Method for separating flavokawain and kavalactone, kavalactone and microencapsulated kavalactone

Abstract

To provide a method for separating flavokawain and kavalactone.SOLUTION: The present application relates to a technical field of separating kavalactone and flavokawain, and in particular, to a method for separating flavokawain and kavalactone, kavalactone, and microencapsulated kavalactone. The separation method includes: S1. grinding root of kava, extracting by supercritical carbon dioxide and collecting a residue for later use, in which an extraction temperature is 45 to 50°C and an extraction pressure is 4 to 8 MPa; S2. extracting the residue by supercritical carbon dioxide, and collecting extracted oil for later use, in which an extraction temperature is 60 to 80°C and an extraction pressure is 20 to 50 MPa; S3. performing re-extraction and adsorption on the extracted oil to obtain a primary product of the kavalactone; and S4. crystallizing the primary product of kavalactone to obtain the kavalactone. The obtained kavalactone is used in the preparation of microencapsulated kavalactone.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present application relates to the technical field of separation of kavalactones and flavokawains, and more particularly to a method for separating kavalactones from flavokawains, kavalactones and microencapsulated kavalactones. [Background technology]

[0002] Kava is a plant in the Capsicum family (Pipe family) native to the South Pacific. Traditionally, kava drink is a suspension of kava root extract and has historically been a religious or celebratory drink. However, due to its relaxing effects, kava gradually developed into an everyday drink for South Pacific Islanders.

[0003] Literature reports suggest that kavalactones contained in kava extracts may reduce stress, improve sleep, manage anxiety, enhance memory, reduce inflammation and regulate the immune system, and prevent cancer. In 1990, the German Federal Office of Health approved kava for the treatment of anxiety disorders, the UK included it in its "Botanical Pharmacopoeia," the US included it in the new edition of the "United States Pharmacopoeia," and many European and US countries list kava as a non-prescription or prescription drug.

[0004] At the same time, the use of kava also poses certain safety risks. In the late 1990s, rare but sometimes severe cases of liver toxicity occurred among kava users, and the reports drew public attention to its safety (Non-Patent Document 1). Recent findings (Non-Patent Document 2) further support this view. The study shows that kava may potentiate paracetamol-induced liver toxicity in vivo through two chalcone-based chemicals, Flavokawains A and B.

[0005] However, in the current method of producing kavalactone, for example, the specific steps of obtaining kavalactone using the supercritical carbon dioxide method are to crush kava root, add it to an extraction vessel, set the temperature to 30-50°C, the pressure to 15-25MPa, and add liquid CO 2 and an additive solvent are introduced to perform supercritical extraction, the extract is analyzed at a temperature of 50-60°C and a pressure of 5-10 MPa, the obtained extract is dissolved in ethyl acetate, passed through a short aluminum peroxide column, ethyl acetate is recovered from the column liquid, and dried at low temperature to obtain the product. For example, the specific steps of obtaining kavalactone by solvent extraction are to crush kava root, add 5-10 times the amount of 60-90% ethanol solution, perform ultrasonic extraction 2-3 times, concentrate the extract under reduced pressure, add the concentrate to a macroporous resin for adsorption, first use 3-5 times the column volume of 40-60% ethanol solution to elute impurities, then use 3-5 times the column volume of 90% ethanol solution to elute, collect the eluate and concentrate under reduced pressure, add alumina to the concentrate and dry it, fill the column, perform gradient elution with ethanol solution, collect the active ingredient, concentrate under reduced pressure and dry it to obtain it.

[0006] However, the above-mentioned solutions focus on the preparation of kavalactone, and do not pay attention to the separation of kavalactone and flavokavain, so that the total amount of flavokavain A and flavokavain B in the obtained kavalactone is relatively large, which is potentially dangerous to human health. Therefore, it is necessary to provide a method for separating kavalactone from flavokavain A and flavokavain B, so as to ensure complete separation of kavalactone from flavokavain A and flavokavain B. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Stevinson C, Huntley A, Ernst E. A systematic review of the safety of kava extract in the treatment of anxiety. Drug Saf. 2002;25(4):251-61. [Non-Patent Document 2] (Narayanapillai SC, Leitzman P, O'Sullivan MG, Xing C. Flavokawains a and B in kava, not dihydromethysticin, potentiate acetaminophen induced hepatotoxicity in C57BL / 6 mice. Chemical research in toxicology. 2014;27(10):1871-6. Summary of the Invention [Problem to be solved by the invention]

[0008] In order to improve the separation effect of kavalactone, flavokawain A and flavokawain B, the present application provides a method for separating flavokawain and kavalactone, kavalactone and microencapsulated kavalactone. [Means for solving the problem]

[0009] In a first aspect, the present application provides a method for separating flavokawains and kavalactones, and adopts the following technical solutions:

[0010] A method for separating flavokawains and kavalactones, comprising the steps of: S1, in which the kava root is crushed and the residue is collected for use after extraction with supercritical carbon dioxide, with an extraction temperature of 45-50°C and a pressure of 4-8Mpa; S2, in which the residue is extracted with supercritical carbon dioxide and the extracted oil is collected for later use, and the extraction temperature is 60-80°C and the pressure is 20-50Mpa; S3, which performs re-extraction and adsorption on the extracted oil to obtain a primary product of kavalactone; and S4 crystallizing the primary product of kavalactone to obtain kavalactone.

[0011] By adopting the above technical solution, the present application first uses low-temperature and low-pressure supercritical carbon dioxide to treat the crushed crude product of kava root in step S1, and since flavoka wine A and flavoka wine B have low polarity and kavalactone has high polarity, in this process, flavoka wine A and flavoka wine B with low polarity are mainly extracted, and kavalactone with high polarity remains in the residue, and then kavalactone and flavoka wine (flavoka wine A and flavoka wine B) are separated. Through this step, a total of about 40-60 wt% of flavoka wine (flavoka wine A and flavoka wine B) and kavalactone can be separated, and the residue contains a lot of kavalactone, and the extract of S1 contains a lot of flavoka wine A and flavoka wine B. Based on S1, proceed to S2. S2 is to treat the residue with high-temperature and high-pressure supercritical carbon dioxide, by which the less polar flavokawains A and B are retained in the residue, the more polar kavalactones are mainly extracted, and the kavalactones and flavokawains (flavokawains A and B) are separated. After S2, the kavalactone content in the obtained extracted oil is 75-85 wt%, and the flavokawains (flavokawains A and B) are 2-5 wt%. The extracted oil is further treated, and a large amount of flavokawein (flavokawein A and flavokawein B) and kavalactones in the extracted oil are first separated by a re-extraction process, and then the flavokawein (flavokawein A and flavokawein B) and kavalactones are further separated by an adsorption process, and the content of flavokawein (flavokawein A and flavokawein B) in the kavalactones obtained after final crystallization is 0.05-0.18 wt%.

