A method for extracting active ingredients of natural products by multi-phase and coaxial liquid centrifugation

Abstract

The present invention belongs to the technical field related to plant extraction and separation, and particularly relates to a method for extracting active ingredients of natural products by multi-phase and coaxial liquid centrifugation. The method includes the pretreatment of natural products, the preparation of multi-phase extraction solvents: preparing a series of aqueous solvents and organic solvents with different densities and different hydrophilic / hydrophobic properties; the construction of a multi-phase and coaxial liquid centrifugation platform; the present invention realizes a multi-liquid-phase extraction system based on a coaxial liquid centrifuge, provides a method for simultaneously extracting different parts of natural products, thereby improving the extraction efficiency of natural products and promoting the development of the pharmaceutical industry.

Description

Technical Field

[0001] The present invention belongs to the technical field related to plant extraction and separation, and particularly relates to a method for extracting active ingredients of natural products by multi-phase and coaxial liquid centrifugation. Technical Background

[0002] Natural products refer to the components or metabolites in extracts of animals, plants or microorganisms. Among them, the main ones are flavonoids, alkaloids, polysaccharides, volatile oils, quinones, terpenoids, saponins, phenolic acids, amino acids and enzymes, etc. Natural products have long played an important role in the medical field: for example, artemisinin extracted from the stems and leaves of Artemisia annua is the best drug for treating malaria drug resistance; ginkgolide B extracted from Ginkgo biloba leaves can inhibit platelet activating factor; cedrene extracted from Platycladus orientalis leaves has obvious inhibitory effects on Clostridium difficile and Clostridium perfringens.

[0003] However, there are numerous natural products. Extracting only one natural product or extracting different natural products step by step will inevitably cause waste of raw materials and reagents. At present, the main methods for extracting and separating natural products in industry and laboratories are still liquid-liquid two-phase extraction or column chromatography. Their steps are cumbersome, consuming a large amount of reagents and time, and having low efficiency. In addition, some new methods for extracting natural products, such as supercritical fluid extraction, are difficult to be widely promoted due to the high cost of equipment; while extraction methods such as microwave and pulsed electric field are not suitable for large-scale industrial production. In the existing extraction technologies using centrifuges, there are problems such as the inability to separate the feed bins and instrument parts of centrifugal extractors and multi-stage centrifugal extraction systems, and difficult cleaning, which is not conducive to the maintenance of instruments and the extraction of other natural products, and they are all based on liquid-liquid two-phase extraction.

[0004] Multi-phase and coaxial liquid centrifugal extraction is a technology that uses multiple extraction liquids to extract raw materials simultaneously. When a multi-liquid phase system is centrifuged, as the rotation speed increases, it will gradually form multiple-layer hollow liquid columns with the same axis. At this time, the contact area between the two liquids is the largest. Substances will be distributed and transferred in each liquid layer according to their own properties and those of the extraction liquid, and the slight oscillation during centrifugation will also accelerate the distribution and transfer of substances, thereby realizing the simultaneous extraction of multi-liquid phases. If petroleum ether and ethyl acetate are used to extract pigments and flavonoid compounds in natural products respectively at the same time, a three-liquid phase system can be adopted; if small-polarity compounds, medium-polarity compounds and large-polarity compounds are to be extracted separately at the same time, a four-liquid phase system can be adopted.

[0005] The multi-phase and coaxial liquid centrifugal extraction technology can greatly reduce the time of the multi-step extraction process and improve the extraction efficiency by using multiple extraction liquids to extract simultaneously, and has broad application prospects in industries such as chemical engineering and pharmaceuticals. Summary of the Invention

[0006] Object of the Invention

[0007] The object of the present invention is to innovatively develop a multi-liquid-phase extraction system based on a coaxial liquid centrifuge in view of the deficiencies of the prior art, and provide a method for simultaneously extracting different parts of natural products, thereby increasing the extraction efficiency of natural products and promoting the development of the pharmaceutical industry.

