Method for extracting triterpenoids from kadsura coccinea

Abstract

The present application belongs to the technical field of natural plant extraction, and particularly relates to a method for extracting triterpenoid compounds from kadsurae. The method comprises the following steps: S1, mixing an extracting agent with kadsurae, and sequentially performing ultrasonic extraction and centrifugation to obtain a crude extract; S2, adsorbing the crude extract with a macroporous resin, and eluting the crude extract with an ethanol aqueous solution with a volume fraction of 50%-80% as an eluent, and collecting the eluate to obtain triterpenoid compounds. The method has high extraction efficiency, high purity of the obtained triterpenoid compounds, and small structural damage to the triterpenoid compounds, which is beneficial to maintaining the pharmacodynamic activity of the triterpenoid compounds, and the operation process is simple, and the method can be used for industrial production and has low production cost.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of extraction of natural plants, and particularly relates to a method for extracting triterpenoid compounds from Sabia japonica Maxim. BACKGROUND

[0002] Sabia japonica Maxim. belongs to the climbing woody liana plant of Sabia of Sabia family, and is mainly distributed in Jiangsu, Anhui, Zhejiang, Fujian, Jiangxi, Guangdong, Guangxi and Guizhou of China. The leaves, roots and stems of Sabia japonica Maxim. can be used as medicine. Since it has good effect of dispelling wind and dredging collaterals, it is often used for treating rheumatism and arthralgia in ancient times. It is recorded in Bencao Gangmu that “Sabia japonica Maxim. is used for treating rheumatism, arthralgia, pruritus, injury and swelling”. It is recorded in Anhui Chinese Herbal Medicine that Sabia japonica Maxim. has the effects of dispelling wind and cold, removing dampness and swelling, and its stems, leaves or roots can be used as medicine. It tastes bitter and pungent, is warm in nature, and belongs to liver meridian. It has the effects of dispelling wind and removing dampness, promoting blood circulation and detoxification, and is used for treating rheumatism and arthralgia, edema, beriberi, contusion and swelling, fracture, deep abscess, osteomyelitis and pruritus. Modern researches have found that Sabia japonica Maxim. has the effects of anti-inflammation, immune regulation, liver protection, antiviral, blood pressure reduction, anti-arrhythmia, antitussive and sedative, and is used for treating various inflammatory diseases, rheumatism, arthritis and other diseases. The chemical components of Sabia japonica Maxim. include triterpenoid compounds, alkaloids, flavonoids, phenylpropanoids, other benzene derivatives, fatty acids and alkanes, etc. Among them, triterpenoid compounds are the most abundant compounds in Sabia japonica Maxim., and are also the key pharmacodynamic components.

[0003] At present, the active ingredients are mainly extracted from natural products by using organic solvent extraction method. However, the use of too much organic solvent and the long extraction time will increase the production cost and cause a series of problems. Therefore, the requirement for green chemical process is higher and higher, especially in the field of natural product extraction, and it is necessary to use a more mild extraction method to reduce the use of organic solvent and shorten the extraction time. At present, there are few reports on the extraction and separation of triterpenoid compounds in Sabia japonica Maxim., and most of them still use traditional methods such as ethanol heating reflux extraction, silica gel column chromatography and gradient elution with petroleum ether-ethyl acetate system to separate the components. However, the use of large amount of organic solvent in this process, the long reflux extraction time, the possible damage to the structure of triterpenoid compounds, the low extraction efficiency and the low purity of triterpenoid compounds still need to be improved.

[0004] Therefore, it is necessary to use a more green and mild method to separate the triterpenoid compounds in Sabia japonica Maxim., and to improve the extraction yield and purity of the triterpenoid compounds in Sabia japonica Maxim. SUMMARY

[0005] The following is a summary of the subject matter of the detailed description herein. This summary is not intended to limit the scope of the claims.