[0012] In addition, kavalactone in this application is a mixture of six specific compounds. The six compounds are 1. Methysticin, 2. Dihydromethystcin, 3. Kavain, 4. Dihydrokavain, 5. Yangonin, and 6. Desmethoxyyangonin. Flavokavain refers to a mixture of two substances, flavokavain A and flavokavain B.

[0013] Optionally, the extraction time of S1 is 60-180 min, the flow rate of supercritical carbon dioxide is 10-50 ml / min, and the extraction time of S2 is 120-180 min, the flow rate of supercritical carbon dioxide is 60-100 ml / min.

[0014] By adopting the above technical solution, the use of low temperature and low pressure processing conditions in S1, and the combination of the above extraction time and supercritical carbon dioxide flow rate to process the raw material is one of the basic conditions for completely separating the less polar flavoka wine (flavoka wine A and flavoka wine B) and the more polar kavalactone. In this process parameter, if the extraction time is too short (less than 60 min) and the flow rate of supercritical carbon dioxide is too slow (less than 10 ml / min), too much flavoka wine remains in the residue, which affects the separation effect of flavoka wine and kavalactone; if the extraction time is too long (more than 180 min) and the flow rate of supercritical carbon dioxide is too fast (more than 50 ml / min), too much kavalactone is extracted together with flavoka wine after S1 processing, and less kavalactone remains in the residue, which affects the yield of kavalactone. Therefore, in S1, to achieve separation of kavalactones and flavokaoin in the residue, an extraction time of 60-180 min, a flow rate of supercritical carbon dioxide of 10-50 ml / min, and low temperature and low pressure processing conditions are required. In S2, the residue is treated under high temperature and high pressure processing conditions combining the above extraction time and flow rate of supercritical carbon dioxide, and finally complete separation of flavokaoin and kavalactones is achieved, with flavokaoin remaining in the residue and kavalactones entering the extract. In this process parameter, if the extraction time is too short (less than 120 min) and the flow rate of supercritical carbon dioxide is too slow (less than 60 ml / min), too much kavalactone will remain in the residue, which will affect the separation effect of flavokawein and kavalactone and the yield of kavalactone. If the extraction time is too long (more than 180 min) and the flow rate of supercritical carbon dioxide is too fast (more than 100 ml / min), when kavalactone is dispersed in the extractant, other substances (meaning highly polar substances other than kavalactone and flavokawein) will be extracted together, which will further affect the yield and purity of the obtained kavalactone crystals. Therefore, in S2, to achieve the separation of kavalactone and flavokawein in the residue, an extraction time of 120-180 min, a flow rate of supercritical carbon dioxide of 60-100 ml / min, and high temperature and pressure processing conditions are required, and the content of flavokawein in the obtained kavalactone crystals is only 0.05-0.09 wt%.

[0015] Optionally, the re-extraction process of S3 includes a step of dissolving the extracted oil in a first solvent, and then extracting with a re-extraction solvent to obtain an extraction mother liquor, the first solvent being an 80-95 Vol.% ethanol solution or an 80-95 Vol.% methanol solution, and the re-extraction solvent being selected from one or more of n-hexane, petroleum ether, and ethyl ether.

[0016] By adopting the above technical solution, kavalactone and flavokawains (flavokawains A and B) can be further separated by utilizing the difference in solubility in solvents due to the difference in polarity between kavalactone and flavokawains (flavokawains A and B), so as to achieve the purpose of retaining more kavalactone in the extraction mother liquor. The above first solvent and re-extraction solvent can be used to achieve a better separation effect. In the solution, the first solvent is an 80 to 95 Vol.% ethanol solution, for example, an 80 Vol.% ethanol solution, an 82 Vol.% ethanol solution, an 85 Vol.% ethanol solution, an 88 Vol.% ethanol solution, a 90 Vol.% ethanol solution, a 93 Vol.% ethanol solution, or a 95 Vol.% ethanol solution, or the first solvent is an 80 to 95 Vol.% methanol solution, for example, an 80 Vol.% methanol solution, an 82 Vol.% methanol solution, an 85 Vol.% methanol solution, an 88 Vol.% methanol solution, a 90 Vol.% methanol solution, a 93 Vol.% methanol solution, or a 95 Vol.% methanol solution.

[0017] Optionally, the adsorption temperature during the adsorption process in S3 is 50-90°C.

[0018] By adopting the above technical solution, the activity of the adsorbent is further increased by the heating operation, the adsorption effect is improved, and the adsorption is more efficient and sufficient by the heating treatment. In the above solution, the adsorption temperature during the adsorption treatment in S3 is 50 to 90°C, and may be, for example, 50°C, 54°C, 58°C, 61°C, 65°C, 69°C, 73°C, 76°C, 80°C, 82°C, 85°C, 88°C, or 90°C.

[0019] Further optionally, the adsorbent in the adsorption process of S3 is selected from one or more of silica gel, alumina, activated carbon, C18, polyamide, and Sephadex LH-20.

[0020] By adopting the above technical solution, flavokawains (flavokawains A and flavokawains B) can be further adsorbed on the adsorbent, and kavalactones and flavokawains (flavokawains A and flavokawains B) can be further separated, and the separation effect can be improved.

[0021] Further optionally, the adsorbent is a mixture of C18, silica gel, and activated carbon, and the weight ratio of the C18, silica gel, and activated carbon is (0.8-1.5):(4.5-5.8):(3-4).

