Extraction method of rhizoma dioscoreae nipponicae total saponins

Abstract

The invention provides a method for extracting total saponins of rhizoma dioscoreae nipponicae. The method comprises the following steps: extracting the rhizoma dioscoreae nipponicae by adopting an ethanol reflux method to obtain a crude extract of the total saponins of the rhizoma dioscoreae nipponicae, and extracting the crude extract by using water-saturated n-butyl alcohol. The method also comprises the step of purifying the obtained extract by adopting a macroporous adsorption resin method. The extraction method disclosed by the invention is low in cost and simple to operate, the extract yield and the purity of the rhizoma dioscoreae nipponicae total saponins can be remarkably improved, and the extraction method disclosed by the invention has stability and reliability and is suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to a method for extracting total saponins from Rhizoma Dioscoreae, and more specifically, to a method for extracting total saponins from Rhizoma Dioscoreae, comprising extracting Rhizoma Dioscoreae by using an ethanol reflux method to obtain a crude extract of total saponins from Rhizoma Dioscoreae, extracting the crude extract by using water-saturated n-butanol to obtain an extract, and optionally purifying the obtained extract by using a macroporous adsorption resin method. Background Art

[0002] Dioscorea ternata, also known as Dioscorea ternata, Dioscorea ternata, Dioscorea ternata, and Dioscorea ternata, is a plant of the Dioscorea family. Dioscorea nipponica Makino ) The main root contains dioscin, diosgenin, diosgenin, smilax glabra glycosides and other ingredients. Rhizoma Dioscoreae has the effects of removing rheumatism, promoting blood circulation and unblocking collaterals, clearing the lungs and resolving phlegm. It is mainly used to treat rheumatic arthralgia, phlegm-heat cough and asthma, chest pain, traumatic injury, carbuncle, sore and so on.

[0003] In order to improve the efficacy of Rhizoma Dioscoreae, reduce its side effects, increase bioavailability, and facilitate the quantitative analysis of its active ingredients, researchers have carried out the extraction of total saponins from Rhizoma Dioscoreae. At present, there are mainly the following methods for the extraction of total saponins: decoction, ethanol reflux extraction, microwave extraction, enzyme extraction, bionic extraction, ultrasonic extraction, supercritical fluid extraction, ultra-high pressure extraction, etc.

[0004] In addition, the extraction process of total saponins from Rhizoma Dioscoreae is accompanied by the precipitation of various chemical substances. According to clinical studies, the content of active ingredients determines the dosage and efficacy of the drug, and how to increase the content of active ingredients has become a research focus. At present, the purification methods of total saponins include macroporous adsorption resin method, column chromatography silica gel method, high-speed countercurrent chromatography method, solvent precipitation method, foam separation method, membrane separation method, etc.

[0005] However, the existing extraction method of total saponins from Rhizoma Dioscoreae cannot meet the application demand of total saponins from Rhizoma Dioscoreae, and there is still a need for an extraction method of total saponins from Rhizoma Dioscoreae with low cost, simple operation, high yield and content of total saponins from Rhizoma Dioscoreae and suitable for large-scale industrial production. Summary of the invention

[0006] 1. Technical issues to be resolved The invention aims at the shortcomings of the existing extraction method of total saponins of Rhizoma Dioscoreae, and provides a method for extracting total saponins of Rhizoma Dioscoreae with low cost, simple operation, high total saponins extract rate and content, and suitable for large-scale industrial production.

[0007] (II) Technical solution The present invention provides a method for extracting total saponins from Rhizoma Dioscoreae, which comprises: using an ethanol reflux method to extract the total saponins from Rhizoma Dioscoreae ( Dioscorea nipponica Makino) is used for extraction to obtain a crude extract of total saponins of Rhizoma Dioscoreae, and the crude extract is extracted with water-saturated n-butanol to obtain an extract, wherein in the ethanol reflux method, an ethanol aqueous solution with a volume concentration of 60-70% is used for extraction, each extraction time is 1.5-2h, the solid-liquid ratio is 1:10 to 1:14, and a total of 2-3 extractions are performed.

[0008] One embodiment of the present invention is a method for extracting total saponins from Rhizoma Dioscoreae, the method comprising: using an ethanol reflux method to extract the total saponins from Rhizoma Dioscoreae ( Dioscorea nipponica Makino ) was used for extraction to obtain a crude extract of total saponins of Rhizoma Dioscoreae, and the crude extract was extracted with water-saturated n-butanol to obtain an extract, and the extract was extracted with an ethanol aqueous solution with a volume concentration of 70% in the ethanol reflux method.

[0009] Preferably, in the ethanol reflux method, each extraction time is 1.5 h.

[0010] Preferably, in the ethanol reflux method, the solid-liquid ratio is 1:12.

[0011] Preferably, the extraction is performed three times in total in the ethanol reflux method.

[0012] Another embodiment of the present invention is a method for extracting total saponins from Rhizoma Dioscoreae, the method comprising: using an ethanol reflux method to extract the total saponins from Rhizoma Dioscoreae ( Dioscorea nipponica Makino ) is used to extract the crude extract of total saponins of Rhizoma Dioscoreae, the crude extract is extracted with water-saturated n-butanol to obtain an extract, and the obtained extract is purified by a macroporous adsorption resin method, wherein in the ethanol reflux method, an ethanol aqueous solution with a volume concentration of 60-70% is used for extraction, each extraction time is 1.5-2h, the solid-liquid ratio is 1:10 to 1:14, and a total of 2-3 extractions are performed.