[0008] Technical Solution

[0009] A method for extracting active ingredients of natural products by multi-phase and coaxial liquid centrifugation, comprising the following steps: a. Pretreatment of natural products: drying and grinding traditional Chinese medicinal materials into powder, sieving, and then pretreating with an aqueous solvent or an organic solvent to obtain a pretreated extract of natural products for standby;

[0010] b. Preparation of multi-phase extraction solvents: preparing a series of aqueous solvents and organic solvents with different densities and different hydrophilic / hydrophobic properties for standby;

[0011] c. Construction of a multi-phase and coaxial liquid centrifugation platform: adding the extraction solvent and the sample solution to the sample bottle in the order of decreasing density to form a three-liquid-phase or multi-liquid-phase system, and radially fixing the sample bottle on the centrifuge;

[0012] The method is characterized in that: in the step a, the sieve mesh number of the natural product powder is 65-200 meshes, and the material-liquid ratio of the natural product powder to the aqueous solvent or the organic solvent is 1:1-1:50 g / mL.

[0013] The method is characterized in that: in the step b, the types of the multi-phase extraction solvents include organic solvents such as petroleum ether, ethyl acetate, n-butanol, tetrachloroethylene, dichloromethane, chloroform, polyethylene glycol, bis(2-ethylhexyl) phosphate, etc., and aqueous solutions including sodium citrate, sodium dihydrogen phosphate, etc., and mixed solvents of the above solvents.

[0014] The multi-phase and coaxial liquid centrifugation technology for extracting active ingredients of natural products is characterized in that: in the step c, the volume ratio of each phase solution is 1:1-1:10, the rotation speed of the centrifuge is 200-20000 rpm, the centrifugation extraction time is 5-360 min, and the sample bottle is radially fixed on the centrifuge.

[0015] The traditional Chinese medicinal material is Platycladus orientalis leaf.

[0016] Key Points of the Present Invention

[0017] 1. Not any active ingredient and organic solvent are suitable for multi-liquid-phase extraction, and the reasons are as follows:

[0018] The three-liquid-phase system can be composed of organic phase A-aqueous phase-organic phase B, aqueous phase A-organic phase-aqueous phase B, or organic phase-bis-aqueous phase.

[0019] In the organic phase A - aqueous phase - organic phase B system or the aqueous phase A - organic phase - aqueous phase B system, the raw materials can be dispersed in the middle phase, and both phase A and phase B extract the active ingredients in the middle phase simultaneously. It can be seen therefrom that there is a competitive relationship between phase A and phase B. Therefore, when their properties are similar, the active ingredients with similar properties will be extracted by phase A simultaneously, and phase B will not play a role. Therefore, for the organic phase A - aqueous phase - organic phase B system or the aqueous phase A - organic phase - aqueous phase B system, the two extraction liquids used should have a large difference in affinity for different active ingredients.

[0020] In the organic phase - aqueous two - phase system, the raw materials are dispersed in the salt solution of the aqueous two - phase system. Existing research shows that the polymer phase in the aqueous two - phase system can extract most natural products with high extraction efficiency. When the organic phase - aqueous two - phase system reacts, the polymer solution in the aqueous two - phase system first extracts the active ingredients in the salt solution, and at the same time the organic phase extracts the active ingredients in the polymer solution until the active ingredients reach a dynamic equilibrium among the three phases of the salt solution - polymer solution - organic phase. Due to the different affinities of the active ingredients and the organic solvents used, the active ingredients with high affinity for the organic solvent will be extracted into the organic phase, and the active ingredients with weak affinity for the organic solvent will remain in the polymer phase, thus realizing the simultaneous extraction of two reagents for different substances. However, if the extraction ability of the organic phase is too weak, the organic phase will not be able to extract the active ingredients from the polymer phase. Therefore, the composition and ratio of the organic solvent and the aqueous two - phase system should be reasonably designed according to the properties of the active ingredients to be extracted.