[0022] By adopting the above technical solution, the separation effect of kavalactone and flavokawains (flavokawains A and flavokawains B) is further improved by using the above preferred adsorbent. In the above solution, the weight ratio of C18, silica gel and activated carbon is 0.8:(4.5~5.8):(3~4), 1.0:(4.5~5.8):(3~4), 1.2:(4.5~5.8):(3~4), 1.5:(4.5~5.8):(3~4), (0.8~1.5):4.5:(3~4), (0.8~1.5):4.7:(3~4), (0. The ratio may be (0.8~1.5):5.1:(3~4), (0.8~1.5):5.3:(3~4), (0.8~1.5):5.6:(3~4), (0.8~1.5):5.8:(3~4), (0.8~1.5):(4.5~5.8):3, (0.8~1.5):(4.5~5.8):3.5 or (0.8~1.5):(4.5~5.8):4.

[0023] More optionally, the amount of the adsorbent added is 1-5 wt% of the extracted oil obtained in S2, and the adsorption time is 4-8 h.

[0024] Optionally, the crystallization process of S4 includes dissolving the primary product of the kavalactone in a second solvent, heating, then adding a third solvent and stirring, stopping the stirring when crystals appear, and then allowing to stand to separate the solid and liquid to obtain crystals.

[0025] By adopting the above technical solution, kavalactone is gradually precipitated by changing the polarity of the solvent, and flavokavian (flavokavian A and flavokavian B) remains in the mother liquor, and the separation of kavalactone and flavokavian (flavokavian A and flavokavian B) is further realized, and the amount of flavokavian (flavokavian A and flavokavian B) remaining in the obtained kavalactone crystals is reduced.

[0026] Further optionally, the heating temperature in S4 is 40 to 80° C., the second solvent is selected from any one or more of methyl acetate, methylbutanone, methanol, ethanol, acetone, and ethyl acetate, and the third solvent is selected from any one or more of petroleum ether, n-hexane, and methylene chloride.

[0027] Further optionally, the volume ratio of the second solvent to the third solvent is (8.5-10):1.8.

[0028] Further optionally, the standing time of S4 is 12 to 24 hours.

[0029] In a second aspect, the present application provides a kavalactone, which adopts the following technical solutions:

[0030] The kavalactone is obtained by the above-mentioned method, and the total amount of flavokawain A and flavokawain B in the kavalactone is 0.05 to 0.18 wt %. Furthermore, the total amount of flavokawain A and flavokawain B in the kavalactone is 0.05 to 0.09 wt %.

[0031] By adopting the above technical solutions, the kavalactone obtained by the present application is a product completely separated from flavokawain A and flavokawain B, and the amount of flavokawain (flavokawain A and flavokawain B) contained therein is very small, only 0.05-0.18 wt%, and in a further optional means, the content is only 0.05-0.09 wt%, so that the safety of the kavalactone is high.

[0032] In a third aspect, the present application provides a microencapsulated kavalactone, which adopts the following technical solutions:

[0033] The microencapsulated kavalactone comprises an inner core and an outer layer, and the raw material of the inner core comprises the above-mentioned kavalactone.

[0034] By adopting the above technical solutions, when the auxiliary materials are added, the oil components still existing in the kavalactone are absorbed, and the kavalactone is in a drier environment; at the same time, after microencapsulation, the direct contact between the kavalactone and the air can be avoided, and the qualitative change of the kavalactone during storage can be prevented, so that the storage period can be extended and the storage and transportation are more convenient; and after microencapsulation, the product becomes water-dispersible, which can promote the digestion and absorption by the human body and improve its utilization rate.

[0035] Optionally, the outer layer material includes an auxiliary material, the auxiliary material being a mixture of emulsified starch and dextrin, and the weight ratio of the emulsified starch to the dextrin is (1-10):1.

[0036] Optionally, the method for preparing the microencapsulated kavalactone comprises the steps of: I. Preparation of oil phase: The kavalactone is dissolved in 85-95 vol.% ethanol solution, then concentrated to obtain an oil, which is then stored at 75-82° C. for later use. Preparation of water phase: The auxiliary materials are added to hot water and dissolved for further use. The weight ratio of the auxiliary material to the kavalactone is (10-200):10. II. The water phase and the oil phase are mixed under high shear and then spray dried to obtain microencapsulated kavalactone.

[0037] The content of the microencapsulated kavalactone prepared in the present application can be adjusted by adding an appropriate amount of auxiliary material as needed. For example, if a higher content of kavalactone is required in the microencapsulated kavalactone, a small amount of auxiliary material can be added, and if a lower content of kavalactone is required in the microencapsulated kavalactone, a larger amount of auxiliary material can be added.

[0038] Further optionally, the temperature of the hot water in which the auxiliary material is dissolved is 80 to 100°C.

[0039] Further optionally, the high-speed shear time in II is 30 to 60 min, the rotation speed is 8000 to 12000 rpm, and the temperature during the spray drying is 80 to 90°C.

[0040] Optionally, the raw material of the microencapsulated kavalactone also includes a film-forming agent, and the weight ratio of the film-forming agent to the auxiliary material is (1-3):1000.

[0041] Further optionally, the film-forming agent is selected from any one or more of gum arabic, xanthan gum, and carrageenan.

[0042] Further optionally, when the raw material of the microencapsulated kavalactone also includes a film-forming agent, the method for preparing the microencapsulated kavalactone comprises the steps of: I. Preparation of oil phase: The kavalactone is dissolved in 85-95 vol.% ethanol solution, then concentrated to obtain an oil, which is then stored at 75-82° C. for later use. Preparation of water phase: The auxiliary materials are added to hot water and dissolved for further use. The weight ratio of the auxiliary material to the kavalactone is (10-200):10. II. The water phase, the oil phase and the film-forming agent are mixed under high shear, followed by spray drying to obtain microencapsulated kavalactone. Effect of the Invention

[0043] In summary, the present application has the following beneficial effects: 1. This application adopts two-stage supercritical extraction, and the first stage of supercritical extraction is mainly to separate most of the flavoka wines (flavoka wines A and B) and kavalactone, during which kavalactone remains in the residue and flavoka wine is present in the extract, and then through the second stage of supercritical extraction, most of the kavalactone and flavoka wine in the residue are further separated, during which kavalactone is present in the extract and some of the flavoka wine is also present in the extract. Then, the kavalactone and flavoka wine in the extract are further separated by re-extraction and adsorption treatment, and the content of flavoka wine in the finally obtained kavalactone crystals is 0.05-0.18wt%. In some solutions, the content of flavoka wine in kavalactone crystals is only 0.05-0.09wt%, which is less than 0.1wt%. 2. In this application, the separation effect of kavalactone and flavokawain is further improved by optimizing the extractant and adsorbent used in the re-extraction and adsorption treatment. 3. In this application, the prepared kavalactone crystals are further microencapsulated to extend the storage time of kavalactone and facilitate its transportation. After microencapsulation, the product becomes water-dispersible, which can promote the human body's digestion and absorption and improve its utilization rate. [Brief description of the drawings]

[0044] [Figure 1] 1 is a liquid chromatogram of flavokawain in kavalactone crystals obtained in Separation Method Example 2. [Diagram 2] 1 is a liquid chromatogram of kavalactone in kavalactone crystals obtained in Separation Method Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] The present invention will be described in more detail below in conjunction with examples.

