Common fenugreek seed total saponins and / or total flavonoids as well as preparation method, application and pharmaceutical composition thereof

Abstract

The invention provides fenugreek total saponins and / or total flavonoids as well as a preparation method and application thereof and a pharmaceutical composition, and relates to the technical field of biological medicines. The method comprises the following steps: extracting fenugreek powder by using a 50-70v / v% ethanol aqueous solution, diluting by using water, separating by using a microfiltration membrane, adsorbing the obtained permeate by using macroporous resin, washing by using water, desorbing by using a 30-35v / v% ethanol aqueous solution to obtain total flavonoids, and desorbing by using a 50-60v / v% ethanol aqueous solution to obtain total saponins. The method of alcohol extraction, microfiltration membrane separation and macroporous resin purification is adopted, the high-purity fenugreek total saponins and total flavonoids can be efficiently prepared at the same time, the process is simple, operation is easy, energy consumption is low, production cost is low, and the method is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a fenugreek total saponin and / or total flavonoids, a preparation method and application thereof, and a pharmaceutical composition. Background Art

[0002] Fenugreek (Trigonella foenum-graecum L), also known as rue, coumarin, bitter bean, fenugreek, and pinellia, is an annual herbaceous plant of the genus Trigonella in the leguminous family, and the monocotyledonous plant class angiosperms. The whole plant has a strong aroma. Fenugreek is warm in nature, bitter in taste, and belongs to the liver and kidney meridians. It can dispel cold and relieve pain. It can be used for kidney deficiency and cold pain in the lower abdomen. At present, fenugreek is mostly used in the treatment of diabetes, hypoglycemic, hypolipidemic and other diseases. In addition, fenugreek also has antioxidant, analgesic and anti-inflammatory, anti-tumor, antibacterial and heart and kidney protection effects. The main chemical components of fenugreek are steroidal saponins, flavonoids, alkaloids, terpenes, fats and oils, amino acids and proteins, coumarins, minerals, etc. In recent years, people have studied more and more natural plant pharmacology, among which fenugreek, as a dual-purpose plant for medicine and food, has received extensive attention.

[0003] Chinese patent CN114288331A discloses a method for simultaneously extracting volatile oil from effective parts of total flavonoids and total saponins of traditional Chinese medicine fenugreek, comprising the following steps: S1, peeling fenugreek seeds to obtain peeled seed kernels, extracting the peeled seed kernels by steam distillation for 5-7 hours, repeating the extraction 2-4 times, and obtaining fenugreek essential oil and fenugreek decoction; S2, standing the fenugreek decoction, filtering, obtaining fenugreek filtered extract, adding ethanol solution, standing for 2-4 hours, obtaining flocculent precipitate and fenugreek water extract; S3, adding ethanol solution to the fenugreek water extract to precipitate, obtaining a first precipitate and a first supernatant; S4, adding ethanol solution with a volume fraction of 60-80% to the first precipitate, soaking for 14-18 hours, and refluxing Extract 2 to 4 times, each extraction time is 1.5 to 2.5 hours, combine the extracts, and concentrate the extracts under reduced pressure to obtain a total extract; S5, add distilled water to the total extract to obtain an aqueous solution of the total extract, filter, elute with polyamide resin and macroporous resin in turn, use pure water as eluent, and collect the eluate; S6, elute the eluate obtained in S5 with polyamide resin and methanol as eluent, continue eluting until colorless, collect the eluate, concentrate the eluate with a multi-stage flash concentrator to an extract, and spray dry the extract to obtain the effective part of total flavonoids; S7, elute the eluate obtained in S5 with polyamide resin and macroporous resin, use ethanol solution as eluent, concentrate the eluate with a multi-stage flash concentrator to an extract, spray dry the extract to obtain the effective part of total saponins. However, the fenugreek decoction in the above method contains a large amount of polysaccharide components (fenugreek gum), the aqueous solution has high viscosity, and filtering is very difficult, and the polysaccharide components are removed by alcohol precipitation to obtain small molecule precipitation, and then extracted with 60-80% ethanol. When the polysaccharide is precipitated by alcohol, the flocculent precipitation may wrap the total flavonoids and total saponin small molecules and precipitate together, thereby causing the loss of flavonoids and saponins; this method adopts two-step resin combination separation, the total flavonoids and total saponin effective components lose too much, and the final product purity is not high. Therefore, it is of great significance to provide an extraction method with simple process, high yield of total flavonoids and total saponins, and high purity. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a total saponin and / or total flavonoids of fenugreek and a preparation method and application thereof, and a pharmaceutical composition. The method provided by the present invention has high purity of total saponin and total flavonoids, high yield, simple process and low energy consumption.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing total saponins and / or total flavonoids of fenugreek, characterized in that it comprises the following steps:

[0007] Mixing fenugreek powder with a first ethanol aqueous solution for extraction to obtain an ethanol extract; wherein the volume fraction of ethanol in the first ethanol aqueous solution is 50-70%;

[0008] diluting the alcohol extract with water, and subjecting the obtained diluted alcohol extract to microfiltration membrane separation to obtain a permeate;

[0009] The permeate is adsorbed by a macroporous resin, and after washing, a first desorption is performed by a second ethanol aqueous solution to obtain a macroporous resin residue and a first desorption solution, respectively; the first desorption solution is dried to obtain total flavonoids; the volume fraction of ethanol in the second ethanol aqueous solution is 30-35%;

[0010] The macroporous resin residue is subjected to a second desorption using a third ethanol aqueous solution, and the obtained second desorption liquid is dried to obtain total saponins; the volume fraction of ethanol in the third ethanol aqueous solution is 50-60%.