[0013] Preferably, in the ethanol reflux method, each extraction time is 1.5 h.

[0014] Preferably, in the ethanol reflux method, the solid-liquid ratio is 1:12.

[0015] Preferably, the extraction is performed three times in total in the ethanol reflux method.

[0016] Preferably, in the macroporous adsorption resin method, AB-8 type resin is used as the purification resin, the sample loading amount is resin: sample = (4.6-5.4): 1, the loading concentration is 0.10-0.20 g / mL of crude drug, the eluent is 60-70% ethanol aqueous solution by volume concentration, and the eluent dosage is 11-14 BV, preferably 12 BV.

[0017] Preferably, in the macroporous adsorption resin method, the loading amount is resin: sample = 5:1.

[0018] Preferably, in the macroporous adsorption resin method, the loading concentration is 0.2 g / mL of the crude drug.

[0019] Preferably, in the macroporous adsorption resin method, the eluent is an ethanol aqueous solution with a volume concentration of 70%.

[0020] (III) Beneficial effects The present invention extracts the Rhizoma Dioscoreae by using an ethanol reflux method, a water-saturated n-butanol extraction method and an optional macroporous adsorption resin method under specific conditions, thereby simplifying the extraction operation of the Rhizoma Dioscoreae total saponins, reducing the extraction cost, and increasing the extract yield and content of the Rhizoma Dioscoreae total saponins. In addition, the extraction method of the present invention has stability and reliability, and is therefore suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the HPLC dioscin standard curve used in determining the dioscin content in Rhizoma Dioscoreae extract.

[0022] Figure 2 This is the UV spectrophotometric dioscin standard curve used in the determination of total saponin content in Rhizoma Dioscoreae.

[0023] Figure 3 It is a graph showing the effect of ethanol concentration on the extract yield and content of total saponins of Rhizoma Dioscoreae in the ethanol reflux method.

[0024] Figure 4 2. It is a graph showing the effect of extraction time on the extract yield and content of total saponins in Rhizoma Dioscoreae in the ethanol reflux method.

[0025] Figure 5 2. It is a graph showing the effect of the number of extractions in the ethanol reflux method on the yield and content of the total saponins of Rhizoma Dioscoreae.

[0026] Figure 6 It is a graph showing the effect of the material-liquid ratio on the extract yield and content of total saponins from Rhizoma Dioscoreae in the ethanol reflux method.

[0027] Figure 7 It is the leakage curve of the macroporous adsorption resin of the present invention.

[0028] Figure 8 This is a graph showing the relationship between the sample concentration and the adsorption rate in the macroporous adsorption resin method.

[0029] Fig. 9 is a graph showing the effect of eluent concentration on desorption rate in a macroporous adsorption resin method.

[0030] Fig.10 This is a graph showing the effect of the amount of eluent used on the resolution in the macroporous adsorption resin method. DETAILED DESCRIPTION

[0031] The present invention will be explained and illustrated in more detail below.

[0032] The extraction method of total saponins of Rhizoma Dioscoreae of the present invention comprises: using ethanol reflux method to extract Rhizoma Dioscoreae ( Dioscorea nipponica Makino ) to obtain a crude extract of total saponins of Rhizoma Dioscoreae, and to extract the crude extract with water-saturated n-butanol to obtain an extract. In the ethanol reflux method, an ethanol aqueous solution with a volume concentration of 60-70% is used for extraction, each extraction time is 1.5-2h, the solid-liquid ratio is 1:10 to 1:14, and a total of 2-3 extractions are performed.

[0033] The ethanol reflux method refers to a method of reflux extraction using ethanol or an aqueous solution containing ethanol as a solvent. Reflux extraction includes reflux hot soaking method and reflux cold soaking method (or circulating reflux method). The present invention adopts reflux hot soaking method, and the extraction temperature is the boiling point of the extraction solvent.

[0034] The material-liquid ratio refers to the ratio of the sample to be extracted to the extraction solvent, and in the present invention, it refers to the ratio of the medicinal material Rhizoma Dioscoreae and the ethanol aqueous solution.

[0035] The invention combines the ethanol reflux method with the water-saturated n-butanol extraction process and optimizes the concentration of the extraction solvent, the extraction time, the solid-liquid ratio and the number of extractions of the ethanol reflux method, so as to improve the extract yield and the total saponin content of the Rhizoma Dioscoreae total saponins.

[0036] The extract yield refers to the mass of the fluid extract or dry extract produced by 100 units of Chinese medicinal materials, generally expressed as a percentage. In the present invention, the extract yield refers to the mass of the dry extract produced by 100 units of Chinese medicinal materials.

[0037] From the perspective of increasing the extraction rate and total saponin content, it is preferred to use an ethanol aqueous solution with a volume concentration of 70% for extraction in the ethanol reflux method. Preferably, in the ethanol reflux method, each extraction time is 1.5 hours. Preferably, in the ethanol reflux method, the solid-liquid ratio is 1:12. Preferably, in the ethanol reflux method, a total of 3 extractions are performed.