[0021] 2. Not any solvent can achieve the purpose of the present invention, and the reasons are as follows:

[0022] When a polymer and a salt are mixed in water in a certain proportion, a layered aqueous two - phase system can be obtained. By superimposing an organic solvent immiscible with water on the aqueous two - phase system, a three - liquid - phase system can be obtained; by superimposing two organic solvents immiscible with water and having a large density difference, a four - liquid - phase system can be obtained. In addition to utilizing the characteristics of the aqueous two - phase system, a three - liquid - phase can also be formed by using two organic solvents with a large density difference and water, and then by superimposing a salt solution with a greater density on this basis, a four - liquid - phase system can be obtained.

[0023] To form a stable multi - liquid - phase system, the solutions of each phase must not be miscible into a homogeneous solution. For example, when forming a three - liquid - phase system of an organic phase plus an aqueous two - phase system, the mass fractions of the salt and polyethylene glycol in the aqueous two - phase system must be higher than the phase - diagram curve of the salt and polyethylene glycol in the aqueous two - phase system; the organic solvent is immiscible with water and will not dissolve polyethylene glycol. When forming a three - liquid - phase system with the composition of organic phase - aqueous phase - organic phase, it is necessary to consider whether the organic solvent is miscible with water and the density of the organic solvent (the two organic solvents must have one density greater than that of the aqueous solution and the other density less than that of the aqueous solution). The factors to be considered for forming a four - liquid - phase system are the same as those for the above - mentioned three - liquid - phase system.

[0024] Therefore, different solvent systems were screened for the present invention, as follows:

[0025] Table 1: Organic phase-aqueous two-phase system

[0026]

[0027] Table 2: Organic phase A-aqueous phase-organic phase B system

[0028]

[0029] The results showed that: in addition to the factors affecting the formation of aqueous two-phase systems, the composition of the organic phase-aqueous two-phase system is also related to the properties of organic solvents.

[0030] If the mass fractions of salt and polymer are below the aqueous two-phase curve, an aqueous two-phase system cannot be formed, but a homogeneous aqueous solution containing both salt and polymer is formed. For example, in the system containing sodium dihydrogen phosphate in Table 1, a polyethylene glycol solution with a mass fraction of 40% is miscible with a sodium dihydrogen phosphate solution with a mass fraction of 20%, but when the mass fraction of sodium dihydrogen phosphate is increased to 40%, a polyethylene glycol solution and a sodium dihydrogen phosphate solution form an aqueous two-phase system. Therefore, the mass fractions of salt and polymer must be above the aqueous two-phase curve.

[0031] When the organic solvent can dissolve polyethylene glycol (such as dichloromethane and n-butanol in Table 1), the polyethylene glycol in the aqueous two-phase system will be extracted into the organic phase, and the mass fraction of polyethylene glycol in the aqueous two-phase system decreases, then the aqueous two-phase system becomes a water phase. Therefore, attention should be paid to whether the selected organic solvent interferes with the formation of the aqueous two-phase system.

[0032] For the organic phase A-aqueous phase-organic phase B system or the aqueous phase A-organic phase-aqueous phase B system, there are two different solvents located on both sides of the middle phase, so there needs to be a significant difference in the densities of solvents A and B. For example, the density of a mixed solution of n-butanol / dichloromethane (2:1, v / v) in Table 2 is 0.98, which is less than the density of water. When the solution is added in sequence to form a three-liquid-phase system, the added aqueous solution will directly pass through the mixed organic solvent to the bottom layer of the system. Therefore, before constructing a multi-phase system, the density of the mixed solution should be measured.

[0033] To obtain flavonoids from Platycladus orientalis leaves, the present invention finally selected the following three conditions:

[0034] The optimal conditions for the three-liquid-phase system for the extraction of flavonoids from Platycladus orientalis leaves are that the volume ratio of ethyl acetate, water and n-butanol / dichloromethane solution is 2:1:2, where the n-butanol / dichloromethane solution is obtained by mixing n-butanol and dichloromethane at a volume ratio of 1:1, the rotation speed is 1000 rpm, and the centrifugal extraction time is 30 min;

[0035] Or the volume ratio of bis(2-ethylhexyl) phosphate / ethyl acetate, polyethylene glycol solution and Platycladus orientalis aqueous solution is 3:1:1, where bis(2-ethylhexyl) phosphate / ethyl acetate is obtained by mixing bis(2-ethylhexyl) phosphate and ethyl acetate at a volume ratio of 1:10, the rotation speed is 3000 rpm, and the centrifugal extraction time is 15 min;