[0006] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes an extraction method of triterpenoid compounds in Kadsura pannata. The extraction method of the present application has high extraction efficiency, high purity of the obtained triterpenoid compounds, small structural damage to the triterpenoid compounds, is conducive to maintaining the pharmacodynamic activity thereof, and has a simple operation process, can be used for industrial production, and has a low production cost.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] The present application provides an extraction method of triterpenoid compounds in Kadsura pannata, comprising the following steps:

[0009] S1, mixing an extractant and Kadsura pannata, and sequentially performing ultrasonic extraction and centrifugation to obtain a crude extract;

[0010] S2, adsorbing the crude extract onto a column with a macroporous resin, and eluting with an ethanol aqueous solution with a volume fraction of 50%-80% as an eluent to collect the eluate to obtain triterpenoid compounds;

[0011] The extractant comprises, in terms of mass percentage, 50%-90% of a deep eutectic solvent and 10%-50% of water, the raw materials for preparing the deep eutectic solvent include a hydrogen bond donor and a hydrogen bond acceptor, the hydrogen bond donor includes at least one of n-octanoic acid, n-decanoic acid and lauric acid, and the hydrogen bond acceptor includes menthol.

[0012] The macroporous resin is selected from AB-8 type and HPD100 type macroporous resins.

[0013] In step S2, the column loading flow rate of the crude extract is 2-4 BV / h, and the elution flow rate of the eluent is 2-4 BV / h.

[0014] The present application constructs a deep eutectic solvent-water homogeneous phase system with low viscosity and appropriate polarity under room temperature conditions through the cooperation of a deep eutectic solvent and water, which has strong biocompatibility, can further be assisted by ultrasonic extraction method, thereby improving the extraction efficiency of triterpenoid compounds in Kadsura pannata, and the process does not require long-term heating reflux, so the structural damage to the triterpenoid compounds is small, which is conducive to maintaining the pharmacodynamic activity thereof.

[0015] Specifically, the present application selects menthol as the hydrogen bond acceptor, which belongs to terpenes, and the cyclohexane part in the molecular structure can increase the hydrophobicity of menthol, avoid the dissolution of the eutectic solvent in water to affect the stability of the molar ratio of the hydrogen bond acceptor and the hydrogen bond donor and the phase volume, and enhance the extraction effect on triterpenoids. In addition, the present application selects at least one of n-octanoic acid, n-decanoic acid and lauric acid as the hydrogen bond donor, which has the advantages of low density and low viscosity (2-14 mPas); and the eutectic solvent formed by the hydrogen bond donor and the above-mentioned hydrogen bond acceptor has good hydrophobicity and is stable in water.

[0016] The triterpenoids in Kadsura pinnata have small polarity, so a weakly polar macroporous resin can be used to improve and optimize the purification and separation effect of triterpenoids and other impurities by using the adsorption of macroporous resin on triterpenoids. In addition, because the adsorption degree of triterpenoids on specific types of macroporous resin is different, too fast or too slow column flow rate and elution flow rate will affect the adsorption and desorption effect of macroporous resin on triterpenoids and the separation effect of triterpenoids and other impurities; therefore, appropriate column flow rate and elution flow rate need to be ensured during the macroporous resin adsorption and elution process.

[0017] In some embodiments of the present application, the hydrogen bond donor is selected from any two of n-octanoic acid, n-decanoic acid and lauric acid, for example, the hydrogen bond donor is selected from n-octanoic acid and n-decanoic acid, n-octanoic acid and lauric acid, and n-decanoic acid and lauric acid.

[0018] In some preferred embodiments of the present application, the hydrogen bond donor is n-decanoic acid and lauric acid. The present application finds that the use of n-decanoic acid and lauric acid in combination can further increase the enrichment purity of triterpenoids compared with the use of a single fatty acid, which may be related to the increase of the hydrophobicity of the eutectic solvent, the decrease of the melting point and the viscosity, etc.

[0019] In some preferred embodiments of the present application, the hydrogen bond acceptor is DL-menthol.

[0020] In some embodiments of the present application, the eutectic solvent is selected from menthol-n-decanoic acid-lauric acid, menthol-n-octanoic acid-lauric acid, menthol-n-octanoic acid-n-decanoic acid, menthol-n-decanoic acid, menthol-n-octanoic acid, and menthol-lauric acid.