[0038] According to one embodiment of the present invention, the extraction method of the total saponins of Rhizoma Dioscoreae of the present invention comprises: extracting Rhizoma Dioscoreae by ethanol reflux method to obtain a crude extract of the total saponins of Rhizoma Dioscoreae, and extracting the crude extract by water-saturated n-butanol to obtain an extract. In the ethanol reflux method, an ethanol aqueous solution with a volume concentration of 70% is used for extraction, each extraction time is 1.5h, the solid-liquid ratio is 1:12, and a total of 3 extractions are performed.

[0039] According to another embodiment of the present invention, the extraction method of the total saponins of Rhizoma Dioscoreae of the present invention comprises: extracting Rhizoma Dioscoreae by ethanol reflux method to obtain a crude extract of the total saponins of Rhizoma Dioscoreae, extracting the crude extract by water-saturated n-butanol to obtain an extract, and purifying the obtained extract by macroporous adsorption resin method, wherein, in the ethanol reflux method, an ethanol aqueous solution with a volume concentration of 60-70% is used for extraction, each extraction time is 1.5-2h, the solid-liquid ratio is 1:10 to 1:14, and a total of 2-3 extractions are performed. By simultaneously using ethanol reflux method extraction, water-saturated n-butanol extraction and macroporous adsorption resin method purification, the present invention can further increase the content of the total saponins of Rhizoma Dioscoreae in the extract, and will not significantly reduce the yield of the total saponins of Rhizoma Dioscoreae.

[0040] The macroporous adsorption resin method is an extraction and refining process that utilizes the adsorption and screening principles of macroporous adsorption resins to separate effective components from samples to be separated and remove impurities.

[0041] From the perspective of increasing the total saponin content, it is preferred to use AB-8 type resin as the purification resin in the macroporous adsorption resin method, the sample loading amount is resin: sample = (4.6-5.4): 1, the loading concentration is 0.10-0.20 g / mL of the crude drug, the eluent is an ethanol aqueous solution with a volume concentration of 60-70%, and the eluent dosage is 11-14 BV, preferably 12 BV.

[0042] The loading amount in the macroporous adsorption resin method refers to the weight ratio of the macroporous adsorption resin to the loaded sample. In the extraction method of the total saponins of Rhizoma Dioscoreae of the present invention, the loading amount refers to the weight ratio of the macroporous adsorption resin to the loaded Rhizoma Dioscoreae extract, expressed as the ratio of resin:sample.

[0043] The loading concentration in the macroporous adsorption resin method of the present invention refers to the mass concentration of the loading solution. In the extraction method of the total saponins of Rhizoma Dioscoreae of the present invention, the loading concentration refers to the mass concentration of the Rhizoma Dioscoreae extract contained in the loading solution, expressed as the concentration of the crude drug content.

[0044] From the perspective of increasing the total saponin content, preferably in the macroporous adsorption resin method, the sample loading is resin: sample = 5: 1. Preferably, in the macroporous adsorption resin method, the loading concentration is 0.2 g / mL of the crude drug. Preferably, in the macroporous adsorption resin method, the eluent is an ethanol aqueous solution with a volume concentration of 70%.

[0045] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

[0046] Example Experimental methods 1. Determination of dioscin content in dioscorea - HPLC (this method is from the HPLC method for Dioscorea zingiberensis in the 2020 edition of the Chinese Pharmacopoeia) Materials: Dioscorea nipponica Makino (purchased from the pharmacy of Shanxi Provincial Hospital of Traditional Chinese Medicine); Dioscorea saponin (China Food and Drug Inspection Institutes, 111707-201703).

[0047] Reagents: methanol, acetonitrile, anhydrous ethanol, n-butanol, perchloric acid, glacial acetic acid, vanillin, etc.

[0048] Instruments and equipment: Intelligent constant temperature electric heating jacket (Beijing Yongguangming Medical Instrument Co., Ltd.); analytical balance (Aux Company, USA); DACW-4 constant temperature water bath (Guangzhou Zhongxing Experimental Instrument Co., Ltd.); water purifier (Hangzhou Tianchuang Environmental Technology Co., Ltd.); rotary evaporator (Yarong Instrument Factory).

[0049] Methods and conditions: The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler; acetonitrile-water (55:45) as the mobile phase; the detection wavelength was 203 nm; and the injection volume was 10uL.

[0050] Preparation of standard solution: Accurately weigh 3.00 mg of diosgenin reference substance and dilute to 10 mL with methanol.

[0051] Preparation of test solution: Accurately weigh 0.25 g of the treated sample, place it in a conical flask, accurately add 25 mL of 65% ethanol, weigh it, ultrasonically treat it for 30 minutes, cool it, weigh it, make up the lost weight with 65% ethanol, shake it well, filter it, and take the filtrate.

[0052] Draw a standard curve: accurately pipette 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 mL of the above standard solution, dissolve in methanol and place in a 5 mL volumetric flask, shake and mix, take 10 μL for injection, and determine its peak area according to the above chromatographic conditions. Draw a standard curve with dioscin concentration as the horizontal axis and peak area as the vertical axis, see Figure 1 The regression equation is Y=36.007X-16.229, r=0.999.