[0036] The optimal conditions for the four-liquid-phase system for extracting flavonoids from Platycladus orientalis are that the volume ratio of bis(2-ethylhexyl) phosphate / ethyl acetate, polyethylene glycol solution, Platycladus orientalis aqueous solution and petroleum ether / tetrachloroethylene is 2:1:1:3, where bis(2-ethylhexyl) phosphate / ethyl acetate is obtained by mixing bis(2-ethylhexyl) phosphate and ethyl acetate at a volume ratio of 1:10, and petroleum ether / tetrachloroethylene is obtained by mixing petroleum ether and tetrachloroethylene at a volume ratio of 1:1, the rotation speed is 8000 rpm, and the centrifugal extraction time is 35 min.

[0037] Beneficial effects

[0038] The present invention can simultaneously obtain flavonoid extracts from two parts of Platycladus orientalis, ethyl acetate and n-butanol, through the organic phase A-aqueous phase-organic phase B system. After three extractions, the residual amounts of the three flavonoid compounds in water are all less than 10%, and the extraction efficiency is high; or obtain extracts from two parts of ethyl acetate and polymer phase or extracts from three parts of ethyl acetate, polymer phase and petroleum ether through the organic phase-aqueous two-phase system.

[0039] The present invention uses a variety of solvents to simultaneously extract different active ingredients of natural products, and can simultaneously obtain products from different parts such as ethyl acetate, n-butanol, polymer phase, petroleum ether, etc.; it can reduce the extraction steps of natural products and significantly reduce the extraction time. Description of the drawings

[0040] Figure 1 Front view (a) and top view (b) of the multi-liquid-phase morphology during coaxial liquid centrifugation;

[0041] Figure 2 、 Three Schematic diagram of the composition of the liquid phase (a) and the four-liquid phase (b);

[0042] Figure 3 Schematic diagram of the radial installation of the sample bottle;

[0043] Figure 4 Effect of different mixing volume ratios of n-butanol and dichloromethane on the extraction of flavonoids from Platycladus orientalis;

[0044] Figure 5 Effect of the volume ratio of each phase on the extraction of Platycladus orientalis flavonoids;

[0045] Figure 6, Effects of different rotation speeds on the extraction of total flavonoids from Platycladus orientalis leaves;

[0046] Figure 7 , Effects of different centrifugal extraction times on the extraction of flavonoids from Platycladus orientalis leaves. Specific embodiments

[0047] The following embodiments are some examples of the present invention and should not be regarded as limitations of the present invention.

[0048] Example 1

[0049] Dry 2 kg of Platycladus orientalis leaves, grind them into powder, and sieve them through an 80-mesh sieve for later use.

[0050] Soak the Platycladus orientalis leaf powder in 75% ethanol overnight, with a solid-liquid ratio of 1:10 g / mL. Extract by gentle boiling under reflux for 6 h, repeat three times, combine the ethanol, and obtain an ethanol extract after rotary evaporation. Disperse the extract in 2 L of water for later use.

[0051] Mix n-butanol and dichloromethane in a volume ratio of 0:1 to 1:1 to obtain n-butanol / dichloromethane solutions with different concentrations for later use.

[0052] Add the n-butanol / dichloromethane solution, the Platycladus orientalis leaf aqueous solution, and ethyl acetate to the sample bottle in the order of decreasing density to form a three-phase liquid system. Fix the sample bottle radially on the centrifuge.

[0053] 1. Set the rotation speed to 1000 rpm, centrifuge and extract for 10 min, with the volume ratio of each solution being 1:1:1. After three-phase separation, the extracts of Platycladus orientalis leaves in both the ethyl acetate and n-butanol parts can be obtained simultaneously. Measure the total flavonoid content in the three phases, and take the n-butanol / dichloromethane mixing volume ratio with the least total flavonoid content in the aqueous phase as the optimal solvent mixing ratio.

