Preparation method for obtaining kaudatin from cynanchum wilfordii extract

Abstract

The present invention relates to a preparation method for kaudatin, especially a method for obtaining kaudatin from a Cynanchum wilfordii extract and, more particularly, to: a Cynanchum wilfordii extract comprising a high concentration of kaudatin prepared by the method; or kaudatin isolated therefrom. By using the preparation method for obtaining kaudatin from a Cynanchum wilfordii extract, according to the present invention, the Cynanchum wilfordii extract comprising a high concentration of kaudatin can be prepared through a high-temperature hot-water extracting or steaming step, and high-yield kaudatin can be purified and isolated from the Cynanchum wilfordii extract.

Description

Manufacturing method for obtaining caudatin from white ginseng extract

[0001] The present invention relates to a method for producing caudatin, and particularly to a method for obtaining caudatin from a Baekhasuo extract, and more particularly to a Baekhasuo extract containing high concentrations of caudatin produced thereby or caudatin separated therefrom.

[0002]

[0003] Baekhasuo is a perennial vine herbaceous plant of the Cucurbitaceae family, and contains oxymethylanthracion derivatives, carbohydrates, an essential oil component called cynanchol, lecithin, rhapontin, a protein component with both hydrophilic and lipophilic groups called phosphatidylcholine, and steroidal glycosides. In particular, caudatin, a steroidal glycoside, has been reported in previous papers to have effects such as inhibition of fat production (non-patent document 1), inhibition of hepatitis B virus (non-patent document 2), and inhibition of breast cancer (non-patent document 3).

[0004] Prior art, Korean Patent No. 10-2105661 (Patent Document 1), discloses a composition of Baekhasuo that exhibits antioxidant activity and NO inhibition activity without cytotoxicity by increasing the phenol content through repeated steaming and drying using an infrared vaporizer. In addition, Korean Patent No. 10-1214347 (Patent Document 2) discloses a method for extracting herbal ingredients that exhibit cytotoxicity reduction, skin irritation relief, and skin whitening effects by steaming a medicinal herb containing Baekhasuo using a white rice steaming method. However, the above patent documents do not recognize caudatin as an effective ingredient of Baekhasuo extract, and therefore, it is difficult to predict whether caudatin can be effectively obtained from Baekhasuo extract prepared as in the above documents.

[0005] Accordingly, the present invention confirmed that a high content of caudatin among various components of Baekhasuo can be obtained after manufacturing a Baekhasuo extract including at least one of a high-temperature extraction and a steaming step, and completed a manufacturing method for obtaining caudatin from a Baekhasuo extract containing a high content of caudatin.

[0006] (Patent Document 1) Republic of Korea Patent No. 10-2105661

[0007] (Patent Document 2) Republic of Korea Patent No. 10-1214347

[0008] (Non-patent Document 1) Jae-Seok Cho, Effect of caudatin on adipogenesis and fat accumulation via hedgehog signaling (2020)

[0009] (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

[0010] (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

[0011]

[0012] The present invention aims to provide a method for obtaining caudatin from a white ginseng extract.

[0013] Another object of the present invention is to provide a white ginseng extract containing high concentration of caudatin prepared therefrom or caudatin isolated therefrom.

[0014]

[0015] The present invention relates to a manufacturing method for obtaining a compound represented by chemical formula 1 from a Baekhasuo extract.

[0016] In the present invention, the compound represented by the following chemical formula 1 has a molecular formula C 28 H 42 It has O7, molecular weight 490.6, CAS No.: 38395-02-7 and is named Caudatin.

[0017] [Chemical Formula 1]

[0018]

[0019] Hereinafter, a manufacturing method for obtaining caudatin from a white ginseng extract according to the present invention will be described in more detail.

[0020] In the present invention, the manufacturing method for obtaining caudatin from Baekhasuo includes a step of adding a solvent to Baekhasuo and extracting it under heat.

[0021] Specifically, the step of extracting by heating may include a step of adding 5 to 20 times (v / w) of a solvent to Baekhasuo and extracting with hot water for 8 to 96 hours to obtain an extract.

[0022] In one embodiment, the hot extraction step may be performed by adding 5 to 20 times (v / w) of water to perform hot water extraction, for example, 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), 17 to 20 times (v / w). Preferably, when 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), the extraction may help increase the caudatin content.

[0023] In one embodiment, the heating 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, 92 to 96 hours. The caudatin content can be increased when extracted for preferably 16 to 92 hours, more preferably 24 to 82, and even more preferably 44 to 78 hours.