[0021] In some embodiments of the present application, the mass percentage of the deep eutectic solvent in the extraction agent is 50%-90%, including but not limited to: 55%-90%, 60%-90%, 65%-90%, 70%-90%, 75%-90%, 80%-90%, 55%-85%, 60%-85%, 70%-85%, 80%-85%, 60%-80%, 65%-80%, 70%-80%.

[0022] In some embodiments of the present application, the mass percentage of water in the extraction agent is 10%-50%, including but not limited to: 10%-45%, 10%-40%, 10%-35%, 10%-30%, 10%-25%, 10%-20%, 15%-45%, 15%-40%, 15%-30%, 15%-25%, 15%-20%, 20%-40%, 20%-35%, 20%-30%.

[0023] In some embodiments of the present application, the volume ratio of the deep eutectic solvent and water in the extraction agent is 3-5:1.

[0024] In some embodiments of the present application, the deep eutectic solvent is prepared by heating raw materials including a hydrogen bond donor and a hydrogen bond acceptor to be clear.

[0025] In some embodiments of the present application, the raw materials of the deep eutectic solvent can also include water.

[0026] In some embodiments of the present application, the molar ratio of the hydrogen bond acceptor and the hydrogen bond donor is 1:0.2-5, including but not limited to: 1:0.2-4, 1:0.2-3, 1:0.2-2, 1:0.2-1, 1:0.2-0.5, 1:0.2-0.4, 1:0.3-4, 1:0.3-3, 1:0.3-2, 1:0.3-1, 1:0.3-0.5, 1:0.4-3, 1:0.4-2, 1:0.4-1, 1:0.5-5, 1:0.5-4, 1:0.5-2, 1:0.5-1, 1:1-5, 1:1-3, 1:1-2.

[0027] The amount ratio of the raw materials of the deep eutectic solvent has an effect on the stability of the deep eutectic solvent, and an improper ratio will cause the stability of the deep eutectic solvent to decrease, and also has a negative impact on the extraction, resulting in a decrease in the extraction yield.

[0028] In some embodiments of the present application, the heating temperature is 60-90℃.

[0029] In some embodiments of the present application, the heating time is 1-10h.

[0030] In some embodiments of the present application, before step S1, the kadsurae cortex is dried and ground into powder of 20-40 mesh.

[0031] In some embodiments of the present application, the solid-liquid ratio of the kadsurae cortex and the extracting agent is 1:5-30 g / mL, including but not limited to 1:5-25 g / mL, 1:5-20 g / mL, 1:5-15 g / mL, 1:5-10 g / mL, 1:10-30 g / mL, 1:10-25 g / mL, 1:10-20 g / mL, 1:10-25 g / mL, 1:15-30 g / mL, 1:15-25 g / mL, 1:15-20 g / mL, 1:20-30 g / mL, 1:20-25 g / mL, 1:10 g / mL, 1:20 g / mL, 1:30 g / mL.

[0032] In some embodiments of the present application, the extraction is performed twice, and the crude extracts obtained after the two extractions, ultrasonic treatment and centrifugation are combined; wherein the extracting agent added in each extraction accounts for 40%-60% of the total mass of the extracting agent.

[0033] In some embodiments of the present application, step S1 is: first, 40%-60% of the extracting agent is mixed with the kadsurae cortex, and then ultrasonic extraction and centrifugation are performed in sequence to obtain a first crude extract and a first precipitate; then the remaining extracting agent is mixed with the obtained first precipitate, and ultrasonic extraction and centrifugation are performed in sequence to obtain a second crude extract and a second precipitate, and the first crude extract and the second crude extract are combined.

[0034] In some embodiments of the present application, the temperature of the ultrasonic treatment is 15-35℃, including but not limited to 15-30℃, 15-25℃, 15-20℃, 20-30℃, 20-25℃.