[0054] The results are as Figure 4 shown. In the n-butanol / dichloromethane mixed solution, the higher the n-butanol content, the less the total flavonoid content in the aqueous phase and the higher the flavonoid extraction rate. Therefore, within the range of volume ratio 0:1 to 1:1, the optimal mixing volume ratio of n-butanol and dichloromethane is 1:1.

[0055] 2. Set the rotation speed to 1000 rpm, centrifuge and extract for 10 min, with n-butanol and dichloromethane mixed in a volume ratio of 1:1, and the volume ratio of each solution being 1 - 2:1:1 - 2. After three-phase separation, the extracts of Platycladus orientalis leaves in both the ethyl acetate and n-butanol parts can be obtained simultaneously. Measure the total flavonoid content in the three phases, and take the three-phase volume ratio with the least total flavonoid content in the aqueous phase as the optimal extraction volume ratio.

[0056] The results are as Figure 5As shown, the larger the volume of the organic phase, the less the total flavonoid content in the aqueous phase and the higher the flavonoid extraction rate. Therefore, within the range of volume ratios of 1 - 2:1:1 - 2, 2:1:2 is the optimal volume ratio for the three liquid phases.

[0057] 3. Set the rotation speed at 500 - 1000 rpm, centrifuge for extraction for 10 min. Mix n-butanol and dichloromethane in a volume ratio of 1:1, and the volume ratio of each solution is 1:1:1. After three-phase separation, the extracts of the ethyl acetate and n-butanol parts of Platycladus orientalis can be obtained simultaneously. Measure the total flavonoid content of the three phases, and the rotation speed with the least total flavonoid content in the aqueous phase is the optimal rotation speed.

[0058] The results are as Figure 6 shown. The greater the rotation speed, the less the total flavonoid content in the aqueous phase and the higher the total flavonoid extraction rate. Therefore, within the range of rotation speeds of 500 - 1000 rpm, 1000 rpm is the optimal rotation speed.

[0059] 4. Set the rotation speed at 1000 rpm, mix n-butanol and dichloromethane in a volume ratio of 1:1, and the volume ratio of each solution is 1:1:1. Centrifuge for extraction for 5 - 35 min. After three-phase separation, the extracts of the ethyl acetate and n-butanol parts of Platycladus orientalis can be obtained simultaneously. Measure the total flavonoid content of the three phases, and the centrifugal extraction time with the least total flavonoid content in the aqueous phase is the optimal centrifugal extraction time.

[0060] The results are as Figure 7 shown. Within the range of centrifugal extraction time of 5 - 30 min, the longer the extraction time, the less the total flavonoid content in the aqueous phase and the higher the flavonoid extraction rate. When the centrifugal extraction time is 30 - 35 min, the change in the total flavonoid content in the three phases is not significant, indicating that the extraction of the total flavonoid reaches equilibrium.

[0061] In summary, the optimal conditions for the three-liquid-phase system for flavonoid extraction from Platycladus orientalis are as follows: the volume ratio of ethyl acetate, water, and n-butanol / dichloromethane solution is 2:1:2, where the n-butanol / dichloromethane solution is obtained by mixing n-butanol and dichloromethane in a volume ratio of 1:1, the rotation speed is 1000 rpm, and the centrifugal extraction time is 30 min.

[0062] Disperse the Platycladus orientalis extract in water, and extract the flavonoids in the aqueous phase of Platycladus orientalis with ethyl acetate and n-butanol / dichloromethane solution according to the above optimal conditions. Repeat the extraction three times, combine the ethyl acetate solution and n-butanol / dichloromethane solution respectively, and measure the contents of myricitrin, quercitrin, and amentoflavone in the three phases. The results are as follows:

[0063] Table 3 Extraction of Myricitrin, Quercitrin, and Amentoflavone from Platycladus orientalis by Multi-phase and Coaxial Liquid Centrifugation

[0064]

[0065] As shown in Table 3, the flavonoid extract of Platycladus orientalis leaves with ethyl acetate and n-butanol fractions was obtained simultaneously. After three extractions, the residual amounts of the three flavonoid compounds in water were all less than 10%, and amentoflavone was completely extracted into the two organic phases.