[0011] Preferably, the solid-to-liquid ratio of the fenugreek powder to the first ethanol aqueous solution is 1 kg: 25-35 L;

[0012] The extraction temperature is 50-70° C., the extraction times are 3-4 times, and the single extraction time is 3-6 hours.

[0013] Preferably, the temperature of the microfiltration membrane separation is 30-70° C., and the pressure is 0.1-0.3 MPa; the mass percentage of the alcohol extract in the diluted alcohol extract is 2-4wt%, and the pH value of the diluted alcohol extract is 6-9.

[0014] Preferably, the model of the macroporous resin includes one or more of HPD-300, HPD-100 and AB-8;

[0015] The volume ratio of the macroporous resin to the permeate is 1:10-20;

[0016] The adsorption time is 3 to 5 hours.

[0017] Preferably, the volume ratio of the macroporous resin to the washing water is 1:4-6, and the washing time is 2.5-4 hours.

[0018] Preferably, the volume ratio of the macroporous resin to the second ethanol aqueous solution is 1:4-6; and the first desorption time is 2.5-3.5 hours.

[0019] Preferably, the volume ratio of the macroporous resin to the third ethanol aqueous solution is 1:6-8; and the second desorption time is 2.5-3.5 hours.

[0020] The present invention provides total saponins and / or total flavonoids of fenugreek seeds prepared by the preparation method described in the above technical scheme.

[0021] The present invention provides a pharmaceutical composition, comprising an active component and a pharmaceutically acceptable excipient; the active component comprises the fenugreek total saponins and / or total flavonoids described in the above technical solution.

[0022] The present invention also provides the use of the total saponins and / or total flavonoids of fenugreek described in the above technical solution or the pharmaceutical composition described in the above technical solution in the preparation of hypoglycemic drugs or anti-Alzheimer's disease drugs.

[0023] The invention uses 50-70 v / v% ethanol aqueous solution to extract fenugreek powder, dilutes with water and separates with a microfiltration membrane, adsorbs the obtained permeate with a macroporous resin, washes with water, and desorbs with a 30-35 v / v% ethanol aqueous solution to obtain total flavonoids, and then desorbs with a 50-60 v / v% ethanol aqueous solution to obtain total saponins. The invention adopts a preparation method of alcohol extraction, microfiltration membrane separation and macroporous resin purification, and can efficiently and simultaneously prepare high-purity fenugreek total saponins and total flavonoids, has a simple process, simple operation, low energy consumption, low production cost, and is suitable for industrial production.

[0024] The present invention studies the pharmacological effects of total saponins and total flavonoids in fenugreek, and animal experiments confirm that the total saponins and total flavonoids of fenugreek prepared by the present invention have obvious hypoglycemic and anti-Alzheimer's effects, and have obvious preventive and therapeutic effects on experimental type 2 diabetes and Alzheimer's disease. The preparations made of the fenugreek and total flavonoids for clinical application can effectively serve patients and have good clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the trend diagram of membrane flux changing with the device operation time at different microfiltration membrane separation temperatures;

[0026] Figure 2 It is the graph of saponin permeability at different microfiltration membrane separation temperatures;

[0027] Figure 3 This is the result diagram of saponin purity at different microfiltration membrane separation temperatures;

[0028] Figure 4 This is a trend chart of membrane flux variation with device operation time at different microfiltration membrane separation pH values;

[0029] Figure 5 The graph of saponin permeability at different microfiltration membrane separation pH values;

[0030] Figure 6 This is the result diagram of saponin purity at different microfiltration membrane separation pH values;

[0031] Figure 7 This is a trend chart of membrane flux changes with device operation time at different alcohol extract concentrations;

[0032] Figure 8 It is the graph of saponin permeability at different alcohol extract concentrations;

[0033] Fig. 9 This is the result diagram of saponin purity at different alcohol extract concentrations;

[0034] Fig.10 The figure shows the effect of different preparation methods on the purity of total saponins and total flavonoids;

[0035] Fig.11 The figure shows the effect of different preparation methods on the yield of total saponins and total flavonoids;

[0036] Fig.12 Schematic diagram of the change trend of saponin concentration in the supernatant after static adsorption of macroporous resin HPD-300 with adsorption time at 28°C and 180 r / min;