[0035] In some embodiments of the present application, the frequency of the ultrasonic treatment is 700-900 W, including but not limited to 700-850 W, 700-800 W, 700-750 W, 750-800 W, 700 W, 750 W, 800 W, 850 W, 900 W.

[0036] In some embodiments of the present application, the total time of the ultrasonic treatment is 20-40 min, further 25-35 min; the ultrasonic treatment is performed twice, therefore, the time of single ultrasonic treatment is 10-20 min, further 12-18 min.

[0037] Ultrasound is a kind of high frequency mechanical wave, which can destroy the cell wall and cell membrane of traditional Chinese medicinal materials, so that the reagent can more easily penetrate into the cell, directly contact with the effective component therein and make the effective component dissolved therein, thus promoting the rapid extraction and extraction of the effective component. The ultrasonic extraction method is matched with the extractant of the present application, so that the extraction rate of triterpenoid compounds can be significantly increased.

[0038] The present application does not limit the rotation speed and time of centrifugation, and the purpose of separation can be achieved, usually: the rotation speed of centrifugation is 4000-6000 rpm, further 4500-5500 rpm. The total centrifugation time is 5-20 min, further 5-15 min; centrifugation is carried out twice, therefore, the single centrifugation time is 2-10 min, further 4-8 min.

[0039] In some embodiments of the present application, after the step S1 is completed, the obtained crude extract is further filtered through a filter membrane with a pore size of 0.22 microns.

[0040] In some embodiments of the present application, before the step S2, a pre-treatment step of macroporous resin is further included, comprising: loading the macroporous resin soaked in 95% ethanol for 24 h into a chromatographic column, allowing it to settle naturally, and after the loading of the resin is completed, washing the chromatographic column with distilled water continuously until there is no alcohol smell.

[0041] In some embodiments of the present application, the crude extract is statically adsorbed by macroporous resin for 2-6 h, and then eluted.

[0042] In some embodiments of the present application, the mass ratio of dry macroporous resin to the crude extract is 2-8:1.

[0043] In some embodiments of the present application, in step S2, before elution with the eluent, 3-6 BV / h of water is used to wash away impurities until the effluent is clear.

[0044] The extraction method of triterpenoid compounds in Sargentodoxa according to the embodiments of the present application at least has the following beneficial effects:

[0045] Compared with the heating reflux extraction technology in the prior art, the operation is complicated, energy consumption is large, energy is wasted, and a series of problems such as loss of activity of target compounds caused by excessively high temperature. The invention uses a specific extractant containing a eutectic solvent and water to extract lindera aggregata korth, the eutectic solvent can destroy the cell wall of biomass, so that the target compound is released, and the extraction effect of the target component is significantly improved, and according to the characteristics that the triterpenoid substances in lindera aggregata korth are small in polarity, the eutectic solvent of the invention can more efficiently dissolve the triterpenoid compounds through intermolecular forces such as hydrophobic interaction and hydrogen bond interaction; in addition, water can reduce the viscosity of the eutectic solvent, which is more conducive to improving the extraction yield, and can avoid the interference of some substances with larger polarity. Combined with the subsequent macroporous resin separation and purification step, the purity of the triterpenoid compounds in the obtained extraction product is further improved.

[0046] In the description of the invention, if the first, second, etc. are described, it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features or the sequence of indicated technical features.

[0047] In the description of the invention, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion, for example, a process, method, system, product or equipment comprising a series of steps or units does not have to be limited to the clearly listed steps or units, but can include other steps or units not clearly listed or inherent to these processes, methods, products or equipment.

[0048] The words "preferably", "more preferably" and the like in the present invention refer to embodiments of the invention that can provide certain advantages in certain situations. However, other embodiments can also be preferred in the same or other situations. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not usable, nor is it intended to exclude other embodiments from the scope of the invention.