[0046] Example of a method for separating flavokawains and kavalactones Separation Method Example 1 The method for separating flavokawains and kavalactones includes the following steps: S1, kava root was crushed, 1 kg of kava root powder was taken and added to the supercritical extraction kettle, then the temperature was set at 50°C, the pressure was set at 8MPa, carbon dioxide was introduced at a flow rate of 10ml / min, the extraction was stopped after 60min, then the extract was discarded and the residue was collected for later use. The yield of extracted oil (weight) was 1.8%, i.e. 18g of extracted oil was obtained, the total content of flavokawain A and flavokawain B in the extracted oil was 8.51wt%, i.e. 1.53g, and the content of kavalactone in the extracted oil was 1.02wt%, i.e. 0.18g. The residue obtained in S2 and S1 was again placed in the supercritical extraction vessel, then the temperature was set at 60°C, the pressure was set at 20MPa, carbon dioxide was introduced at a flow rate of 60ml / min, and the extraction was stopped after 180min of extraction treatment, then the residue was discarded, and the extracted oil was collected for later use. The yield of the extracted oil was 12.4wt%, i.e. 24g of extracted oil was obtained, the content of kavalactone in the extracted oil obtained was 75.43wt%, i.e. 94g, and the content of flavokawains (flavokawains A and B) in the extracted oil obtained was 2.02wt%, i.e. 2.50g. The extracted oil obtained in S3 and S2 was subjected to re-extraction and adsorption. The specific steps are as follows: The extracted oil was dissolved in 80Vol.% methanol solution with a volume twice that of the extracted oil, and then n-hexane was added with an equal volume to the 80Vol.% methanol solution to perform re-extraction, and the mixture was thoroughly shaken and mixed, and then allowed to stand to further separate kavalactone and flavokawains (flavokawains A and flavokawains B). The alcohol phase was collected to form an extraction mother liquor, which was then concentrated into an oily liquid weighing 120g for later use. After adding 80Vol.% methanol solution to the concentrated extraction mother liquor to dissolve it, the solution was heated to 50°C, 1.2g of adsorbent was added, and the mixture was stirred for 4h to allow adsorption, and then filtered. The resulting separated liquid was concentrated into an ointment of about 90g, which was then stored for later use. Here, the adsorbent was Sephadex LH-20, i.e., hydroxypropyl dextran gel. S4, the ointment concentrate obtained in S3 was dissolved in ethyl acetate, and the amount of ethyl acetate added was 0.5 times the weight of the ointment concentrate, i.e., 45 g. After heating the solution, petroleum ether was slowly added and stirred at 25 rpm. The volume ratio of ethyl acetate to petroleum ether was 8.5:1.8. When crystals precipitated, the stirring was stopped and the solution was left to stand for 12 hours to crystallize, and then filtered to obtain kavalactone crystals.

[0047] Separation Method Example 2 The method for separating flavokawains and kavalactones, the specific steps of which are as follows: S1, kava root was crushed, 1 kg of kava root powder was taken and added to the supercritical extraction kettle, then the temperature was set at 48°C, the pressure was set at 6MPa, carbon dioxide was introduced at a flow rate of 30ml / min, and the extraction was stopped after 120min extraction process, then the extract was discarded and the residue was collected for later use. The yield of extracted oil (weight) was 2%, i.e. 20g of extracted oil was obtained, the total content of flavokawain A and flavokawain B in the extracted oil was 10.04wt%, i.e. 2g, and the content of kavalactone in the extracted oil was 1.5wt%, i.e. 0.3g, and this step can remove about 50% of the flavokawains (flavokawain A and flavokawain B). The residue obtained in S2 and S1 was again placed in the supercritical extraction vessel, then the temperature was set at 70°C, the pressure was set at 35MPa, carbon dioxide was introduced at a flow rate of 80ml / min, the extraction was stopped after 150min, then the residue was discarded, and the extracted oil was collected for later use. The yield of the extracted oil was 13wt%, i.e. 130g of extracted oil was obtained, the content of kavalactone in the extracted oil obtained was 85.62wt%, i.e. 111.3g, and the content of flavokawains (flavokawains A and B) in the extracted oil obtained was 2.53wt%, i.e. 3.3g. The extracted oil obtained in S3 and S2 was subjected to re-extraction and adsorption. The specific steps are as follows: The extracted oil was dissolved in a 90Vol.% ethanol solution with a volume 3.5 times that of the extracted oil, and then an equal volume of petroleum ether was added to the 90Vol.% ethanol solution to perform re-extraction, and the mixture was thoroughly shaken and then allowed to stand to further separate kavalactone and flavokawains (flavokawains A and flavokawains B). The alcohol phase was collected to form an extraction mother liquor, which was then concentrated into an oily liquid weighing 125g for later use. After adding and dissolving the concentrated extraction mother liquor with a 90Vol.% ethanol solution, the solution was heated to 70°C, 4.6g of adsorbent was added, and the mixture was stirred for 5h to adsorb, and then filtered. The resulting separated liquid was concentrated into an ointment of about 98g, which was then stored for later use. Here, the adsorbent was a mixture of C18, silica gel, and activated carbon in a weight ratio of 1.2:5.3:3.5. The ointment concentrates obtained in S4 and S3 were dissolved in methanol, the amount of methanol added was 98 g, the solution was heated, dichloromethane was slowly added, and the mixture was stirred at 25 rpm, the volume ratio of methanol to dichloromethane was 9.2:1.8, and when crystals precipitated, the stirring was stopped and the mixture was left for 20 hours to crystallize, and then filtered to obtain kavalactone crystals. The liquid chromatographs of flavokawain and kavalactone in the kavalactone crystals are shown in Figures 1 and 2, respectively.