[0037] Fig.13 It is a schematic diagram showing the trend of saponin concentration in the supernatant after desorption of 90v / v% ethanol aqueous solution with adsorption time at 28°C and 180r / min;

[0038] Fig.14 It is the static adsorption capacity test graph of macroporous resins AB-8, D-101, HP20, ADS-7, HPD-300 and HPD-100;

[0039] Fig.15 The total saponin concentration in the desorption solution of ethanol aqueous solution with different concentrations is shown in the figure;

[0040] Fig.16 This is the result graph of total saponin purity after desorption of ethanol aqueous solutions with different concentrations. DETAILED DESCRIPTION

[0041] The present invention provides a method for preparing total saponins and / or total flavonoids of fenugreek, comprising the following steps:

[0042] Mixing fenugreek powder with a first ethanol aqueous solution for extraction to obtain an ethanol extract; wherein the volume fraction of ethanol in the first ethanol aqueous solution is 50-70%;

[0043] diluting the alcohol extract with water, and subjecting the obtained diluted alcohol extract to microfiltration membrane separation to obtain a permeate;

[0044] The permeate is adsorbed by a macroporous resin, and after washing, a first desorption is performed by a second ethanol aqueous solution to obtain a macroporous resin residue and a first desorption solution, respectively; the first desorption solution is dried to obtain total flavonoids; the volume fraction of ethanol in the second ethanol aqueous solution is 30-35%;

[0045] The macroporous resin residue is subjected to a second desorption using a third ethanol aqueous solution, and the obtained second desorption liquid is dried to obtain total saponins; the volume fraction of ethanol in the third ethanol aqueous solution is 50-60%.

[0046] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0047] The invention mixes fenugreek powder with a first ethanol aqueous solution to perform extraction to obtain an ethanol extract; the volume fraction of ethanol in the first ethanol aqueous solution is 50-70%.

[0048] In the present invention, the fenugreek powder is preferably fenugreek seed powder, and the particle size of the fenugreek seed powder is preferably ≥60 meshes.

[0049] In the present invention, the volume fraction of ethanol in the first ethanol aqueous solution is preferably 60-70%, more preferably 65-70%.

[0050] In the present invention, the solid-to-liquid ratio of the fenugreek powder to the first ethanol aqueous solution is preferably 1 kg:25-35 L, more preferably 1 kg:25-30 L.

[0051] In the present invention, the extraction temperature is preferably 50-70°C, more preferably 60-70°C, and further preferably 65-70°C; the number of extractions is preferably 3-4 times; the single extraction time is preferably 3-6h, more preferably 3.5-5h, and further preferably 3.5-4h; the extraction preferably includes oscillation extraction, and the oscillation extraction speed is preferably 60-80r / min, more preferably 70r / min.

[0052] After the extraction, the present invention preferably further comprises centrifuging the obtained extraction system and concentrating the obtained supernatant to obtain an alcohol extract. In the present invention, the rotation speed of the centrifugal separation is preferably 8000-10000 r / min, more preferably 9000 r / min; the time of the centrifugal separation is preferably 3-5 min, more preferably 4 min. In the present invention, the volume of the alcohol extract is preferably 1 / 4-1 / 3 of the total volume of the first ethanol aqueous solution.

[0053] After obtaining the alcohol extract, the present invention dilutes the alcohol extract with water, and separates the obtained diluted alcohol extract with a microfiltration membrane to obtain a permeate.

[0054] In the present invention, the mass percentage of the alcohol extract in the diluted alcohol extract is preferably 2-4wt%, more preferably 2-3wt%; the pH value of the diluted alcohol extract is preferably 6-9, more preferably 7-8.

[0055] In the present invention, the temperature of the microfiltration membrane separation is preferably 30-70°C, more preferably 40-70°C, and further preferably 50-60°C; the pressure of the microfiltration membrane separation is preferably 0.1-0.3MPa, more preferably 0.15-0.2MPa; the membrane pore size of the microfiltration membrane separation is preferably 50-200nm, more preferably 100-200nm; the number of microfiltration membrane separations is preferably 2-3 times, more preferably 3 times.

[0056] In the present invention, the specific operation of the microfiltration membrane separation preferably includes: adding the alcohol extract to the storage tank of the microfiltration membrane separation equipment for microfiltration membrane separation, adding water to maintain the liquid level when the residual liquid from the membrane separation accounts for less than one-third of the diluted alcohol extract, the water level in the storage tank after the last water addition is the same as the water level before microfiltration membrane separation, collecting the permeate and concentrating it to obtain a concentrated permeate.

[0057] After obtaining the permeate, the present invention uses a macroporous resin to adsorb the permeate, and after washing with water, uses a second ethanol aqueous solution to perform a first desorption to obtain a macroporous resin residue and a first desorption liquid, respectively; the first desorption liquid is dried to obtain total flavonoids; the volume fraction of ethanol in the second ethanol aqueous solution is 30-35%.