[0066] Example 2

[0067] 2 kg of Platycladus orientalis leaves were dried, ground into powder, and sieved through a 100-mesh sieve for later use.

[0068] The Platycladus orientalis leaf powder was soaked in 75% ethanol overnight with a solid-liquid ratio of 1:10 g / mL, and extracted by gentle boiling under reflux for 6 h. This process was repeated three times, and the ethanol was combined and rotary evaporated to obtain an ethanol extract. The extract was dispersed in an aqueous solution of sodium citrate with a mass fraction of 35% for later use.

[0069] An aqueous solution of polyethylene glycol 2000 with a mass fraction of 40% was prepared for later use.

[0070] Bis(2-ethylhexyl) phosphate and ethyl acetate were mixed at a volume ratio of 0:10 - 1:10 to obtain bis(2-ethylhexyl) phosphate / ethyl acetate solutions with different concentrations for later use.

[0071] The Platycladus orientalis leaf extract solution, polyethylene glycol solution, and ethyl acetate solution were added to the sample bottle in the order of decreasing density to form a three-phase system. The sample bottle was radially fixed on a centrifuge.

[0072] The rotation speed was set at 1000 rpm, and centrifugal extraction was carried out for 10 min. Bis(2-ethylhexyl) phosphate and ethyl acetate were mixed at a volume ratio of 0:10 - 1:10, and the volume ratio of each solution was 1:1:1. After three-phase separation, the Platycladus orientalis leaf extract with ethyl acetate and polyethylene glycol fractions could be obtained simultaneously. The content of quercetin in the three phases was measured, and the best mixing ratio of bis(2-ethylhexyl) phosphate / ethyl acetate was the one that could extract the most quercetin in ethyl acetate. In the range of 0:10 - 1:10, the best mixing volume ratio of bis(2-ethylhexyl) phosphate to ethyl acetate was 1:10.

[0073] The rotation speed was set at 1000 rpm, and centrifugal extraction was carried out for 10 min. Bis(2-ethylhexyl) phosphate and ethyl acetate were mixed at a volume ratio of 1:10, and the volume ratio of each phase solution was 1 - 10:1 - 10:1. After three-phase separation, the Platycladus orientalis leaf extract with ethyl acetate and polyethylene glycol fractions could be obtained simultaneously. The content of quercetin in the three phases was measured, and the best ratio of the three-phase volume was the one that could extract the most quercetin in ethyl acetate. In the range of the volume ratio of each phase solution of 1 - 10:1 - 10:1, the best ratio was 3:1:1.

[0074] Set the rotation speed to 400 - 10000 rpm, centrifuge for extraction for 10 min. Mix bis(2-ethylhexyl) phosphate and ethyl acetate in a volume ratio of 1:10. The volume ratio of each phase solution is 3:1:1. After three-phase separation, the extracts of ethyl acetate and polyethylene glycol parts of Platycladus orientalis can be obtained simultaneously. Measure the content of quercitrin in the three phases. The rotation speed at which the most quercitrin can be extracted in ethyl acetate is the optimal rotation speed. In the range of 400 - 10000 rpm, 3000 rpm is the best.

[0075] Set the centrifugal extraction time to 10 - 60 min, rotation speed 3000 rpm. Mix bis(2-ethylhexyl) phosphate and ethyl acetate in a volume ratio of 1:10. The volume ratio of each phase solution is 3:1:1. After three-phase separation, the extracts of ethyl acetate and polyethylene glycol parts of Platycladus orientalis can be obtained simultaneously. Measure the content of quercitrin in the three phases. The rotation speed at which the most quercitrin can be extracted in ethyl acetate is the optimal extraction time. In the range of 10 - 60 min, 15 min is the best.

[0076] In summary, the optimal mixing ratio of bis(2-ethylhexyl) phosphate and ethyl acetate is 1:10, the optimal rotation speed is 3000 rpm, the centrifugal extraction time is 15 min, and the volume ratio of bis(2-ethylhexyl) phosphate / ethyl acetate, polyethylene glycol solution and Platycladus orientalis aqueous solution is 3:1:1.