[0049] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum value and the maximum value of the range, and each value between the minimum value and the maximum value. Further, when the range refers to an integer, each integer between the minimum value and the maximum value of the range is included. In addition, when multiple ranges are provided to describe characteristics or properties, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0050] "Quality parts" refers to the basic unit of measurement indicating the mass ratio relationship of multiple components, 1 part can represent any unit mass, such as 1g, 2.689g, etc. If we say that the mass of component A is a parts, and the mass of component B is b parts, it means that the mass ratio of component A to component B is a:b. Alternatively, the mass of component A is aK, and the mass of component B is bK (K is an arbitrary number, indicating a multiple factor). It should not be misunderstood that unlike the mass fraction, the sum of the mass fractions of all components is not limited to 100 parts.

[0051] "and / or" is used to indicate that one or both of the described cases can occur, for example, A and / or B includes (A and B) and (A or B). DETAILED DESCRIPTION

[0052] The concept and technical effects of the present application will be described below in conjunction with examples to fully understand the purpose, features and effects of the present application. Obviously, the described examples are only a part of the examples of the present application, not all examples, and other examples obtained by those skilled in the art based on the examples of the present application without creative labor are within the scope of protection of the present application.

[0053] In the present application, unless specified, all equipment and raw materials can be purchased from the market or commonly used in the industry. The methods in the following examples are conventional methods in the art, unless otherwise specified.

[0054] In the specific embodiment of the present application, vanillin-glacial acetic acid method is used to determine the content of triterpenoid compounds by ultraviolet spectrophotometry. The specific experimental method is as follows: take 1mL of extract in a beaker, add to a 25mL volumetric flask, dilute to volume, dissolve with a small amount of anhydrous ethanol, and test the absorbance of the test solution at 550nm wavelength using ultraviolet spectrophotometry. The absorbance value is substituted into the corresponding standard curve to calculate the content of triterpenoid compounds obtained.

[0055] The calculation formula is: triterpenoid content (mg / g) = absorbance value corresponding to triterpenoid content in standard curve x dilution factor / sample mass of flos sinopodophyllum.

[0056] The purity of triterpenoid compounds (%) = (the content of total triterpenoid compounds in the obtained final extract / the mass of the obtained final extract) x 100%.

[0057] EXAMPLE

[0058] Example 1

[0059] The present embodiment provides a method for extracting triterpenoid compounds from flos sinopodophyllum, comprising the following steps:

[0060] S1, dry Sabia japonica Maxim. is crushed, passed through a 30 mesh sieve to obtain a powder;

[0061] S2, a certain amount of lauric acid, n-decanoic acid and DL-menthol are accurately weighed in a flask in a molar ratio of 1:1:2, then heated and stirred at 80°C until a transparent homogeneous liquid is formed to obtain a eutectic solvent;

[0062] S3, the eutectic solvent obtained in step S2 is mixed with water in a volume ratio of 4:1 as an extraction solvent, and the powder obtained in step S1 is fully stirred with the extraction solvent. The powder obtained in step S1 is extracted under room temperature and ultrasonic conditions (ultrasonic frequency is 850W). The extraction is carried out twice, each time for 15 minutes. During each extraction process, 10 times the amount of extraction solvent is used, i.e. the mass of the powder to the volume of the extraction solvent is 1g:10mL. After the extraction process is completed, centrifugation is carried out at 5000rpm for 5min, and a liquid-liquid-solid three-phase system is obtained. The uppermost layer is a eutectic solvent phase enriched with triterpenoids, the middle layer is an aqueous phase, and the lowermost layer is a Sabia japonica Maxim. powder. The uppermost layer is collected to obtain a crude extract;

[0063] S4, the crude extract obtained in step S3 is filtered through a filter membrane with a pore size of 0.22 microns to remove impurities;

[0064] S5, AB-8 type macroporous adsorption resin is selected to separate and purify triterpenoids. The specific process is as follows: AB-8 macroporous resin soaked in 95% ethanol for 24h is loaded into a chromatography column and allowed to settle naturally. After the column resin is loaded, the chromatography column is washed with distilled water until there is no alcohol smell. The filtered crude extract of S4 is poured into the AB-8 macroporous resin at a flow rate of 3BV / h. The weight ratio of dry resin to crude extract is 6:1, and the static adsorption time is 4h. Then, the macroporous resin column is washed with distilled water at a flow rate of 4BV / h to remove impurities until the effluent is clear. Then, 75% ethanol aqueous solution is used to clean the column at a flow rate of 3BV / h. The eluate is collected and concentrated to obtain a triterpenoid extract.