[0024] The above heating extraction step can be advantageous in terms of efficiency in the extraction, concentration or drying process of obtaining caudatin from Baekhasuo by adding a solvent 5 to 20 times (v / w) compared to Baekhasuo for 8 to 96 hours.

[0025] Prior art patents, such as Korean Patent No. 10-2105661 (Patent Document 1) and Korean Patent No. 10-1214347 (Patent Document 2), do not recognize caudatin as an effective ingredient of the extract of Baekhasuo, and therefore, do not present specific conditions for effectively obtaining caudatin from the extract of Baekhasuo prepared as in the above documents.

[0026] On the other hand, the present invention was able to obtain a Baekhasuo extract with increased caudatin content by performing the hot water extraction step of Baekhasuo at a high temperature of 95°C or higher, preferably 100°C or higher, and confirmed that caudatin is not sufficiently extracted at temperatures lower than 95°C.

[0027] The above temperature may be pressurized, and more specifically may be performed under a pressure of 1 to 3 atmospheres.

[0028] The above-mentioned warm extraction step can use a mixture of water and an organic solvent as an extraction solvent. For example, the extraction solvent can be 50% or more, 70% or more, 90% or more, 95% or more, 99% or more, 99.9% or more, or 100% water. Preferably, using 100% water is advantageous in terms of increasing the yield of caudatin. In particular, when using alcohols as an organic solvent, for example, when using a mixed extraction solvent containing 50% or more ethanol, there is a problem that caudatin is not detected in the extract because the hot water extraction temperature does not reach 95°C (Experimental Example 1).

[0029] The step of obtaining the above first extract may further include one or more of the steps of steaming and drying the Baekhasuo as a pretreatment process.

[0030] The above steaming step can use steam to soften the cell walls and tissues of Baekhasuo to promote the release of caudatin and increase the content of the active ingredient in the final extract.

[0031] The steaming step according to the present invention can be performed at a temperature of 90 to 130°C. The steaming temperature can 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, and preferably, by performing the steaming at 95 to 125°C, a Baekhasuo extract with an increased content of caudatin can be obtained (Experimental Example 2).

[0032] In the above steaming step, the steaming time can be performed for 1 to 80 hours. The steaming time may be, for example, 1 to 80 hours, more than 1 to 80 hours, more than 2 to 80 hours, more than 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, and preferably more than 4 to 80 hours, 5 to 80 hours, 6 to 80 hours, 7 to 80 hours, 8 to 80 hours, 16 to 80 hours at 100°C. By performing the process for 24 to 80 hours, or 72 to 80 hours, or more than 2 to 80 hours, 3 to 80 hours, or 4 to 80 hours at 110°C, or more than 1 to 80 hours, more than 1 to 80 hours, 2 to 80 hours, 3 to 80 hours, 4 to 80 hours, or 5 to 80 hours at 120°C, an extract of Baekhasuo with increased content of caudatin can be obtained (Experimental Example 2).

[0033] The above steaming step can be performed by applying pressure to reach a high temperature, and the pressure can be, for example, 1 atm or more, 2 atm or more, or 3 atm or more.

[0034] At this time, if the above conditions are exceeded, caudatin may not be sufficiently extracted or caudatin may be destroyed, and thus the extraction yield may not be increased.

[0035] The manufacturing method of the present invention may further include a step of concentrating the extract obtained in the heating extraction step as a drying step to obtain a concentrate.

[0036] The step of obtaining the above concentrate may include a filtration step for removing solids and foreign substances, and the filtration step may use a filtration membrane of 0.01 to 1.5 μm.

[0037] In the step of obtaining the above concentrate, the solvent is evaporated to prepare the concentrate so that it has a brix of 10 to 20.

[0038] The drying step may further include a step of powdering the concentrate, and the powdering step may use a conventional method used in the art, for example, a spray dryer may be used, but is not limited thereto.

[0039] The above drying step may further include a purification step.

[0040] The above purification step is a process for removing other substances from the Baekhasuo extract and obtaining caudatin. The above purification step can be performed using a purification method commonly used in the art, such as separation using differences in solubility, separation using a filtration membrane, or separation using various chromatographies (designed for separation by size, charge, hydrophobicity, or affinity).