[0058] In the present invention, the macroporous resin preferably includes one or more of HPD-300, HPD-100 and AB-8, more preferably HPD-300. In the present invention, the volume ratio of the macroporous resin to the permeate is preferably 1:10-20, more preferably 1:12-15.

[0059] In the present invention, the adsorption time is preferably 3 to 5 hours, more preferably 4 to 5 hours; the adsorption temperature is preferably room temperature.

[0060] In the present invention, the volume ratio of the macroporous resin to the washing water is preferably 1:4-6, more preferably 1:4.5-5.5, and further preferably 1:5. In the present invention, the washing time is preferably 2.5-4h, more preferably 3-4h, and further preferably 3.5-4h.

[0061] In the present invention, the volume fraction of ethanol in the second ethanol aqueous solution is preferably 30-34%, more preferably 30-32%. In the present invention, the volume ratio of the macroporous resin to the second ethanol aqueous solution is preferably 1:4-6, more preferably 1:4.5-5.5, and further preferably 1:5. In the present invention, the time of the first desorption is preferably 2.5-3.5h, more preferably 3h; the temperature of the first desorption is preferably room temperature.

[0062] In the present invention, the drying preferably includes freeze drying. The present invention has no particular limitation on the conditions of the freeze drying as long as the solvent can be completely removed.

[0063] After obtaining the macroporous resin residue, the invention uses a third ethanol aqueous solution to perform a second desorption on the macroporous resin residue, dries the obtained second desorption liquid, and obtains total saponins; the volume fraction of ethanol in the third ethanol aqueous solution is 50-60%.

[0064] In the present invention, the volume ratio of the macroporous resin to the third ethanol aqueous solution is preferably 1:6-8, more preferably 1:6.5-8, and further preferably 1:7-7.5. In the present invention, the volume fraction of ethanol in the third ethanol aqueous solution is preferably 50-55%, more preferably 50-52%.

[0065] In the present invention, the second desorption time is preferably 2.5 to 3.5 hours, more preferably 3 hours; the second desorption temperature is preferably room temperature.

[0066] The present invention provides total saponins and / or total flavonoids of fenugreek seeds prepared by the preparation method described in the above technical solution. In the present invention, the purity of the total saponins is preferably ≥90%, and the purity of the total flavonoids is preferably ≥40%.

[0067] The present invention provides a pharmaceutical composition, comprising an active component and a pharmaceutically acceptable excipient; the active component comprises the fenugreek total saponins and / or total flavonoids described in the above technical solution.

[0068] The present invention provides the use of the total fenugreek saponins and / or total flavonoids described in the above technical solution or the pharmaceutical composition described in the above technical solution in the preparation of hypoglycemic drugs or anti-Alzheimer's disease drugs.

[0069] In order to further illustrate the present invention, the total saponins and / or total flavonoids of fenugreek and the preparation method thereof are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0070] In the following examples and comparative examples, the total saponin content was determined using anisaldehyde-concentrated sulfuric acid colorimetric method, and the total flavonoid content was determined using aluminum nitrate-sodium nitrite colorimetric method.

[0071] Example 1

[0072] (1) Crude extraction: 600 g of fenugreek seeds were crushed and passed through a 60-mesh sieve. The fenugreek seed powder (the portion below the sieve) was placed in an extraction tank. 15 L of 70 v / v% ethanol aqueous solution was added. The mixture was extracted three times at 70 r / min and 70° C. The single extraction time was 4 h. The extracts were combined and centrifuged at 8000 r / min for 5 min. The supernatant was concentrated to 15 L to obtain an alcohol extract (based on the amount of fenugreek seeds, the concentration of the alcohol extract was 4 w / v%). The saponin concentration in the alcohol extract was determined to be 5.24 mg / mL, and the total saponin purity was 22.46%.

[0073] (2) Microfiltration membrane filtration: Take alcohol extract, add pure water to dilute, obtain diluted alcohol extract (15L, alcohol extract concentration 2w / v%, pH=7), pour the gained diluted alcohol extract into the storage tank of microfiltration membrane device, the device membrane pore size is 200nm, start the microfiltration membrane device, control the film temperature to be 40 ℃, the film pressure to be 0.15MPa, use a material bucket to access the permeate, the device internal membrane separation residual liquid accounts for less than one-third of the diluted alcohol extract (i.e., there are 5L of feed liquid left in the storage tank), in the storage tank, add 10L of pure water, repeat 3 times. After the last tank volume is one-third of the diluted alcohol extract, stop the running device, merge the permeate filtered by microfiltration membrane 3 times, and be concentrated to 15L. It is 4.31mg / mL to measure the saponin concentration in the gained concentrated permeate, and the total saponin purity is 34.19%.