[0053] Measurement and calculation of dioscin content: The samples were measured by high performance liquid chromatography to obtain the peak area. The concentration of dioscin in the test samples was calculated based on the standard curve and regression equation, and further converted into the content of dioscin (a major component of the total saponins of Dioscorea schrenkiana) in the extract (mg / g).

[0054] 2. Determination method of total saponin content in Rhizoma Dioscoreae - UV spectrophotometry (this method comes from the UV spectrophotometry method for Rhizoma Dioscoreae in the 2020 edition of the Chinese Pharmacopoeia) (1) Preparation of standard solution Accurately weigh 4.00 mg of dioscin standard, dissolve it in methanol to make up to 25 mL, and ultrasonicate for 15 min to obtain a dioscin standard solution with a concentration of 0.16 mg / mL, which was stored in a refrigerator at 4°C.

[0055] (2) Determination of detection wavelength Accurately pipette 1.0 mL of standard solution and test solution into stoppered test tubes, open the lid and place in a 70°C water bath. After the solvent in the test tube is evaporated, add 0.2 mL of 5% vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid into the test tubes, shake well, cover the stopper, and continue to react in a 70°C water bath for 20 min. Cool in ice water for 5 min, add glacial acetic acid to make the volume 10 mL, shake well, let stand for 20 min, and perform spectral scanning at 200-800 nm. The absorption curves of the reference and test samples are basically the same, and both have maximum absorption peaks at 468 nm. The blank solution has no absorption at this wavelength, so 468 nm is selected as the detection wavelength.

[0056] (3) Drawing of standard curve Accurately pipette 0.10, 0.20, 0.40, 0.80, 1.00, 1.60, and 2.00 mL into stoppered test tubes respectively, and perform the same operation as (2). Detect the absorbance at a wavelength of 468 nm. Draw a standard curve with the mass of dioscin as the horizontal axis and the absorbance as the vertical axis, see Figure 2 .

[0057] The regression equation obtained is: Y=1.9689X+0.1904, r=0.999, Y is the absorbance, X is the mass of diosgenin (mg), and the linear range is 0.016--0.320mg.

[0058] (4) Measurement and calculation of the total saponin content of Rhizoma Dioscoreae: Measure the absorbance of the test solution at a wavelength of 468 nm, and calculate the content of the total saponin content of Rhizoma Dioscoreae in the Rhizoma Dioscoreae extract based on the standard curve and regression equation.

[0059] Example 1 and Comparative Examples 1 to 6 Example 1: Weigh 20g of Rhizoma Dioscoreae medicinal slices, crush them, use 70% ethanol as solvent for reflux extraction twice, use 200mL extraction solvent each time, extract for 1.5h, and combine the extracts. Take 1 / 2 of the weight of the extract, extract twice with water-saturated n-butanol (400ml×2), and use the above-mentioned high-performance liquid chromatography to determine the content after extraction; the remaining 1 / 2 of the extract is directly determined by the above-mentioned high-performance liquid chromatography, and the results are shown in Table 1 below.

[0060] Comparative Example 1: Weigh 20 g of Rhizoma Dioscoreae medicinal slices, crush, add 200 mL of 70% ethanol, reflux extract for 1.5 h, extract once. The content of the obtained extract was determined using the above-mentioned high performance liquid chromatography method, and the results are shown in Table 1 below.

[0061] Comparative Example 2: Weigh 20 g of Rhizoma Dioscoreae medicinal slices, crush, add 200 mL of purified water, reflux extract for 1.5 h, extract once. The content of the obtained extract was determined using the above-mentioned high performance liquid chromatography method, and the results are shown in Table 1 below.

[0062] Comparative Example 3: Weigh 20g of the decoction piece of Rhizoma Cynoglossi, crush it, add 200mL of 70% ethanol, reflux extract for 1.5h, extract once; after filtering, add 200mL of purified water, continue reflux extraction for 1.5h; combine the two filtrates and dry them. The content of the obtained extract was determined using the above-mentioned high performance liquid chromatography method, and the results are shown in Table 1 below.

[0063] Comparative Example 4: Weigh 20 g of Rhizoma Dioscoreae medicinal slices, crush them, and use 70% ethanol as solvent for reflux extraction twice, using 200 mL of extraction solvent each time, extracting for 1.5 hours, and combine the extracts. Use the above-mentioned high performance liquid chromatography method to determine the content of the obtained extracts, and the results are shown in Table 1 below.

[0064] Comparative Example 5: Extraction was carried out in the same manner as in Comparative Example 2, except that 1 / 2 weight of the extract was taken from the obtained extract and extracted twice with water-saturated n-butanol (200 ml x 2). After extraction, the content was determined using the above-mentioned HPLC method; the remaining 1 / 2 of the extract was directly determined using the above-mentioned HPLC method, and the results are shown in Table 1 below.

[0065] Comparative Example 6: Extraction was carried out in the same manner as in Comparative Example 3, except that 1 / 2 weight of the extract was taken from the obtained extract and extracted twice with water-saturated n-butanol (400 ml x 2). After extraction, the content was determined using the above-mentioned HPLC method; the content of the remaining 1 / 2 extract was directly determined using the above-mentioned HPLC method, and the results are shown in Table 1 below.