[0048] Separation Method Example 3 The method for separating flavokawains and kavalactones includes the following steps: S1, kava root was crushed, 1 kg of kava root powder was taken and added to the supercritical extraction kettle, then the temperature was set at 45°C, the pressure was set at 4MPa, carbon dioxide was introduced at a flow rate of 50ml / min, the extraction was stopped after 180min, then the extract was discarded and the residue was collected for later use. The yield of extracted oil (weight) was 2.5%, i.e. 25g of extracted oil was obtained, the total content of flavokawain A and flavokawain B in the extracted oil was 13.07wt%, i.e. 3.27g, and the content of kavalactone in the extracted oil was 2.07wt%, i.e. 0.5g. The residue obtained in S2 and S1 was again placed in the supercritical extraction vessel, then the temperature was set at 80°C, the pressure was set at 50MPa, carbon dioxide was introduced at a flow rate of 100ml / min, the extraction was stopped after 120min, then the residue was discarded, and the extracted oil was collected for later use. The yield of the extracted oil was 11wt%, i.e. 110g of extracted oil was obtained, the content of kavalactone in the extracted oil obtained was 88.16wt%, i.e. 97.0g, and the content of flavokawains (flavokawains A and B) in the extracted oil obtained was 3.07wt%, i.e. 3.4g. The extracted oil obtained in S3 and S2 was subjected to re-extraction and adsorption. The specific steps are as follows: The extracted oil was dissolved in 95Vol.% ethanol solution with a volume 5 times that of the extracted oil, and then ethyl ether was added in an equal volume to the 95Vol.% ethanol solution to perform re-extraction, and the mixture was thoroughly shaken and then allowed to stand to further separate kavalactone and flavokawains (flavokawains A and flavokawains B). The alcohol phase was collected to form an extraction mother liquor, which was then concentrated into an oily liquid weighing 122g for later use. After adding and dissolving 95Vol.% ethanol solution into the concentrated extraction mother liquor, the solution was heated to 90°C, 6.7g of adsorbent was added, and the mixture was stirred for 8h to adsorb, and then filtered. The resulting separated liquid was concentrated into an ointment of about 103g, which was then stored for later use. Here, the adsorbent was obtained by mixing silica gel and activated carbon in a weight ratio of 5.3:3.5. S4, the ointment concentrate obtained in S3 was dissolved in acetone, and the weight of acetone added was twice that of the ointment concentrate product, i.e., 206 g. After heating the solution, n-hexane was slowly added and stirred at a rotation speed of 25 rpm. The volume ratio of acetone to n-hexane was 10:1.8. When crystals precipitated, the stirring was stopped and the solution was left to stand for 24 hours to crystallize, and then filtered to obtain kavalactone crystals.

[0049] Separation Method Examples 4 to 11 Compared with Example 2, the difference between the following examples is that when step S1 or S2 reacts in the supercritical extraction kettle, the reaction temperature or pressure is set differently, for details, see Table 1.

[0050] Table 1. Reaction temperatures and pressures in S1 and S2 in different embodiments JPEG0007625756000001.jpg64160

[0051] Separation Method Example 12 Compared with Example 2, this Example is different in that the adsorbent selected in step S3 is different. Specifically, the adsorbent in this Example is a mixture of C18, silica gel, and activated carbon in a weight ratio of 0.8:4.5:3, and the rest is the same as Example 2.

[0052] Separation Method Example 13 Compared with Example 2, this Example is different in that the adsorbent selected in step S3 is different. Specifically, the adsorbent in this Example is a mixture of C18, silica gel, and activated carbon in a weight ratio of 1.5:5.8:4, and the rest is the same as Example 2.

[0053] Separation method comparison examples 1 to 8 Compared with Example 2, the following Comparative Examples are distinguished by the set reaction time or the flow rate of supercritical carbon dioxide when reacting in the supercritical extraction vessel in step S1 or S2, and for details, see Table 2.

[0054] Table 2. Reaction times and supercritical carbon dioxide flow rates in S1 and S2 in different embodiments JPEG0007625756000002.jpg75165

[0055] Separation method comparison example Separation method comparison examples 1 to 7 Compared with Example 2, the following Comparative Examples are different in that in step S1 or S2, when the reaction is carried out in a supercritical extraction vessel, the reaction temperature or pressure is set differently; for details, see Table 3.

[0056] Table 3. Reaction parameters of S1 and S2 in different comparative embodiments JPEG0007625756000003.jpg69147

[0057] Compared with Example 2, the difference of this comparative example is that step S1 is not performed in the separation method, and the separation method is specifically as follows. S1, kava root was crushed, 1 kg of kava root powder was taken and added to the supercritical extraction kettle, then the temperature was set at 70°C, the pressure was set at 35 MPa, carbon dioxide was introduced at a flow rate of 80 ml / min, the extraction was stopped after 150 min, then the residue was discarded and the extracted oil was collected for later use. S2, the specific step is similar to step S3 in the second embodiment. S3, the specific steps are the same as step S4 in Example 2, and finally, kavalactone crystals are obtained. The kavalactone content in the kavalactone crystals is 93.64 wt%, and the total content of flavokawein A and flavokawein B is 1.21 wt%.

[0058] Separation method comparison example 9 Compared with Example 2, the difference of this comparative example is that in step S3 of the separation method, only the re-extraction process was performed, and the separation method is specifically as follows. S1 and S2 were the same as S1 and S2 in Example 2, and 130 g of extracted oil was obtained. The extracted oils obtained in S3 and S2 were subjected to re-extraction treatment. The specific steps were as follows: the extracted oil was dissolved in 90 Vol.% ethanol solution with a volume 3.5 times that of the extracted oil, then the same volume of petroleum ether as that of the 90 Vol.% ethanol solution was added for re-extraction, and the mixture was thoroughly shaken and then allowed to stand to further separate kavalactones and flavokawains (flavokawains A and B). The alcohol phase was collected to form the extraction mother liquor, which was then concentrated into an oily liquid weighing 125 g for later use. The oily concentrated extract mother liquor obtained in S4 and S3 was dissolved in 125 g of methanol, and the subsequent steps were the same as in Example 2 to obtain kavalactone crystals.