[0074] (3) Purification by macroporous resin: 40 mL of HPD-300 wet resin and 500 mL of concentrated permeate were added to the resin tank for mixed adsorption for 5 h, the liquid phase was discarded, the resin was rinsed with 200 mL of pure water for 4 h, the liquid phase was discarded, and the resin was desorbed with 200 mL of 30 v / v% ethanol aqueous solution for 3 h, the desorbed liquid was collected and freeze-dried to constant weight to obtain the total flavonoid component, and the total flavonoid purity was determined to be 45.14%. The resin was desorbed with 300 mL of 50 v / v% ethanol aqueous solution for 3 h, the desorbed liquid was collected and freeze-dried to constant weight to obtain the total saponin component, and the total saponin purity was 90.57%.

[0075] Example 2

[0076] (1) Crude extraction: 600 g of fenugreek seeds were crushed and passed through a 60-mesh sieve. The fenugreek seed powder (the portion below the sieve) was placed in an extraction tank. 15 L of a 70 v / v% ethanol aqueous solution was added. The mixture was extracted three times at 70 r / min and 70° C. The single extraction time was 3.5 h. The extracts were combined and centrifuged at 8000 r / min for 5 min. The supernatant was concentrated to 15 L to obtain an alcohol extract (concentration of 4 w / v%). The saponin concentration in the alcohol extract was determined to be 5.18 mg / mL, and the total saponin purity was 21.08%.

[0077] (2) Microfiltration membrane filtration: Take the alcohol extract pure water to dilute, obtain the diluted alcohol extract (15L, alcohol extract concentration is 2w / v%, pH=7), pour the obtained diluted alcohol extract into the storage tank of the microfiltration membrane device, the device membrane pore size is 200nm, start the microfiltration membrane device, control the membrane temperature to 60°C, the membrane pressure to 0.15MPa, use a bucket to receive the permeate, after the membrane separation residual liquid in the device accounts for less than one-third of the diluted alcohol extract (i.e., there are 5L of feed liquid left in the storage tank), add 10L of pure water in the storage tank, repeat 3 times. After the last tank volume is one-third of the diluted alcohol extract, stop the operation device to obtain the permeate. The permeate is concentrated to 15L, and the saponin concentration in the obtained concentrated permeate is measured to be 4.52mg / mL, and the total saponin purity is 37.24%.

[0078] (3) Purification by macroporous resin: 40 mL of HPD-300 wet resin and 500 mL of concentrated permeate were added to the resin tank for mixed adsorption for 5 h, the liquid phase was discarded, the resin was rinsed with 200 mL of pure water for 4 h, the liquid phase was discarded, and the resin was desorbed with 200 mL of 30 v / v% ethanol aqueous solution for 3 h, the desorbed liquid was collected and freeze-dried to constant weight to obtain the total flavonoid component, and the total flavonoid purity was determined to be 41.02%. The resin was desorbed with 300 mL of 50 v / v% ethanol aqueous solution for 3 h, the desorbed liquid was collected and freeze-dried to constant weight to obtain the total saponin component, and the total saponin purity was 92.43%.

[0079] Example 3

[0080] (1) Crude extraction: 3 kg of fenugreek seeds were crushed and passed through a 60-mesh sieve. The fenugreek seed powder (the portion below the sieve) was placed in an extraction tank, and 75 L of a 70 v / v% ethanol aqueous solution was added. The extraction was performed three times at 70 r / min and 70° C. The single extraction time was 3.5 h. The extracts were combined and centrifuged at 8000 r / min for 5 min. The supernatant was concentrated to 15 L to obtain an alcohol extract (concentration of 20 w / v%). The saponin concentration in the alcohol extract was determined to be 4.96 mg / mL, the total saponin purity was 22.64%, and the total flavonoid purity was 3.15%.

[0081] (2) Microfiltration membrane filtration: Take the alcohol extract, dilute with pure water, obtain the diluted alcohol extract (15L, alcohol extract concentration is 2w / v%, pH=7), pour the obtained diluted alcohol extract into the storage tank of the microfiltration membrane device, the device membrane pore size is 200nm, start the microfiltration membrane device, control the membrane temperature to 50°C, the membrane pressure to 0.15MPa, use a bucket to receive the permeate, after the membrane separation residual liquid in the device accounts for less than one-third of the diluted alcohol extract (i.e., there are 5L of feed liquid left in the storage tank), add 10L of pure water to the storage tank, repeat 3 times. After the last tank volume is one-third of the diluted alcohol extract, stop the operation of the device to obtain the permeate. The permeate is concentrated to 15L, and the saponin concentration in the obtained concentrated permeate is measured to be 4.19mg / mL, the total saponin purity is 36.18%, and the total flavonoids purity is 8.69%.

[0082] (3) Purification by macroporous resin: 200 mL of HPD-300 wet resin was taken, 3 L of concentrated permeate was added to the resin tank for mixed adsorption for 5 h, the liquid phase was discarded, the resin was rinsed with 1 L of pure water for 4 h, the liquid phase was discarded, and the resin was desorbed with 1 L of 30 v / v% ethanol aqueous solution for 3 h, the desorbed liquid was collected and freeze-dried to constant weight to obtain the total flavonoid component, and the total flavonoid purity was 38.26%. The resin was desorbed with 1.5 L of 50 v / v% ethanol aqueous solution for 3 h, the desorbed liquid was collected and freeze-dried to constant weight to obtain the total saponin component, and the total saponin purity was 94.86%.