[0066] [Table 1] Results of content determination of Example 1 and Comparative Examples 1 to 6 using high performance liquid chromatography Standard peak area: 948.6

[0067] Referring to Table 1, by comparing Comparative Example 1 with Comparative Example 2, and by comparing Comparative Example 3 with Comparative Example 4, it can be seen that when ethanol aqueous solution is used as a solvent for extraction, the content of dioscin in the obtained Dioscorea sibiricum extract is significantly higher than the crude extract obtained by using water as the extraction solvent. By comparing Example 1 with Comparative Examples 1 to 6, it can be seen that the content of dioscin in the Dioscorea sibiricum extract can be significantly increased by extracting with ethanol reflux method and then extracting with water-saturated n-butanol. Since dioscin is the main component of the total saponins in Dioscorea sibiricum, the increase in the content of dioscin indicates the increase in the content of total saponins in the Dioscorea sibiricum extract.

[0068] In order to determine the operating conditions for ethanol reflux extraction of total saponins from Rhizoma Dioscoreae, the extraction rate and total saponins content were used as investigation criteria. Single-factor experiments and orthogonal experiments were used to determine the optimal ranges of ethanol concentration, extraction time, number of extractions and solid-liquid ratio. The accuracy and stability of the process and its parameters were verified through verification experiments.

[0069] Single factor experiment (1) Effect of ethanol concentration on the yield and content of total saponins in Rhizoma Dioscoreae Weigh 5 portions of Rhizoma Dioscoreae medicinal slices, 20 g each, crush, and extract with ethanol reflux once for 90 min each time. Add 200 mL of ethanol solutions with concentrations of 50%, 60%, 70%, 80%, and 90% respectively for extraction. After the extract is dried, add 60 mL of purified water to fully dissolve it, extract the supernatant with water-saturated n-butanol (30 mL × 2), combine the n-butanol layers, and evaporate to dryness. The content of total saponins in Rhizoma Dioscoreae was determined by the above-mentioned ultraviolet spectrophotometry, and the results are as follows: Figure 3 shown.

[0070] Depend on Figure 3 It can be seen that with the increase of ethanol concentration, the content of total saponins continues to increase. When the ethanol concentration is 60-70%, the total saponin content and the cream yield in the obtained extract are relatively high. In particular, when the ethanol concentration is 70%, the total saponin content and the cream yield reach the maximum value. When the ethanol concentration exceeds 70%, it may be because when the ethanol concentration is too high, the dissolution of water-soluble saponins decreases, while the dissolution of fat-soluble impurities increases, thereby reducing the total saponin extraction amount.

[0071] (2) Effect of extraction time on the yield and content of total saponins in Rhizoma Dioscoreae Weigh 5 portions of Rhizoma Dioscoreae medicinal slices, 20 g each, crush them, add 200 mL of 70% ethanol to each, extract once, and the extraction time is 0.5 h, 1 h, 1.5 h, 2 h, and 2.5 h respectively. After the extract is dried, add 60 mL of purified water to fully dissolve it, take the supernatant and extract it with water-saturated n-butanol (30 mL × 2), combine the n-butanol layers, and evaporate to dryness. The content of total saponins of Rhizoma Dioscoreae was determined by the above-mentioned ultraviolet spectrophotometry, and the results are as follows: Figure 4 shown.

[0072] Depend on Figure 4 It can be seen that when the extraction time of Rhizoma Dioscoreae is 1.5-2h, the total saponin content and the yield of the extract are higher. When the extraction time of Rhizoma Dioscoreae is less than 1.5h, due to the short extraction time, some components have not been extracted in time, which will cause incomplete extraction of total saponins, and then lead to low total saponin content. With the increase of extraction time, the total saponin content increases accordingly, and the yield of the extract and the total saponin content reach the maximum value at 2h.

[0073] (3) Effect of extraction times on the yield and content of total saponins in Rhizoma Dioscoreae Weigh 3 portions of Rhizoma Dioscoreae medicinal slices, 20 g each, crush them, add 200 mL of 70% ethanol to each portion, extract once, twice, and three times, 90 min each time. After the extract is dried, add 60 mL of purified water to fully dissolve, extract the supernatant with water-saturated n-butanol (30 mL × 2), combine the n-butanol layers, and evaporate to dryness. The content of total saponins in Rhizoma Dioscoreae was determined by the above-mentioned UV spectrophotometry method. The results are as follows: Figure 5 shown.

[0074] Depend on Figure 5 It can be seen that the content of total saponins in Rhizoma Dioscoreae increases with the increase of extraction times. Considering the economy, time consumption, paste yield and total saponin content, the extraction is 2 to 3 times, preferably 3 times.

[0075] (4) Effect of the ratio of solid to liquid on the yield and content of total saponins in Rhizoma Dioscoreae Weigh 5 portions of Radix Dioscoreae medicinal slices, 20 g each, crush, extract three times, 90 min each time, and select the solid-liquid ratio of 1:6, 1:8, 1:10, 1:12, and 1:14 to extract Radix Dioscoreae total saponins from the 5 portions of medicinal materials. After the extract is dried, add 60 mL of purified water to fully dissolve it, take the supernatant and extract it with water-saturated n-butanol (30 mL × 2), combine the n-butanol layers, and evaporate to dryness. The content of Radix Dioscoreae total saponins was determined according to the above-mentioned ultraviolet spectrophotometry method, and the results are as follows: Figure 6 shown.