[0065] The content of the obtained triterpenoid is 15.2mg / g, and the purity of triterpenoid in the final triterpenoid extract is 83.9%.

[0066] Example 2

[0067] The present embodiment provides a method for extracting triterpenoids from Sabia japonica Maxim., comprising the following steps:

[0068] S1, dry Sabia japonica Maxim. is crushed, passed through a 30 mesh sieve to obtain a powder;

[0069] S2, a certain amount of n-decanoic acid and DL-menthol were accurately weighed in a molar ratio of 1:1 in a flask, and then heated and stirred at 80°C until a transparent homogeneous liquid was formed to obtain a eutectic solvent;

[0070] S3, the eutectic solvent obtained in step S2 was mixed with water in a volume ratio of 4:1 as an extraction solvent, and the powder obtained in step S1 was fully stirred and mixed with the extraction solvent, and the powder obtained in step S1 was extracted under room temperature and ultrasonic conditions (ultrasonic frequency 800W), and the extraction was carried out twice, each time for 12 min, and in each extraction process, 10 times the amount of extraction solvent was used, that is, the mass of the powder was 1g and the volume of the extraction solvent was 10mL; after the extraction process was completed, centrifugation was carried out at 5500rpm for 4min, and a liquid-liquid-solid three-phase system was obtained, in which the upper layer was the eutectic solvent phase enriched with triterpenoids, the middle layer was the water phase, and the lower layer was the Sabia japonica Maxim. powder; the upper layer was collected to obtain a crude extract;

[0071] S4, the crude extract obtained in step S3 was filtered through a filter membrane with a pore size of 0.22 microns to remove impurities;

[0072] S5, AB-8 type macroporous adsorption resin was selected to separate and purify triterpenoids, and the specific process was as follows: AB-8 macroporous resin soaked in 95% ethanol for 24h was loaded into a chromatography column and allowed to settle naturally, and after the column loading resin was completed, the chromatography column was washed with distilled water until there was no alcohol smell; the filtered crude extract of S4 was poured into the AB-8 macroporous resin at a flow rate of 2.5BV / h, wherein the weight ratio of dry resin to crude extract was 8:1, and the static adsorption time was 3h, then the macroporous resin column was washed with distilled water at a flow rate of 3BV / h to remove impurities until the effluent was clear; then 50% ethanol by volume was used to clean the column at a flow rate of 2.5BV / h, and the eluate was collected, and after concentration, the triterpenoid extract was obtained.

[0073] The content of the obtained triterpenoid was measured to be 11.1mg / g, and the purity of triterpenoids in the finally obtained triterpenoid extract was 80.4%.

[0074] Example 3

[0075] The present embodiment provides a method for extracting triterpenoids from Sabia japonica Maxim., comprising the following steps:

[0076] S1, dry Sabia japonica Maxim. was crushed and passed through a 30-mesh sieve to obtain a powder;

[0077] S2, a certain amount of lauric acid and DL-menthol were accurately weighed in a flask at a molar ratio of 1:1, and then heated and stirred at 80°C until a transparent homogeneous liquid was formed to obtain a eutectic solvent;

[0078] S3, the eutectic solvent obtained in step S2 was mixed with water as an extraction solvent at a volume ratio of 4:1, and the powder obtained in step S1 was fully stirred and mixed with the extraction solvent, and the powder obtained in step S1 was extracted under room temperature and ultrasonic conditions (ultrasonic frequency 800W), and the extraction was carried out twice, each time for 12 min, and in each extraction process, 10 times the amount of extraction solvent was used, i.e. the mass of the powder was 1g and the volume of the extraction solvent was 10mL; after the extraction process was completed, centrifugation was carried out at 5000rpm for 6min, and a liquid-liquid-solid three-phase system was obtained, in which the upper layer was the eutectic solvent phase enriched with triterpenoids, the middle layer was the water phase, and the lower layer was the lindera powder; the upper layer was collected to obtain a crude extract;