[0059] Separation method comparison example 10 The difference between this comparative example and Example 2 is that in the separation method, only the adsorption treatment was performed in step S3, and the specific separation method is as follows. S1 and S2 were the same as S1 and S2 in Example 2, and 130 g of extracted oil was obtained. The extracted oil obtained in S3 and S2 was subjected to adsorption treatment. The specific steps were as follows: the extracted oil was dissolved in 90Vol.% ethanol solution, heated to 70℃, 4.6g of adsorbent was added, stirred for 5h to adsorb, filtered, and the resulting separated liquid was concentrated into an ointment and stored for later use. Here, the adsorbent was a mixture of C18, silica gel, and activated carbon in a weight ratio of 1.2:5.3:3.5. S4 is the same as S4 in the second embodiment.

[0060] Separation method comparison example 11 Compared with Example 2, the difference of this comparative example is that in step S3 of the separation method, first, an adsorption process is performed, and then a re-extraction process is performed. The specific separation method is as follows. S1 and S2 were the same as S1 and S2 in Example 2, and 130 g of extracted oil was obtained. The extracted oil obtained in S3 and S2 was subjected to adsorption treatment. The specific steps were as follows: the extracted oil was dissolved in a 90 Vol.% ethanol solution with a volume 3.5 times that of the extracted oil, heated to 70°C, 4.6 g of adsorbent was added, stirred for 5 hours to allow adsorption, and then filtered. The resulting separated liquid was concentrated into an ointment and stored for later use. Here, the adsorbent was a mixture of C18, silica gel, and activated carbon in a weight ratio of 1.2:5.3:3.5. The concentrated ointment-like separated liquid was dissolved in 90Vol.% ethanol solution, and the amount of 90Vol.% ethanol added was 3.5 times the volume of the ointment-like separated liquid, and then an equal volume of petroleum ether was added to the 90Vol.% ethanol solution to perform re-extraction, and the mixture was thoroughly shaken and then allowed to stand to further separate kavalactones and flavokawains (flavokawains A and B). The alcohol phase was collected to form an extraction mother liquor, which was then concentrated into an oil for later use. The oily concentrated extract mother liquor obtained in S4 and S3 was dissolved in methanol, and the subsequent steps were the same as in Example 2 to obtain kavalactone crystals.

[0061] Performance Detection Test The method for detecting the content of kavalactone, flavokawain A and flavokawain B was based on the journal article Piper methysticum Rootstock Dry Extract published in the Herbal Medicines Compendium, and the content was detected by high performance liquid chromatography. The specific results are shown in Table 4.

[0062] Table 4. Content of kavalactones and flavocarboxaldehyde in the products of different stages obtained from different embodiments. JPEG0007625756000004.jpg244162JPEG0007625756000005.jpg244162JPEG0007625756000006.jpg238162

[0063] From the data results of Example 2, Examples 4-5 and Comparative Examples 1-2, it is recommended to set the extraction pressure in the range of 4-8Mpa in step S1. If the pressure is set to less than 4Mpa (Comparative Example 1), the separation effect of flavoka wines (flavoka wines A and flavoka wines B) in step S1 becomes poor, and a large amount of flavoka wines (flavoka wines A and flavoka wines B) exists in the residue, and a large amount of flavoka wines (flavoka wines A and flavoka wines B) and kavalactone are extracted together in step S2. However, in the subsequent re-extraction and adsorption operation, flavoka wines (flavoka wines A and flavoka wines B) and kavalactone cannot be completely separated, so the content of flavoka wines (flavoka wines A and flavoka wines B) in the obtained kavalactone crystals is relatively high. If the pressure is set higher than 8Mpa (Comparative Example 2), a large amount of kavalactone and flavokavain (flavokavain A and flavokavain B) will be extracted together in step S1 and discarded as waste liquid, which will directly lead to excessive loss of kavalactone; at the same time, excessive extraction pressure will cause great damage to the cells of the raw material in the S1 extraction process, making it easier to extract the cell contents, so that the content of flavokavain (flavokavain A and flavokavain B) in the extracted oil after re-extraction in S2 will be high, as well as the content of other impurities. Therefore, considering the utilization rate of kavalactone and the content of flavokavain in kavalactone crystals, it is not recommended to use an extraction pressure higher than 8Mpa in step S1.

[0064] From the data results of Example 2, Examples 6-7 and Comparative Examples 3-4, it can be seen that in step S1, it is recommended to set the extraction temperature in the range of 45-50°C. When the temperature is set below 45°C (Comparative Example 3), a large amount of flavoka wines (flavoka wine A and flavoka wine B) also exists in the residue, which is then extracted together with kavalactone in step S2, and the content of flavoka wines (flavoka wine A and flavoka wine B) in the finally obtained kavalactone crystals is relatively high. Setting the temperature in step S1 higher than 50°C (Comparative Example 4) also causes problems such as excessive loss of kavalactone and high content of flavoka wines (flavoka wine A and flavoka wine B), so it is not recommended to use an extraction temperature higher than 50°C in step S1.

[0065] From the data results of Example 2, Examples 8-9 and Comparative Example 5, it can be seen that in step S2, it is recommended to set the extraction pressure in the range of 20-50 MPa. If the pressure is set lower than 20 MPa (Comparative Example 5), a large amount of kavalactone will exist in the residue of step S2, which will be discarded as waste, so in consideration of the utilization rate of kavalactone, it is not recommended to use an extraction pressure lower than 20 MPa in step S2, and there is a problem that, when the extraction pressure is lower than 20 MPa, flavokavian (flavokavian A and flavokavian B) is easily extracted, so that the content of flavokavian (flavokavian A and flavokavian B) in the obtained kavalactone crystals is relatively high. At the same time, it is not recommended to use a pressure higher than 50 MPa due to the pressure that the device can withstand.