[0076] Depend on Figure 6 It is known that with the increase of the solid-liquid ratio, the paste yield and content of total saponins of Rhizoma Dioscoreae increased. The reason is that the increase of the solid-liquid ratio will make the contact between 70% ethanol and the medicinal materials more complete. At 1:12, the total saponin content of Rhizoma Dioscoreae was the highest. When the solid-liquid ratio exceeded 1:12, more impurities were precipitated, which reduced the content of total saponins.

[0077] Orthogonal experiment Based on the single factor experiment, ethanol concentration (factor A), extraction time (factor B), extraction times (factor C), and solid-liquid ratio (factor D) were selected as influencing factors, and each factor was taken at 3 levels. 4)Orthogonal test, see Table 2.

[0078] [Table 2] Orthogonal factor table

[0079] The results of the orthogonal experiment are shown in Table 3, and the results of the variance analysis are shown in Table 4.

[0080] [Table 3] Orthogonal experiment results

[0081] [Table 4] ANOVA results

[0082] According to the intuitive analysis of the range R value in Table 3, the influence of each factor is B>D>C>A, and the optimal extraction condition is A2B1C3D2. The variance analysis results in Table 4 show that there is no significant difference between the factors. Therefore, the optimal conditions for ethanol reflux of total saponins of Rhizoma Dioscoreae determined in this experiment are 70% ethanol concentration, 3 extractions, 1.5h each time, and a solid-liquid ratio of 1:12.

[0083] Verification test In order to ensure the accuracy and reliability of the experiment, the experiment needs to be repeated three times.

[0084] Take 20g of the medicinal slices of Rhizoma Cynoglossi, crush them, and perform ethanol reflux extraction under the following conditions: the extraction solvent is 70% ethanol, the solid-liquid ratio is 1:12, and the extraction times are 3 times, each time for 1.5 hours. Set up three sets of parallel experiments. The experimental results are shown in Table 5.

[0085] [Table 5] Verification test results

[0086] The results in Table 5 show that the content of total saponins in the crude extract of Rhizoma Dioscoreae was 58.24% after three extractions with 70% ethanol concentration, each for 1.5 h, and a solid-liquid ratio of 1:12. The experiment proved that the total saponin content extracted by this method was higher than that of other extraction conditions, indicating that the results were stable and reproducible.

[0087] Example 2 and Comparative Example 7 Example 2: After extracting the medicinal material Rhizoma Dioscoreae according to the extraction method of Example 1, the obtained extract was dissolved with a small amount of purified water, and the supernatant was purified with D101 macroporous adsorption resin. The loading solution concentration was 0.1 g / mL, and eluted with 5 BV of purified water and 50% ethanol in succession. After the impurities were removed, eluted with 5 BV65% ethanol, and the eluate was collected. After the eluate was dried, the above-mentioned ultraviolet spectrophotometry was used for content determination, and the measured Rhizoma Dioscoreae total saponin content was 90.24%.

[0088] Comparative Example 7: After extracting the medicinal material Rhizoma Cynoglossi according to the extraction method of Example 1, the obtained extract was dissolved with a small amount of organic solvent, dripped into 200-300 mesh silica gel, and ground evenly in a mortar at 60°C. After the liquid was completely adsorbed, it was taken out and dried for standby use. When loading the column, the diameter-to-height ratio was 1:18, the eluent was ethyl acetate: ethanol: water = 6:1:2, and the elution rate was 0.6mL / min. The column liquid was collected, dried, and the content was determined by the above-mentioned ultraviolet spectrophotometry, and the total saponin content measured was 82.57%.

[0089] According to the comparison between Example 2 and Comparative Example 7, the purification efficiency of the extract of Rhizoma Dioscoreae by the macroporous adsorption resin is higher than that by the column chromatography silica gel method. Moreover, the purification by the macroporous adsorption resin is simple to operate.

[0090] In order to determine the operating conditions of macroporous adsorption resin purification, the effects of macroporous adsorption resin type, detection wavelength, sample loading amount, sample loading concentration, eluent concentration and eluent dosage on the purification effect were studied, and the optimal operating conditions were determined accordingly.

[0091] (1) Screening of macroporous adsorption resin According to the physicochemical properties of total saponins from Rhizoma Dioscoreae, several resins suitable for purifying total saponins from Rhizoma Dioscoreae were selected from numerous macroporous resins, and further screening was carried out after comprehensive consideration from the two perspectives of adsorption rate and desorption rate.

[0092] The specific parameters of the three macroporous adsorption resins of different polarities, AB-8, D101 and HPD100, used in the experiment are shown in Table 6.

[0093] [Table 6] Parameters of three macroporous adsorption resins

[0094] Soak the AB-8 macroporous resin in 95% ethanol for 24 hours to allow it to fully swell. First, wash it with 100% ethanol until the effluent is free of white turbidity when an appropriate amount of distilled water is added. Then wash it with distilled water until there is no alcohol smell and set aside.