[0079] S4, the crude extract obtained in step S3 was filtered through a filter membrane with a pore size of 0.22 microns to remove impurities;

[0080] S5, AB-8 type macroporous adsorption resin was selected to separate and purify triterpenoids, and the specific process was as follows: AB-8 macroporous resin soaked in 95% ethanol for 24h was loaded into a chromatography column and allowed to settle naturally, and after the column loading resin was completed, the chromatography column was washed with distilled water until there was no alcohol smell; the crude extract after filtration in S4 was poured into the AB-8 macroporous resin at a flow rate of 4BV / h, wherein the weight ratio of dry resin to crude extract was 5:1, and the static adsorption time was 5h, then the macroporous resin column was washed with distilled water at a flow rate of 2BV / h to remove impurities until the effluent was clear; then 50% volume concentration of ethanol was used to clean at a flow rate of 4BV / h, and the eluate was collected, and after concentration, triterpenoid extract was obtained.

[0081] The content of the obtained triterpenoid was measured to be 10.9mg / g, and the purity of triterpenoid in the finally obtained triterpenoid extract was 81.8%.

[0082] Comparative Example 1

[0083] This comparative example provides a method for extracting triterpenoids from lindera, which is carried out according to the method of Example 1, with the only difference being that:

[0084] Step S2 is omitted; and,

[0085] Step S3 is:

[0086] The powder obtained in step S1 was mixed with an aqueous ethanol solution with a volume fraction of 75%, and the powder obtained in step S1 was extracted under the condition of room temperature and ultrasonic (ultrasonic frequency was 850 W), the extraction was carried out twice, each extraction time was 15 min, and the mass of the powder to the volume of the extraction solvent was 1 g:10 mL in each extraction process; after the extraction process was completed, centrifugation was carried out at 5000 rpm for 5 min, and the supernatant was collected to obtain a crude extract.

[0087] The content of the obtained triterpenoid compounds was 6.0 mg / g, and the purity of the triterpenoid compounds in the finally obtained triterpenoid extract was 76.6%.

[0088] Comparative Example 2

[0089] This comparative example provides a method for extracting triterpenoid compounds in lindera glauca, which is carried out by referring to example 1, and the only difference is that:

[0090] Step S2: a certain amount of choline chloride and ethylene glycol were accurately weighed in a molar ratio of 1:1 in a flask, and then heated and stirred at 80°C until a transparent homogeneous liquid was formed to obtain a deep eutectic solvent;

[0091] The deep eutectic solvent obtained in this comparative example was used in step S3.

[0092] The content of the obtained triterpenoid compounds was 8.5 mg / g, and the purity of the triterpenoid compounds in the finally obtained triterpenoid extract was 79.4%.

[0093] The results show that the content of triterpenoid compounds is lower than that in the example, and the possible reason is that water existing in the extraction system can destroy the hydrogen bonds between the components of the choline chloride-ethylene glycol deep eutectic solvent to different degrees due to its strong hydrogen bonding ability. Therefore, the choline chloride-ethylene glycol deep eutectic solvent is not suitable for extracting triterpenoid compounds with water in this application.

[0094] Comparative Example 3

[0095] This comparative example provides a method for extracting triterpenoid compounds in lindera glauca, which is carried out by referring to example 1, and the only difference is that:

[0096] Step S2: a certain amount of DL-menthol and lactic acid were accurately weighed in a molar ratio of 1:1 in a flask, and then heated and stirred at 80°C until a transparent homogeneous liquid was formed to obtain a deep eutectic solvent;

[0097] The deep eutectic solvent obtained in this comparative example was used in step S3.

[0098] The content of the obtained triterpenoid compounds is 8.9 mg / g, and the purity of the triterpenoid compounds in the finally obtained triterpenoid extract is 78.5%.