[0083] Example 4

[0084] Effect of Microfiltration Membrane Separation Temperature on Saponin Separation

[0085] In the alcohol extract prepared in embodiment 3, add pure water to dilute, obtain dilute alcohol extract (concentration is 4w / v%, pH=7, volume is 15L), the gained dilute alcohol extract is poured into the storage tank of microfiltration membrane device, the device membrane pore size is 200nm, start the microfiltration membrane device, control the film temperature to be respectively 30 ℃, 40 ℃, 50 ℃, 60 ℃ and 70 ℃, the film pressure is 0.15MPa, cross the film and use the material bucket to access the permeate, after the device inner membrane separation residual liquid accounts for the dilute alcohol extract within one-third (i.e., there are 5L feed liquid left in the storage tank), in the storage tank, add 10L pure water, repeat 3 times. After the last tank volume is one-third of the dilute alcohol extract, stop the running device, obtain permeate. The permeate is concentrated to 15L, and concentrated permeate is obtained. The total saponin content in the concentrated permeate was determined by anisaldehyde-concentrated sulfuric acid colorimetry, and then the saponin permeability was calculated: saponin permeability = saponin mass in the concentrated permeate / saponin mass in the extract × 100%. 10 mL of the concentrated permeate was freeze-dried, the solid mass was weighed, and the saponin purity was calculated: saponin purity = saponin purity = saponin mass in the concentrated permeate / total solid mass in the concentrated permeate × 100%. The saponin purity before microfiltration membrane separation was 22.64%.

[0086] Figure 1 The diagram below shows the variation trend of membrane flux with device operation time at different microfiltration membrane separation temperatures. Figure 2 is the saponin permeability at different microfiltration membrane separation temperatures, Figure 3 The results of saponin purity at different microfiltration membrane separation temperatures are shown in the figure. As can be seen from the figure, when the microfiltration membrane separation temperature is 30℃, 40℃, 50℃, 60℃ and 70℃, the saponin permeability is 76.04%, 79.58%, 85.03%, 82.8% and 82.81% respectively, and the saponin permeability is basically stable at more than 80%; when the microfiltration membrane separation temperature is 30℃, 40℃, 50℃, 60℃ and 70℃, the saponin purity is 33.54%, 36.82%, 37.06%, 35.67% and 33.36% respectively, and the saponin purity is increased by 10.90%, 14.18%, 14.42%, 13.03% and 10.72% respectively. Among them, when the microfiltration membrane separation temperature is 50℃, the saponin permeability and purity are the highest, and the saponin purification effect is the best.

[0087] Example 5

[0088] Effect of pH value on the separation of saponins

[0089] Pure water was added to the alcohol extract prepared in Example 3 for dilution to obtain a diluted alcohol extract (concentration of 4w / v%, volume of 15L, pH values ​​of 4, 7 and 10 respectively), and the obtained diluted alcohol extract was poured into the storage tank of the microfiltration membrane device, the device membrane pore size was 200nm, the microfiltration membrane device was started, the membrane temperature was controlled to be 30°C, the membrane pressure was 0.15MPa, and the permeate was taken using a bucket. After the device reached the minimum circulation volume (i.e., 5L of feed liquid was left in the storage tank), 10L of pure water was added to the storage tank, and repeated 3 times. After the minimum circulation volume was reached for the last time, the device was stopped to obtain the permeate. The permeate was concentrated to 15L to obtain a concentrated permeate. The saponin permeability and purity were calculated.

[0090] Figure 4 The diagram below shows the change trend of membrane flux with device operation time under different microfiltration membrane separation pH values. Figure 5 is the saponin permeability at different microfiltration membrane separation pH values, Figure 6 The results of saponin purity under different microfiltration membrane separation pH values ​​are shown in the figure. As can be seen from the figure, when the microfiltration membrane separation pH values ​​are 4, 7 and 10, the saponin permeability is 82.14%, 76.04% and 75.23%, respectively, and the saponin purity is 33.4%, 37.06% and 30.02%, respectively, indicating that acidity will increase the saponin permeability, but acidic precipitation will be produced under acidic conditions, thereby reducing the purity of saponin. Considering all factors, pH = 7 is selected as the acidity and alkalinity condition for microfiltration membrane filtration.