[0066] From the data results of Example 2, Examples 10-11 and Comparative Examples 6-7, it can be seen that in step S2, it is recommended to set the extraction temperature in the range of 60-80° C. If the temperature is set below 60° C. (Comparative Example 6), a large amount of kavalactone will exist in the residue in step S2 and will be discarded as waste, and the lower the extraction temperature, the more suitable it is for extracting flavokavian (flavokavian A and flavokavian B), and the poorer the separation effect of kavalactone and flavokavian (flavokavian A and flavokavian B), so considering the utilization rate of kavalactone and the separation effect of kavalactone and flavokavian (flavokavian A and flavokavian B), it is not recommended that the temperature in step S2 be below 60° C. If the temperature is set higher than 80°C (Comparative Example 7), other impurities will be extracted along with kavalactone and flavokawain (flavokawain A and flavokawain B) in step S2, which directly leads to an increase in impurities and a decrease in the relative content of kavalactone in the obtained kavalactone crystals.

[0067] In this process, the extraction pressure and temperature have a relatively large impact on the separation effect, but at the same time, the extraction time and the flow rate of supercritical carbon dioxide are also one of the parameters that ensure the technical effect.

[0068] From the data results of Example 2 and Comparative Examples 1-2, it is recommended to control the extraction time of step S1 within the range of 60-180 min. If the extraction time is less than 60 min (Comparative Example 1), it is difficult to fully separate flavowains (flavowains A and flavowains B) from kavalactone in the residue, and the content of flavowains (flavowains A and flavowains B) obtained in the extracted oil after step S1 is low, so that the content of flavowains (flavowains A and flavowains B) in the finally obtained kavalactone crystals is relatively high. When the extraction time exceeds 180 min (Comparative Example 2), the separation effect of kavalactone and flavokawein is also reduced, and the content of flavokawein (flavokawein A and flavokawein B) in the obtained kavalactone crystals is relatively high. The reason for this may be that, even if flavokawein (flavokawein A and flavokawein B) and kavalactone in the residue can be completely separated by S1, the extraction time is too long, so that the damage to kava cells in this processing step is large, and therefore the substances in the cells are easily extracted. After the residue is treated by S2, more flavokawein (flavokawein A and flavokawein B) and other impurities are extracted together with kavalactone, so that the content of flavokawein (flavokawein A and flavokawein B) in the finally obtained kavalactone crystals is relatively high.

[0069] The results of Example 2 and Comparative Examples 3-4 show that it is recommended to control the flow rate of supercritical carbon dioxide during extraction in S1 within the range of 10-50 ml / min, and exceeding this range will also affect the separation effect of kavalactones and flavokawains.

[0070] In addition, Comparative Examples 5 to 8 show the effects of the extraction time and the flow rate of supercritical carbon dioxide on the separation effect in step S2. In Comparative Example 5, if the extraction time is too short, the yield of the extracted oil is directly affected, and at the same time, the extraction to kavalactone is insufficient, so that the proportion of flavoka wines (flavoka wine A and flavoka wine B) in the extracted oil is relatively high, and finally, the content (0.09 wt%) of flavoka wines (flavoka wine A and flavoka wine B) in the obtained kavalactone crystals is slightly high. In Comparative Example 6, if the extraction time is too long, other impurities are extracted together with kavalactone and flavoka wines (flavoka wine A and flavoka wine B), so the relative content of kavalactone is reduced, and the content (0.09 wt%) of flavoka wines (flavoka wine A and flavoka wine B) in the finally obtained kavalactone crystals is slightly high. Similarly, the flow rate of supercritical carbon dioxide has a similar effect on the separation effect. In Comparative Example 7, when the flow rate of supercritical carbon dioxide is less than 60 ml / min or more than 100 ml / min, there exists a situation where the content of flavokawains (flavokawains A and flavokawains B) in the kavalactone crystals is slightly higher.

[0071] However, among the four extraction parameters (temperature, pressure, time, and flow rate of supercritical carbon dioxide) in steps S1 and S2, pressure and temperature have a relatively large effect on the separation effect, while time and flow rate of supercritical carbon dioxide have a relatively small effect on the separation effect.

[0072] The data results of Example 2 and Comparative Example 8 show that when kavalactone and flavokawein (flavokawein A and flavokawein B) are directly separated by high-temperature and high-pressure extraction, it is difficult to achieve the purpose of effective separation, and the obtained kavalactone crystals still contain 1.21 wt% flavokawein.

[0073] In addition, further combining with the data of Comparative Examples 9-11, it can be seen that re-extraction and adsorption treatment are also important for the separation of kavalactone and flavocarnitine. First, kavalactone and flavocarnitine are further separated by re-extraction, and then kavalactone and flavocarnitine are further separated by adsorption. In this solution, it is not recommended to first adsorb and then re-extract, otherwise it is difficult to achieve the separation effect, and the content of flavocarnitine in the obtained kavalactone crystals is still more than 0.1wt%.

[0074] Examples of Microencapsulated Kavalactones Example 1 of Microencapsulated Kavalactone The microencapsulated kavalactone includes an inner core and an outer layer, and the raw materials for preparing the inner core are kavalactone crystals and 85 vol.% ethanol solution, and the raw materials for preparing the outer layer are auxiliary materials, a film-forming agent, and water. The weight ratio of the auxiliary materials to kavalactone is 1:1, and the auxiliary materials are obtained by mixing emulsified starch and dextrin in a weight ratio of 1:1. The film-forming agent is xanthan gum, and the weight ratio of the film-forming agent to the auxiliary materials is 1:100. The kavalactone crystals are prepared using separation method Example 1.

[0075] The method for preparing microencapsulated kavalactone is as follows. I. Preparation of oil phase: 200 g of kavalactone crystals were dissolved in 85 Vol.% ethanol solution, concentrated into an oil, and stored at 75° C. for later use. Preparation of water phase: 200g of prepared auxiliary materials are added to 1600g of hot water at 80°C to dissolve, and used later. II. The oil phase was added to the water phase under high-speed shear at 8000 rpm, then 0.2 g of xanthan gum was added, and the mixture was subjected to high-speed shear for 30 minutes, followed by spray drying at 80° C. to obtain microencapsulated kavalactone. The microencapsulated kavalactone can be stored at room temperature for 24 months.