[0099] The results show that the content of the obtained triterpenoid compounds is lower than that in the embodiment, which may be related to the hydrophobicity of the eutectic solvent formed by lactic acid and DL-menthol.

[0100] Comparative Example 4

[0101] This comparative example provides an extraction method of triterpenoid compounds in Litsea Coreana Levl. var. sinensis Hemsl., which is performed according to the method of Example 1, with the only difference being that:

[0102] Step S5 is to separate and purify the triterpenoid compounds by selecting AB-8 type macroporous adsorption resin, and the specific process is as follows: AB-8 macroporous resin soaked in 95% ethanol for 24 h is loaded into a chromatographic column and allowed to settle naturally, and after the loading of the resin is completed, the chromatographic column is washed with distilled water until there is no alcohol smell; the filtered crude extract of S4 is poured into the AB-8 macroporous resin at a flow rate of 8 BV / h, wherein the weight ratio of dry resin to crude extract is 6:1, and the static adsorption time is 40 min; then the macroporous resin column is washed with distilled water at a flow rate of 4 BV / h until the effluent is clear; and then the macroporous resin column is washed with 75% ethanol aqueous solution at a flow rate of 8 BV / h, and the eluate is collected, concentrated, and then the triterpenoid extract is obtained.

[0103] The content of the obtained triterpenoid compounds is 9.3 mg / g, and the purity of the triterpenoid compounds in the finally obtained triterpenoid extract is 61.2%.

[0104] The possible reason is that the too high flow rate significantly affects the purification and enrichment effect of the triterpenoid compounds.

[0105] The above describes the embodiments of the present application in detail, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for extracting triterpenoids from Kadsura pannata, comprising the following steps: S1, mixing an extractant and Kadsura pannata, and sequentially performing ultrasonic extraction and centrifugation to obtain a crude extract; S2, adsorbing the crude extract onto a macroporous resin column, and eluting the crude extract with an eluent of 50%-80% ethanol aqueous solution to obtain triterpenoids; wherein the extractant comprises, in mass percentage, 50%-90% of a deep eutectic solvent and 10%-50% of water, the deep eutectic solvent is prepared from a hydrogen bond donor and a hydrogen bond acceptor, the hydrogen bond donor comprises at least one of n-octanoic acid, n-decanoic acid and lauric acid, and the hydrogen bond acceptor comprises menthol; a solid-liquid ratio of the Kadsura pannata to the extractant is 1:5-30 g / mL; The macroporous resins are selected from the group consisting of AB 8 type, HPD 100 type macroporous resins; in step S2, a column loading flow rate of the crude extract is 2-4 BV / h, and an elution flow rate of the eluent is 2-4 BV / h.

2. The extraction method according to claim 1, characterized in that, the hydrogen bond donor is selected from any two of n-octanoic acid, n-decanoic acid and lauric acid.

3. The extraction method of claim 1, wherein, a molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:0.2-5.

4. The extraction method of claim 1, wherein, before step S1, the method further comprises drying and crushing the Kadsura pannata to pass through a 20-40 mesh sieve to obtain Kadsura pannata powder.

5. The extraction method of claim 1, wherein, the ultrasonic extraction is performed at a temperature of 15-35 ℃, a frequency of 700-900 W and for a time of 20-40 min.

6. The extraction method of claim 1, wherein, the centrifugation is performed at a rotation speed of 4000-6000 rpm and for a time of 2-10 min.

7. The extraction method of claim 1, wherein, after step S1, the method further comprises filtering the crude extract through a filter membrane with a pore size of 0.22 microns before performing step S2.

8. The extraction method of claim 1, wherein, the crude extract is statically adsorbed onto the macroporous resin for 2-6 h before elution.

9. The extraction method of claim 8, wherein, a mass ratio of the macroporous resin to the crude extract is 2-8:

1.

10. The extraction method of claim 1, wherein, in step S2, 3-6 BV / h of water is used to wash off impurities before elution with the eluent.