[0091] Example 6

[0092] Effect of alcohol extract concentration (mass percentage) on saponin separation

[0093] Pure water was added to the alcohol extract prepared in Example 3 for dilution to obtain a diluted alcohol extract (concentrations were 1 w / v%, 2 w / v% and 4 w / v%, pH=7, and a volume of 15 L). The resulting diluted alcohol extract was poured into a storage tank of a microfiltration membrane device, the device membrane pore size was 200 nm, the microfiltration membrane device was started, the membrane temperature was controlled to be 30 ° C, the membrane pressure was 0.15 MPa, and a barrel was used to receive the permeate. After the residual liquid in the device membrane separation accounted for less than one-third of the diluted alcohol extract (i.e., 5 L of feed liquid was left in the storage tank), 10 L of pure water was added to the storage tank, and repeated 3 times. After the last tank volume was one-third of the diluted alcohol extract, the device was stopped to obtain the permeate. The permeate was concentrated to 15 L to obtain a concentrated permeate. The transmittance and purity of saponins were calculated.

[0094] Figure 7 The diagram below shows the variation trend of membrane flux with device operation time under different alcohol extract concentrations. Figure 8 is the saponin permeability at different alcohol extract concentrations, Fig. 9 The figure shows the results of saponin purity under different alcohol extract concentrations. It can be seen from the figure that when the alcohol extract concentration is 1wt%, 2wt% and 4wt%, the saponin permeability is 77.6%, 87.06% and 85.85% respectively, and the saponin purity is 32.52%, 37.07% and 37.06% respectively, indicating that before and after passing through the membrane, the saponin permeability is basically stable at more than 80%. Considering the comprehensive saponin purity, 2wt% is selected as the extraction solution concentration condition.

[0095] Comparative Example 1

[0096] Total saponins and total flavonoids were prepared according to the method of Example 3, which differed from Example 1 only in that the microfiltration membrane separation step was omitted, and the purity of total saponins was 70.62%, and the purity of total flavonoids was 35.33%.

[0097] Fig.10 The figure is an effect diagram of different preparation methods on the purity of total saponins and total flavonoids, wherein the preparation methods are: crude extraction (Example 3), crude extraction + microfiltration membrane separation (Example 3), crude extraction + macroporous resin purification (Comparative Example 1) and crude extraction + microfiltration membrane separation + macroporous resin purification (Example 3). It can be seen from the figure that the present invention adopts the method of crude extraction + microfiltration membrane separation + macroporous resin purification to prepare total saponins and total flavonoids, which can significantly improve the purity of total saponins and total flavonoids, so that the purity of saponins is stabilized at 30-40%.

[0098] Fig.11The figure is an influence diagram of different preparation methods on the yield of total saponins and total flavonoids, wherein the preparation methods are: crude extraction (Example 3), crude extraction + microfiltration membrane separation (Example 3), crude extraction + macroporous resin purification (Comparative Example 1) and crude extraction + microfiltration membrane separation + macroporous resin purification (Example 3). It can be seen from the figure that the present invention adopts the method of crude extraction + microfiltration membrane separation + macroporous resin purification to prepare total saponins and total flavonoids, and the yield of total saponins is as high as 6.32%, and the yield of total flavonoids is as high as 4.98%, and the yield of total saponins and total flavonoids is high.

[0099] Example 7

[0100] Static adsorption of macroporous resin (HPD-300)

[0101] Take 200mL of HPD-300 wet resin, concentrate 3L of permeate, add it to the resin tank, adsorb it at 28℃ and shaker speed of 180r / min, and then desorb it using 90v / v% ethanol aqueous solution at 28℃ and shaker speed of 180r / min; from the beginning of adsorption (or desorption), take 100μL of supernatant every 30min, measure the saponin concentration, and obtain the change trend of static adsorption / desorption of macroporous resin over time.

[0102] Fig.12 This is a schematic diagram showing the trend of saponin concentration in the supernatant after static adsorption of macroporous resin HPD-300 with adsorption time at 28°C and 180 r / min. Fig.13 The figure is a schematic diagram of the change trend of saponin concentration in the supernatant after desorption of 90v / v% ethanol aqueous solution at 28℃ and 180r / min. It can be seen from the figure that the macroporous resin HPD-300 requires 250~300min for complete adsorption and 150~200min for complete desorption, which is the time condition for adsorption using macroporous resin HPD-300 and desorption using different concentrations of ethanol aqueous solution.

[0103] Example 8

[0104] Effect of macroporous resin types on the quality of adsorbed saponins

[0105] 1 g of each of the macroporous resins AB-8, D-101, HP20, ADS-7, HPD-300 and HPD-100 was accurately weighed, and each resin was placed in a conical flask containing 10, 20, 30, 40, 50 and 60 mL of fenugreek stock solution (i.e., the concentrated permeate prepared in Example 3), placed on a 28°C constant temperature water bath shaker, shaken at a speed of 180 r / min for 8 hours, and allowed to stand for 4 hours to achieve saturated adsorption, and the supernatant was taken to determine the saponin content. The adsorption capacity of the macroporous resin was used as an index to obtain a macroporous resin adsorption capacity curve, the abscissa being the volume of the fenugreek stock solution of different volumes, the ordinate being the mass of saponin that the macroporous resin can adsorb, and the mass of adsorbed saponin = (saponin concentration in the fenugreek stock solution before adsorption - saponin concentration in the supernatant after adsorption) × the volume of the fenugreek stock solution.