[0077] Example 3

[0078] Dry 2 kg of Platycladus orientalis, grind it into powder and sieve it with a 100-mesh sieve, and set aside.

[0079] Soak the Platycladus orientalis powder in 75% ethanol overnight, with a material-liquid ratio of 1:10 g / mL, reflux extract at a gentle boil for 6 h, repeat three times, combine the ethanol, and obtain an ethanol extract after rotary evaporation. Disperse the extract in an aqueous solution of sodium citrate with a mass fraction of 35%, and set aside.

[0080] Prepare an aqueous solution of polyethylene glycol 2000 with a mass fraction of 40%, and set aside.

[0081] Mix bis(2-ethylhexyl) phosphate and ethyl acetate in a volume ratio of 0:10 - 1:10 to obtain bis(2-ethylhexyl) phosphate / ethyl acetate solutions with different concentrations, and set aside.

[0082] Mix petroleum ether and tetrachloroethylene in a volume ratio of 0:1 - 1:1 to obtain petroleum ether / tetrachloroethylene solutions with different concentrations, and set aside.

[0083] Add the tetrachloroethylene solution, Platycladus orientalis extract solution, polyethylene glycol solution, and ethyl acetate solution to the sample bottle in the order of decreasing density to form a four-liquid phase system. Fix the sample bottle radially on the centrifuge.

[0084] Set the rotation speed to 10000 rpm, centrifuge and extract for 10 min. Di(2-ethylhexyl) phosphate and ethyl acetate are mixed at a volume ratio of 0:10 to 1:10, petroleum ether and tetrachloroethylene are mixed at a volume ratio of 0:1. The volume ratio of each phase solution is 1:1:1:1. After four-phase separation, the extracts of Platycladus orientalis leaves in three parts of ethyl acetate, polyethylene glycol and tetrachloroethylene can be obtained simultaneously. Measure the content of quercitrin in the four phases. The best mixing ratio is the volume ratio of di(2-ethylhexyl) phosphate / ethyl acetate that can extract the most quercitrin in ethyl acetate. In the range of 0:10 to 1:10, 1:10 is the best mixing ratio of di(2-ethylhexyl) phosphate and ethyl acetate.

[0085] Set the rotation speed to 10000 rpm, centrifuge and extract for 10 min. Di(2-ethylhexyl) phosphate and ethyl acetate are mixed at a volume ratio of 1:10, petroleum ether and tetrachloroethylene are mixed at a volume ratio of 0:1 to 1:1. The volume ratio of each phase solution is 1:1:1:1. After four-phase separation, the extracts of Platycladus orientalis leaves in three parts of ethyl acetate, polyethylene glycol and tetrachloroethylene can be obtained simultaneously. Measure the content of α-pinene in tetrachloroethylene. The best mixing ratio is the volume ratio of petroleum ether / tetrachloroethylene that can extract the most α-pinene in tetrachloroethylene. In the range of 0:1 to 1:1, 1:1 is the best mixing ratio of petroleum ether and tetrachloroethylene.

[0086] Set the rotation speed to 10000 rpm, centrifuge and extract for 10 min. The volume ratio of each solution is 1 to 10:1 to 10:1:1 to 10. After four-phase separation, the extracts of Platycladus orientalis leaves in three parts of ethyl acetate, polyethylene glycol and tetrachloroethylene can be obtained simultaneously. Measure the content of quercitrin and α-pinene in the two organic solvents of ethyl acetate and tetrachloroethylene. The best four-phase volume ratio is the one with the largest separation degree of quercitrin and α-pinene. In the range of the volume ratio of each solution being 1 to 10:1 to 10:1:1 to 10, 2:1:1:3 is the best ratio.

[0087] Set the rotation speed to 5000 - 15000 rpm, centrifuge and extract for 10 min. Di(2-ethylhexyl) phosphate and ethyl acetate are mixed at a volume ratio of 1:10, petroleum ether and tetrachloroethylene are mixed at a volume ratio of 1:1. The volume ratio of each phase solution is 2:1:1:3. After four-phase separation, the extracts of Platycladus orientalis leaves in three parts of ethyl acetate, polyethylene glycol and tetrachloroethylene can be obtained simultaneously. Measure the content of quercitrin in ethyl acetate and the content of α-pinene in tetrachloroethylene. The best rotation speed is the one with the largest separation degree of quercitrin and α-pinene. In the range of 5000 - 15000 rpm, 8000 rpm is the best rotation speed.