[0041] In a specific example of the present invention, it was confirmed that the caudatin content of the Baekhasuo extract increased through the high-temperature hot water extraction and / or steaming steps (Tables 1 to 3). These results indicate that a high content of caudatin can be obtained from the Baekhasuo extract under the high-temperature hot water extraction and / or steaming conditions of the present invention.

[0042]

[0043] The present invention provides a white ginseng extract containing high concentration of caudatin, manufactured by the above manufacturing method, or caudatin isolated therefrom.

[0044] In one embodiment, the extract of Baekhasuo is provided in a concentrated or powdered form.

[0045] Specifically, the caudatin content may be included in an amount of 0.001 to 10 mg / g based on the total weight of the Baekhasuo extract concentrate.

[0046] Specifically, the caudatin content may be included in an amount of 0.01 to 10 mg / g based on the total weight of the Baekhasuo extract powder.

[0047]

[0048] In addition, the present invention provides a composition comprising a Baekhasuo extract manufactured by the above manufacturing method as an effective ingredient.

[0049] The composition according to the present invention may be a food composition for alleviating menopausal symptoms, which includes a Baekhasuo extract as an active ingredient.

[0050] In the present invention, "menopausal symptoms" may refer to "menopausal osteoporosis" caused by a decrease in hormone production due to menopause or other factors in women. Menopausal osteoporosis refers to osteoporosis symptoms caused by an imbalance between osteoblasts, which are involved in bone formation, and osteoclasts, which are involved in tissue destruction and absorption, due to a decrease in hormone production in postmenopausal women.

[0051] As used herein, "alleviation" refers to any action that improves or beneficially alters a desired symptom by administering the composition of the present invention. Those skilled in the art to which this invention pertains will be able to accurately determine the criteria for a disease and determine the degree of improvement, enhancement, and treatment by referencing materials provided by the Korean Medical Association and other sources.

[0052] The above 'food' refers to a natural product or processed product containing one or more nutrients, and for example, refers to a product that has gone through a certain process and has become directly edible, and in its conventional sense, it includes all health functional foods, functional foods, beverages, food additives, and beverage additives. The above 'food additive' or 'beverage additive' is in a form that includes or is made of a compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof, so that the food containing the additive includes an effective amount of the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof. The term 'effective amount' refers to the amount of an extract that has the effect of improving or beneficially changing menopausal symptoms or menopausal osteoporosis, or promoting the differentiation and proliferation of osteoblasts.

[0053] Specifically, in the present invention, the extract of Baekhasuo may be included in an amount of, for example, 0.001 to 30 wt%, 0.001 to 28 wt%, or 0.001 to 26 wt% relative to the total weight of the food composition.

[0054] If each active ingredient is included below the lower limit, the efficacy of alleviating female menopausal symptoms may not be achieved, and if it is included above the upper limit, the physical properties, color, unique flavor, and unique characteristics of the active ingredient itself may have a negative effect on the product.

[0055] The above food includes all foods in the conventional sense, and may be, for example, various foods, beverages, gum, tea, vitamin complexes, health functional foods, etc. In addition, in the present invention, the food includes, but is not limited to, special nutritional foods (e.g., formulated milk, infant and baby food, etc.), processed meat products, fish products, tofu, starch jelly, noodles (e.g., ramen, noodles, etc.), health supplements, seasoned foods (e.g., soy sauce, soybean paste, red pepper paste, mixed sauce, etc.), sauces, confectionery (e.g., snacks), processed dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi, pickled vegetables, etc.), beverages (e.g., fruit and vegetable beverages, soy milk, fermented beverages, etc.), and natural seasonings (e.g., ramen soup, etc.).

[0056] The above foods, functional foods, health functional foods, beverages, food additives, and beverage additives can be manufactured by conventional manufacturing methods. The term "functional food" is the same as "food for special health use (FoSHU)," and refers to foods with high medical and therapeutic effects that are processed to efficiently exhibit bioregulatory functions in addition to providing nutrition. Here, "functionality" means obtaining a useful effect for health purposes, such as regulating nutrients for the structure and function of the human body or physiological functions. The functional food of the present invention can be manufactured by a method conventionally used in the art, and can be manufactured by adding raw materials and ingredients conventionally added in the art during the manufacturing process. In addition, the formulation of the functional food can be manufactured without limitation as long as it is a formulation recognized as a functional food. The food composition of the present invention can be manufactured into various forms of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long period of time as it uses food as a raw material, and has excellent portability, so the functional food of the present invention can be consumed as a supplement to enhance the alleviation effect of menopausal symptoms or menopausal osteoporosis.