[0076] Example 2 of Microencapsulated Kavalactone The microencapsulated kavalactone includes an inner core and an outer layer, and the raw materials for preparing the inner core are kavalactone crystals and a 90 vol.% ethanol solution, and the raw materials for preparing the outer layer are auxiliary materials, a film-forming agent, and water. Here, the weight ratio of the auxiliary materials to kavalactone is 22:10, and the auxiliary materials are obtained by mixing emulsified starch and dextrin in a weight ratio of 4:1. The film-forming agent is gum arabic, and the weight ratio of the film-forming agent to the auxiliary materials is 2:100. The kavalactone crystals were prepared using the separation method Example 2.

[0077] The method for preparing microencapsulated kavalactone is as follows. I. Preparation of oil phase: 200 g of kavalactone crystals were dissolved in 90 Vol.% ethanol solution, concentrated into an oil, and stored at 80° C. for later use. Preparation of water phase: 440g of the prepared auxiliary materials are added to 3960g of hot water at 90°C to dissolve, and used later. II. The oil phase was added to the water phase under high-speed shear at a rotation speed of 10,000 rpm, then 0.88 g of gum arabic was added, and high-speed shear was performed for 45 minutes, followed by spray drying at 85°C to obtain microencapsulated kavalactone. The microencapsulated kavalactone can be stored at room temperature for 26 months. The content of kavalactone in the microencapsulated kavalactone was 30.52 wt%.

[0078] Example 3 of Microencapsulated Kavalactone The microencapsulated kavalactone includes an inner core and an outer layer, and the raw materials for preparing the inner core are kavalactone crystals and a 95Vol.% ethanol solution, and the raw materials for preparing the outer layer are auxiliary materials, a film-forming agent, and water. Here, the weight ratio of the auxiliary materials to kavalactone is 20:1, and the auxiliary materials are obtained by mixing emulsified starch and dextrin in a weight ratio of 10:1. The film-forming agent is carrageenan, and the weight ratio of the film-forming agent to the auxiliary materials is 3:100. The kavalactone crystals are prepared using the separation method of Example 3.

[0079] The method for preparing microencapsulated kavalactone is as follows. I. Preparation of oil phase: 200 g of kavalactone crystals were dissolved in 95 Vol.% ethanol solution, concentrated into an oil, and stored at 82° C. for later use. Preparation of aqueous phase: 4 kg of the prepared auxiliary materials are added to 40 kg of hot water at 100°C to dissolve, and then used later. II. The oil phase was added to the water phase under high-speed shear at a rotation speed of 12000 rpm, then 12 g of carrageenan was added, and the mixture was subjected to high-speed shear for 60 minutes, followed by spray drying at 90°C to obtain microencapsulated kavalactone. The microencapsulated kavalactone can be stored at room temperature for 25 months.

[0080] Example 4 of Microencapsulated Kavalactone The difference between this embodiment and Example 2 of the microencapsulated kavalactone is that in this embodiment, the raw materials for preparing the outer layer do not contain a film-forming agent, and the rest are the same as Example 2 of the microencapsulated kavalactone.

[0081] When preparing the microencapsulated kavalactone, step 1 is the same as in Example 2 of the microencapsulated kavalactone, and step II specifically includes: II. The oil phase was added to the water phase under high-speed shear at a rotation speed of 10,000 rpm, and the mixture was subjected to high-speed shear for 45 minutes, followed by spray drying at 85°C to obtain microencapsulated kavalactone. The microencapsulated kavalactone could be stored at room temperature for 26 months.

[0082] If the kavalactone crystals are not microencapsulated, the shelf life of the kavalactone crystals is shortened to 5 to 7 months. This is because the obtained kavalactone crystals contain some oil, and the untreated oil is easily spoiled, shortening the shelf life of the kavalactone crystals. After microencapsulation, the oil contained in the kavalactone is adsorbed by the auxiliary material, making it less likely to come into contact with air, and the shelf life is improved.

[0083] This specific embodiment is merely illustrative of the present application and does not limit the present application. After reading this specification, those skilled in the art can make modifications to this embodiment as necessary without making any creative contribution, but as long as they are within the scope of the claims of this application, they will be protected by patent law.

Claims

1. A method for separating flavokawains and kavalactones, comprising the steps of: Step S1, in which the kava root is crushed and the residue is collected for use after extraction with supercritical carbon dioxide, the extraction temperature being 45-50°C and the pressure being 4-8 MPa; Step S2, extracting the residue with supercritical carbon dioxide and then collecting the extracted oil for later use, the extraction temperature is 60-80°C and the pressure is 20-50Mpa; Step S3 of subjecting the extracted oil to a re-extraction process and an adsorption process to obtain a primary product of kavalactone; and step S4 of crystallizing the primary product of kavalactone to obtain kavalactone, The method is characterized in that the extraction time of S1 is 60 to 180 min, the flow rate of supercritical carbon dioxide is 10 to 50 ml / min, and the extraction time of S2 is 120 to 180 min, and the flow rate of supercritical carbon dioxide is 60 to 100 ml / min.

2. The method according to claim 1, wherein the re-extraction process of S3 includes a step of dissolving the extracted oil in a first solvent and then extracting with a re-extraction solvent to obtain an extraction mother liquor, the first solvent being an 80-95 vol. % ethanol solution or an 80-95 vol. % methanol solution, and the re-extraction solvent is selected from one or more of n-hexane, petroleum ether, and ethyl ether.

3. The method according to claim 1, wherein the adsorption temperature during the adsorption treatment in S3 is 50 to 90°C.

4. The method according to claim 1, characterized in that the adsorbent in the adsorption treatment of S3 is selected from one or more of silica gel, alumina, activated carbon, C18, polyamide, and hydroxypropyl dextran gel.

5. 5. The method of claim 4, wherein the adsorbent is a mixture of C18, silica gel, and activated carbon, and the weight ratio of C18, silica gel, and activated carbon is (0.8-1.5):(4.5-5.8):(3-4).

6. The method according to claim 1, characterized in that the crystallization treatment in step S4 includes the steps of dissolving the primary product of the kavalactone in a second solvent, heating the solution, adding a third solvent and stirring the solution, stopping the stirring when crystals appear, and allowing the solution to stand for solid-liquid separation to obtain kavalactone crystals.

7. The method according to claim 6, wherein the heating temperature in S4 is 40 to 80° C., the second solvent is selected from any one or more of methyl acetate, methylbutanone, methanol, ethanol, acetone, and ethyl acetate, and the third solvent is selected from any one or more of petroleum ether, n-hexane, and methylene chloride.

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