[0088] Set the rotation speed to 8000 rpm, the centrifugal extraction time to 5 - 60 min. Mix bis(2-ethylhexyl) phosphate and ethyl acetate in a volume ratio of 1:10, and mix petroleum ether and tetrachloroethylene in a volume ratio of 1:1. The volume ratio of each phase solution is 2:1:1:3. After four-phase separation, the extracts of three parts of Platycladus orientalis can be obtained simultaneously, namely ethyl acetate, polyethylene glycol, and tetrachloroethylene. Determine the content of quercitrin in ethyl acetate and the content of α-pinene in tetrachloroethylene. The rotation speed with the largest separation degree of quercitrin and α-pinene is the optimal extraction time. In the range of 5 - 60 min, 35 min is the optimal centrifugal extraction time.

[0089] In summary, the optimal mixing volume ratio of bis(2-ethylhexyl) phosphate and ethyl acetate is 1:10, the optimal mixing volume ratio of petroleum ether and tetrachloroethylene is 1:1, the optimal rotation speed is 8000 rpm, the centrifugal extraction time is 35 min, and the volume ratio of bis(2-ethylhexyl) phosphate / ethyl acetate, polyethylene glycol solution, Platycladus orientalis aqueous solution, and petroleum ether / tetrachloroethylene is 2:1:1:3.

Claims

1. A method for extracting active ingredients of natural products by multi-phase and coaxial liquid centrifugation, characterized in that: a. Pretreatment of natural products: The Platycladus orientalis powder is soaked in 75% ethanol overnight, with a solid-liquid ratio of 1:10 g / mL, and extracted by refluxing at a gentle boil for 6 h. This is repeated three times, and the ethanol is combined and rotary evaporated to obtain an ethanol extract; the extract is dispersed in 2 L of water to obtain a sample solution for standby. b. Preparation of multi-phase extraction solvents: Prepare a series of aqueous solvents and organic solvents with different densities and different hydrophilic / hydrophobic properties for standby. c. Construction of a multi-phase and coaxial liquid centrifugation platform: The extraction solvent and the sample solution are added to the sample bottle in the order of decreasing density to form a three-liquid or four-liquid phase system, and the sample bottle is radially fixed on the centrifuge. For the three-liquid phase system for extracting flavonoids from Platycladus orientalis: The volume ratio of ethyl acetate, water, and n-butanol / dichloromethane solution is 2:1:2, where the n-butanol / dichloromethane solution is obtained by mixing n-butanol and dichloromethane in a volume ratio of 1:1, the rotation speed is 1000 rpm, and the centrifugal extraction time is 30 min; or the volume ratio of bis(2-ethylhexyl) phosphate / ethyl acetate, polyethylene glycol solution, and Platycladus orientalis aqueous solution is 3:1:1, where bis(2-ethylhexyl) phosphate / ethyl acetate is obtained by mixing bis(2-ethylhexyl) phosphate and ethyl acetate in a volume ratio of 1:10, the rotation speed is 3000 rpm, and the centrifugal extraction time is 15 min. For the four-liquid phase system for extracting flavonoids from Platycladus orientalis: The volume ratio of bis(2-ethylhexyl) phosphate / ethyl acetate, polyethylene glycol solution, Platycladus orientalis aqueous solution, and petroleum ether / tetrachloroethylene is 2:1:1:3, where bis(2-ethylhexyl) phosphate / ethyl acetate is obtained by mixing bis(2-ethylhexyl) phosphate and ethyl acetate in a volume ratio of 1:10, and petroleum ether / tetrachloroethylene is obtained by mixing petroleum ether and tetrachloroethylene in a volume ratio of 1:1, the rotation speed is 8000 rpm, and the centrifugal extraction time is 35 min.

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