[0057] The above food may contain food additives that are food-scientifically acceptable, and may further contain suitable carriers, excipients, and diluents commonly used in the manufacture of health functional foods.

[0058] The composition may include additional ingredients commonly used in food compositions to improve odor, taste, appearance, etc. For example, it may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr), etc. In addition, it may include amino acids such as lysine, tryptophan, cysteine, and valine.

[0059] The above composition may include food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder and high-purity bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), colorants (tar colorants, etc.), color developers (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG, monosodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, D-potassium hydrogen tartrate, etc.), reinforcing agents, emulsifiers, thickeners (pastes), film-forming agents, gum-forming agents, foam suppressants, solvents, and improvers. The above additives can be selected according to the type of food and used in an appropriate amount.

[0060] Each of the above-mentioned ingredients included in the food composition according to the present invention may preferably be included in the food composition of the present invention within a range not exceeding the maximum usage amount specified in the regulations related to “food use / approval” prescribed by the government of each country.

[0061]

[0062]

[0063] By using the manufacturing method for obtaining caudatin from the extract of Baekhasuo according to the present invention, a Baekhasuo extract containing a high concentration of caudatin can be manufactured through high-temperature hot water extraction and steaming steps, and a high yield of caudatin can be purified and separated from the Baekhasuo extract.

[0064]

[0065] The numerical values ​​set forth in this specification are to be interpreted as including the equivalent range unless otherwise specified.

[0066]

[0067] Hereinafter, the present invention will be described in detail with examples to aid understanding. However, the examples according to the present invention may be modified in various different 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 more fully explain the present invention to those of average skill in the art.

[0068]

[0069] Experimental Example 1. Obtaining Caudatin according to extraction solvent, extraction temperature, and extraction time.

[0070] [extraction]

[0071] A test was conducted to determine whether the yield of caudatin was affected by extraction solvent, extraction temperature, or extraction time. 1 g of dried white ginseng was added to 10 mL of each solvent as shown in Table 1, and extraction was performed under the specified temperature and time conditions.

[0072] [Quantitative analysis of caudatin]

[0073] First, 1 g of the extracted sample was collected and transferred to a 10 mL volumetric flask, then methanol was added to the volume to 20 mL, and ultrasonicated for 1 minute at room temperature. Next, a portion of this was placed in a vial and quantitatively analyzed by high-performance liquid chromatography (HPLC). HPLC used a C18 column as the stationary phase and water containing 0.1% phosphoric acid and acetonitrile as the mobile phase, and detection was performed using a UV detector at 222 nm. The quantification was calculated by comparing the results with the quantification curve obtained using 1 ppm, 10 ppm, 100 ppm, and 500 ppm samples prepared with caudatin standards. The results are shown in Table 1.

[0074] Extraction solventExtraction temperatureExtraction timeCaudatin (ug / g)Comparative example 1Ethanol70℃16 hours0Comparative example 2Ethanol / water (50:50 v / v)80℃16 hours0Comparative example 3Water90℃16 hours0Example 1Water100℃16 hours190Example 2Water100℃24 hours272Example 3Water105℃16 hours460

[0075] From the above results, it was confirmed that caudatin could be obtained only if extraction was performed at a temperature exceeding 90℃. In addition, since the boiling point of ethanol is 78℃, it was confirmed that it is not desirable to include an ethanol content of more than 50% because it is difficult to raise the extraction temperature above 90℃ when ethanol is included as a solvent. In addition, it was confirmed that it is desirable to use water with a boiling point of 100℃ as a solvent when obtaining caudatin from Baekhasuo.

[0076] Therefore, it is preferable that the heated extraction be performed at a temperature of 95°C or higher for 8 to 96 hours using water as a solvent.

[0077]

[0078] Experimental Example 2. Obtaining Caudatin by Steaming

[0079] An experiment was conducted to determine the change in the yield of caudatin depending on whether or not Baekhasuo was steamed. First, 1 kg of dried Baekhasuo was placed in a steamer and steamed at different temperatures and times. After steaming was completed, the steamed Baekhasuo was dried in a dryer at 40°C for 24 hours. Then, Baekhasuo of each example was used as a sample and caudatin was quantified using the same method as the quantitative analysis of caudatin in Experimental Example 1, and the results are shown in Table 2.

