Polygonum multiflorum according to claim 9, further comprising the step of powdering the concentrated Polygonum multiflorum extract.

11. Caudatin obtained from Polygonum multiflorum, produced by the manufacturing method described in any one of claims 1 to 10.

12. Caudatin obtained from Polygonum multiflorum extract according to claim 11, wherein the caudatin content is 0.001 to 10 mg / g based on the total weight of the Polygonum multiflorum extract.

13. Caudatin obtained from Polygonum multiflorum extract powder according to claim 11, wherein the caudatin content is 0.01 to 10 mg / g based on the total weight of the Polygonum multiflorum extract powder.

14. White Polygonum cuspidatum extract, produced by the manufacturing method described in any one of claims 1 to 10.

15. A food composition containing Polygonum multiflorum extract as an active ingredient, manufactured by the manufacturing method described in any one of claims 1 to 10.

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