[0095] Take 3 portions of the treated AB-8 macroporous resin, 20 g each, and place them in 250 mL stoppered conical bottles, add 100 mL of the sample solution 0.2 g / mL (including crude drug amount), shake for 30 seconds every 10 minutes, continue for 2 hours, and then stand for 24 hours to allow the resin to fully adsorb and filter. Collect the filtrate after being adsorbed by the resin, determine the ultraviolet absorbance value, and bring it into the standard curve to calculate the content of total saponins to examine the static adsorption effect of the resin. The results are shown in Table 7.

[0096] The resin after static adsorption was filtered out, and each filtered resin was placed in a 250 mL stoppered conical flask, and 150 mL of 70% ethanol was accurately added. It was shaken for 30 seconds every 10 minutes for the first 2 hours, and then left to stand for 24 hours. The desorption liquid after desorption was collected, and the ultraviolet absorbance value was measured, and the total saponin content was calculated by the standard curve to examine the static elution performance of the resin. The results are shown in Table 8.

[0097] [Table 7] Comparison of static adsorption effects of three macroporous resins

[0098] [Table 8] Comparison of static elution effects of three macroporous resins

[0099] By comparing the adsorption and desorption properties of different types of macroporous adsorption resins, it was found that the performance of AB-8 was better than the other two resins, so AB-8 resin was selected as the purification resin for total saponins of Rhizoma Dioscoreae, and this resin was used in subsequent experiments.

[0100] (2) Determination of detection wavelength Take 20g of the treated AB-8 resin, wet-load the column, load 200mL of the sample solution containing 0.2g / mL of crude drug, take the column liquid for spectral scanning at 200-800nm, the absorption curves of the standard and the test sample are basically the same, both have the maximum absorption peak at 300nm, the blank solution has no absorption at this wavelength, so 300nm is selected as the detection wavelength.

[0101] (3) Sample loading amount investigation Take 20g of resin to load the column, and load 200mL of sample solution containing 0.2g / mL crude drug. The first 30mL is collected into one tube for every 5mL, for a total of 6 tubes, 30-50mL is collected into one tube for every 10mL, for a total of 2 tubes, and 50-200mL is collected into one tube for every 25mL, for a total of 6 tubes. Accurately transfer an appropriate amount from each tube, measure the absorption value at 300nm and calculate the leakage rate, and draw a leakage curve (such as Figure 7 to determine the optimal loading volume.

[0102] Research shows that when the leakage rate reaches about 10%, it can be considered a leak point, see Table 9. Figure 7 It can be seen that the leakage rate of the fourth portion is about 10%, and the sample volume at this time is 20 mL. Therefore, the preferred ratio of resin: sample is about (4.6-5.4):1, and more preferably 5:1.

[0103] [Table 9] Sample loading inspection

[0104] (4) Sample concentration investigation Take 6 portions of resin column, each portion is 20g, accurately absorb 20 mL of the drug solution containing 0.2g / mL of crude drug, make solutions containing 0.05, 0.10, 0.15, 0.20, 0.25, 0.30g / mL of crude drug, collect the column liquid, and determine the content of total saponin. The content determination method adopts the above-mentioned ultraviolet spectrophotometry, and calculates the adsorption rate according to the following formula to examine the loading concentration.

[0105] M0--the content of total saponins before adsorption (mg); M1--the content of total saponins after adsorption (mg); M2--Content of total saponins after desorption (mg).

[0106] The adsorption rate of each loading concentration is shown in Table 10. The optimal loading concentration curve is drawn according to Table 10. Figure 8 .

[0107] [Table 10] Sample concentration investigation

[0108] From Table 10 and Figure 8It can be seen that when the loading concentration is 0.10-0.20g / mL, the adsorption rate is significantly higher. When the concentration is less than 0.2g / mL (including crude drug amount), the adsorption rate slowly increases with the increase of concentration. This may be because as the concentration increases, the specific gravity of total saponins increases, and the probability of adsorption increases. When the loading solution concentration is 0.2g / mL (including crude drug amount), the adsorption performance of the resin is close to saturation, the adsorption force of the resin on total saponins also reaches its peak, and the adsorption rate reaches the maximum value. When the concentration is greater than 0.2g / mL (including crude drug amount), the concentration is too high and has exceeded the maximum limit that the resin can adsorb. Correspondingly, the concentration is too high, and the content of impurities in the loading solution also increases. During the adsorption process, impurities and total saponins produce competitive adsorption, which affects the adsorption efficiency of total saponins and reduces the adsorption rate of total saponins. Therefore, the loading concentration can be selected as 0.10-0.20g / mL (including crude drug amount), and 0.2g / mL is more preferred.

[0109] (5) Effect of eluent concentration on desorption rate Take 20g of resin to load the column, take 20mL of 0.2g / mL (including crude drug amount) drug solution, and load it twice continuously. After loading, wash away impurities with 50mL of water, and elute with 50mL (5 times column volume) of 30%, 40%, 50%, 60%, 70%, 80%, and 95% ethanol in turn. Collect the eluate from each part and determine the content of total saponin therein. The above-mentioned ultraviolet spectrophotometry method is used for content determination, and the desorption rate is calculated. The calculation formula is shown in "(4) Sample concentration investigation". The results are shown in Table 11 and Fig. 9 shown.