[0080] Steaming temperature Steaming time Caudatin (ug / g) Example 4 100 ℃ 8 hours 87 Example 5 100 ℃ 24 hours 706 Example 6 100 ℃ 72 hours 1427 Example 7 110 ℃ 4 hours 96 Example 8 121 ℃ 2 hours 152 Example 9 121 ℃ 5 hours 800

[0081] From the above results, it was confirmed that the content of caudatin obtained from Baekhasuo changed depending on the steaming conditions. In particular, it was confirmed that the yield of caudatin increased as the steaming time increased, and that the yield of caudatin could be increased in a shorter period of time by increasing the steaming temperature.

[0082] Therefore, it is efficient and preferable to perform the steaming for a time of more than 4 to 80 hours at 100°C, more than 2 to 80 hours at 110°C, or 1 to 80 hours at 120°C.

[0083]

[0084] Experimental Example 3. Obtaining Caudatin by Combination of Hot Water Extraction and Steaming

[0085] In the above Experimental Examples 1 and 2, it was confirmed that pretreatment by high-temperature hot water extraction or steaming was necessary to obtain caudatin from Baekhasuo. Next, it was confirmed whether the effect was enhanced when these were combined to obtain caudatin.

[0086] The steamed white ginseng root of Example 5 of Experimental Example 2 was extracted with water at room temperature (25°C) or 100°C for 24 hours. After the extraction was completed, caudatin was quantified using the same method as the quantitative analysis of caudatin in Experimental Example 1, and the results are shown in Table 3.

[0087] Condition Caudatin (ug / g) Comparative Example 4 Extraction at room temperature after steaming 0 Example 10 Extraction at 100℃ after steaming 835

[0088] From the above results, it was confirmed that even if Baekhasuo contains a large amount of caudatin through steaming, extraction must be performed at a temperature exceeding 90℃ to produce an extract containing a large amount of caudatin. In addition, it was confirmed that steaming Baekhasuo before hot water extraction increases the yield of caudatin compared to when hot water extraction alone is used.

Claims

1. A manufacturing method for obtaining caudatin from Baekhasuo, comprising the step of adding a solvent to Baekhasuo and performing a heating extraction.

2. In paragraph 1, A method for obtaining caudatin from Baekhasuo, wherein the above heating extraction step is performed at a temperature of 95 to 110°C.

3. In paragraph 1, A method for obtaining caudatin from Baekhasuo, wherein the above-mentioned heating extraction step is performed by adding a solvent 5 to 20 times (v / w) compared to Baekhasuo and extracting for 8 to 96 hours.

4. In paragraph 1, A method for obtaining caudatin from Baekhasuo, wherein the above heating extraction step is performed by extracting with less than 50% ethanol as a solvent.

5. In paragraph 1, In the first paragraph, A method for obtaining caudatin from Baekhasuo, wherein the above heating extraction step is an extraction step including water as a solvent.

6. A manufacturing method for obtaining caudatin from Baekhasuo, comprising a step of steaming Baekhasuo.

7. In paragraph 6, A method for producing caudatin from Baekhasuo, wherein the above steaming step is performed at a temperature of 90 to 130°C for 2 to 72 hours.

8. Steaming the Baekhasuo; and A manufacturing method for obtaining caudatin from Baekhasuo, comprising a step of adding a solvent to steamed Baekhasuo and performing a heating extraction.

9. In paragraph 8, A manufacturing method for obtaining caudatin from Baekhasuo, further comprising a step of concentrating the Baekhasuo extract obtained by the above-mentioned heating extraction step.

10. In paragraph 9, A manufacturing method for obtaining caudatin from Baekhasuo, further comprising a step of powdering the above-mentioned concentrated Baekhasuo extract.

11. Caudatin obtained from Baekhasuo, manufactured by a manufacturing method according to any one of claims 1 to 5, 6 to 7, or 8 to 10.

12. In paragraph 11, Caudatin obtained from Baekhasuo, having a caudatin content of 0.001 to 10 mg / g based on the total weight of Baekhasuo extract.

13. In paragraph 11, Caudatin obtained from Baekhasuo, having a caudatin content of 0.01 to 10 mg / g based on the total weight of Baekhasuo extract powder.

14. An extract of Baekhasuo, manufactured by a manufacturing method according to any one of claims 1 to 5, 6 to 7, or 8 to 10.

15. A food composition comprising a Baekhasuo extract as an active ingredient, manufactured by a manufacturing method according to any one of claims 1 to 5, 6 to 7, or 8 to 10.

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