[0106] Fig.14 The static adsorption capacity test graph of macroporous resins AB-8, D-101, HP20, ADS-7, HPD-300 and HPD-100 is shown in the figure. As can be seen from the figure, the macroporous resin HPD-300 has the best adsorption effect on saponin.

[0107] Example 9

[0108] Choice of desorbent

[0109] Using Example 1 to concentrate the permeate

[0110] The macroporous resin HPD-300 adsorbed with the concentrated permeate prepared in Example 1 (total saponin concentration of 4.31 mg / mL, resin volume: concentrated permeate volume ratio = 1:15) was desorbed using ethanol aqueous solutions of different concentrations (ethanol volume fractions of 10%, 30%, 50%, 70% and 90%, respectively) for the same time, and the desorbed liquid was collected and tested for total saponin concentration and purity.

[0111] Fig.15 The total saponin concentration in the desorption solution of ethanol aqueous solution with different concentrations is shown in the figure. Fig.16 The figure is the result of the total saponin purity after desorption of different concentrations of ethanol aqueous solution. It can be seen from the figure that when the volume fraction of ethanol in the desorbent ethanol aqueous solution is 10%, 30%, 50%, 70% and 90%, the total saponin concentration in the desorption liquid is 0.07%, 0.93%, 2.92%, 0.3% and 0.13%, respectively, and the total saponin purity is 22.04%, 49.19%, 92.37%, 66.16% and 52.02%, respectively. With the increase of the concentration of ethanol aqueous solution, the concentration and purity of total saponin first increase and then decrease. When the volume fraction of ethanol in the ethanol aqueous solution is 50%, the concentration and purity of total saponin are the highest, and thus its extraction rate is also the highest.

[0112] The invention adopts macroporous resin HPD-300 to absorb and separate total flavonoids and total saponins. The purity of total saponins can reach 90-95% of the total mass of the extract, and the purity of total flavonoids can reach more than 40%, which can be directly used in clinical practice.

[0113] Although the above-mentioned embodiments have made a detailed description of the present invention, they are only some embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on the embodiments of the present invention without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for preparing total saponins and / or total flavonoids of fenugreek, characterized in that: The following steps are involved: Mixing the fenugreek powder with the first ethanol aqueous solution for extraction to obtain an ethanol extract; The volume fraction of ethanol in the first ethanol aqueous solution is 50-70%; diluting the alcohol extract with water, and subjecting the obtained diluted alcohol extract to microfiltration membrane separation to obtain a permeate; The permeate is adsorbed by a macroporous resin, and after washing, a first desorption is performed by a second ethanol aqueous solution to obtain a macroporous resin residue and a first desorption liquid, respectively; the first desorption liquid is dried to obtain total flavonoids; The volume fraction of ethanol in the second ethanol aqueous solution is 30-35%; The macroporous resin residue is subjected to a second desorption using a third ethanol aqueous solution, and the obtained second desorption liquid is dried to obtain total saponins; the volume fraction of ethanol in the third ethanol aqueous solution is 50-60%.

2. The preparation method according to claim 1, characterized in that: The solid-to-liquid ratio of the fenugreek powder to the first ethanol aqueous solution is 1 kg: 25-35 L; The extraction temperature is 50-70° C., the extraction times are 3-4 times, and the single extraction time is 3-6 hours.

3. The preparation method according to claim 1, characterized in that: The temperature of the microfiltration membrane separation is 30-70°C, and the pressure is 0.1-0.3MPa; the mass percentage of the alcohol extract in the diluted alcohol extract is 2-4wt%, and the pH value of the diluted alcohol extract is 6-9.

4. The preparation method according to claim 1, characterized in that: The model of the macroporous resin includes one or more of HPD-300, HPD-100 and AB-8; The volume ratio of the macroporous resin to the permeate is 1:10-20; The adsorption time is 3 to 5 hours.

5. The preparation method according to claim 1 or 4, characterized in that: The volume ratio of the macroporous resin to the washing water is 1:4-6, and the washing time is 2.5-4 hours.

6. The preparation method according to claim 1 or 4, characterized in that: The volume ratio of the macroporous resin to the second ethanol aqueous solution is 1:4-6; and the first desorption time is 2.5-3.5 hours.

7. The preparation method according to claim 1 or 4, characterized in that: The volume ratio of the macroporous resin to the third ethanol aqueous solution is 1:6-8; and the second desorption time is 2.5-3.5 hours.

8. Total saponins and / or total flavonoids of fenugreek seeds obtained by the preparation method according to any one of claims 1 to 7.

9. A pharmaceutical composition, characterized in that The invention comprises active components and pharmaceutically acceptable excipients; the active components comprise the total saponins and / or total flavonoids of fenugreek as claimed in claim 8.

10. Use of the total saponins and / or total flavonoids of fenugreek according to claim 8 or the pharmaceutical composition according to claim 9 in the preparation of hypoglycemic drugs or anti-Alzheimer's disease drugs.

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