[0110] [Table 11] Effect of eluent concentration on desorption rate

[0111] M0:2.2393 M1:0.4126 M0-M1=1.8267mg Depend on Fig. 9 It can be seen that with the increase of eluent concentration, the desorption rate of total saponins of Rhizoma Dioscoreae increases accordingly. When the eluent concentration is 70%, the desorption rate is the largest. At this time, the elution effect is the best, which is beneficial to increase the content of total saponins of Rhizoma Dioscoreae. Therefore, ethanol concentration of 60-70% can be selected as the eluent concentration, and ethanol concentration of 70% is more preferred.

[0112] (6) Effect of eluent dosage on resolution Take 20g of resin to load the column, take 20mL of 0.2g / mL (including crude drug amount), and load it twice continuously. After loading, elute with water and 50% ethanol to remove impurities, and then elute with 200mL of 70% ethanol. Each 4mL is 1 fraction, collect the eluate, and measure the absorbance at a wavelength of 300nm.

[0113] The UV absorbance values ​​of each eluent volume gradient are shown in Table 12. A curve is drawn with the sample number as the horizontal axis and the measured absorbance value as the vertical axis. The curve is shown in Fig.10 shown.

[0114] [Table 12] Effect of eluent dosage on resolution

[0115] From Table 12 and Fig.10 It can be seen that as the volume of the eluent increases, the absorbance value becomes smaller and smaller, and the content of the total saponins of Rhizoma Dioscoreae in the eluent becomes less and less. When the number is 30 (the amount of eluent is 12 BV), the absorbance value of the sample is infinitely close to zero, indicating that the adsorbed total saponins are basically eluted completely at this time. Therefore, the amount of eluent can be selected as 11-14BV, preferably 12BV. Among them, BV (bed volume) refers to the volume of the resin loaded in the resin column. For example, 12 BV means that the ratio of the volume of the eluent to the bed volume of the resin column is 12.

[0116] (7) Verification test Take 20g of resin to load the column, take 20mL of 0.2g / mL (including crude drug amount), and load the sample twice continuously. After the loading is completed, remove impurities with water and 50% ethanol respectively, and then elute with 70% ethanol 12BV. Collect the desorbed liquid, dry it, and determine the content of total saponins in it. The content determination uses the above-mentioned ultraviolet spectrophotometry method, and the experiment is performed in three groups in parallel.

[0117] Purification was performed according to the optimal purification process, the desorption liquid was collected, concentrated to an extract state by rotary evaporation, and then freeze-dried into a powder by vacuum freeze-drying. The content was determined by ultraviolet spectrophotometry, and three parallel determinations were performed. The results are shown in Table 13.

[0118] [Table 13] Verification test results

[0119] After ethanol reflux extraction and water-saturated n-butanol extraction, the total saponin yield of Rhizoma Dioscoreae was 15.15% and the total saponin content was 58.24%. After purification by macroporous adsorption resin method, the total saponin yield was 5.2%. Although the yield was reduced, the total saponin content increased from the original 58.24% to 94.47%.

Claims

1. A method for extracting total saponins from Rhizoma Dioscoreae, characterized in that: The method comprises: The ethanol reflux method was used to Dioscorea nipponica Makino ) to obtain a crude extract of total saponins from Rhizoma Dioscoreae, and extracting the crude extract using water-saturated n-butanol to obtain an extract, Among them, in the ethanol reflux method, an ethanol aqueous solution with a volume concentration of 60-70% is used for extraction, each extraction time is 1.5-2h, the solid-liquid ratio is 1:10 to 1:14, and a total of 2-3 extractions are performed.

2. The method for extracting total saponins of Rhizoma Dioscoreae according to claim 1, characterized in that: In the ethanol reflux method, an ethanol aqueous solution with a volume concentration of 70% is used for extraction.

3. The method for extracting total saponins of Rhizoma Dioscoreae according to claim 1, characterized in that: In the ethanol reflux method, each extraction time is 1.5 h.

4. The method for extracting total saponins of Rhizoma Dioscoreae according to claim 1, characterized in that: In the ethanol reflux method, the solid-liquid ratio is 1:

12.

5. The method for extracting total saponins of Rhizoma Dioscoreae according to claim 1, characterized in that: In the ethanol reflux method, extraction was performed three times in total.

6. The method for extracting total saponins of Rhizoma Dioscoreae according to claim 1 or 5, characterized in that: The method further comprises purifying the extract by using a macroporous adsorption resin method.

7. The method for extracting total saponins of Rhizoma Dioscoreae according to claim 1, characterized in that: In the macroporous adsorption resin method, AB-8 type resin is used as the purification resin, the sample loading amount is resin: sample = (4.6-5.4): 1, the loading concentration is 0.10-0.20 g / mL of crude drug, the eluent is an ethanol aqueous solution with a volume concentration of 60-70%, and the eluent dosage is 11-14 BV, preferably 12 BV.

8. The method for extracting total saponins of Rhizoma Dioscoreae according to claim 1, characterized in that: In the macroporous adsorption resin method, the loading amount is resin: sample = 5:

1.

9. The method for extracting total saponins of Rhizoma Dioscoreae according to claim 1, characterized in that: In the macroporous adsorption resin method, the loading concentration is 0.2 g / mL of crude drug.

10. The method for extracting total saponins of Rhizoma Dioscoreae according to claim 1, characterized in that: The eluent used was an ethanol aqueous solution with a volume concentration of 70%.

Images