CN1151164C - Prepn of epimedium general flavone with osteoporosis resisting function - Google Patents

Abstract

The present invention relates to a method for preparing epimedium total flavonoid with an osteoporosis resisting function, which is characterized in that the method comprises the following steps: extracting with water, concentrating the water extracting solution until a state that the density is from 1.05 to 1.11, and then extracting with normal butyl alcohol. The method has the advantages of no application of macroporous resin, simplified operation steps and simplified operation in the steps. The content of total flavonoid is measued by icariin in the range of 55 to 65%, and the metastasis rate of the total flavonoid can reach 75 to 80%. The osteoporsis resisting function of the epimedium total flavonoid is relevant to the content of the total flavonoid, and high-content total flavonoid preparations have good curative effects.

Description

Preparation method of epimedium total flavonoids with anti-osteoporosis effect

technical field

The invention belongs to a preparation method of effective parts of traditional Chinese medicines, in particular to a preparation method of epimedium total flavonoids with anti-osteoporosis effect.

Background technique

Epimedium is a traditional Chinese medicine for nourishing the kidney and strengthening the bones. "Compendium of Materia Medica" records that it has the functions of "benefiting essence, strengthening bones, strengthening waist and knees, and strengthening heart". Modern pharmacological studies have proved that Epimedium can enhance the functions of the hypothalamus-pituitary-gonad axis, adrenal cortex axis, thymus axis and other endocrine systems. The growth of bone cells, and has a direct inhibitory effect on osteoclasts, so it can resist osteoporosis caused by ovariectomized, testicular animals and long-term high-dose administration of adrenal cortex hormones. Epimedium is used as the main drug in many compound preparations for the treatment of osteoporosis in clinical use, that is, they are all Chinese medicine compound preparations.

There have been many reports in the literature about the preparation process of the total flavonoids of Epimedium, but most of them are extracted with ethanol. Such as: Ma Huiping et al. in the "Extraction and Separation Technology Research of Epimedium Total Flavonoids" West China Pharmaceutical Journal 2002, 17 (1): 1-3, extracting 3 times with 10 times the amount of 70% ethanol with crude powder of the material, each time 1.5 h; The best separation process is: apply acid-base precipitation method, and the extract is purified by methanol. Shen Qingliang etc. in "Orthogonal Design Method Study on Epimedium Extraction Process Conditions", Chinese Journal of Experimental Formulas, 2001, 7 (5): 5-6, the coarse powder of the medicinal materials was added with 10 times the amount of 70% ethanol, and the reflux extraction was performed twice. , 40 minutes each time. Wang Changli et al. in "Research on the Extraction and Separation Technology of Epimedium Total Flavonoids", Chinese patent medicine, 1996, 18 (5): 1, reported that the coarse powder of the medicinal material was soaked in 20 times the amount of water, soaked for 1.5 hours, extracted 2 times, each time 1 Hour.

The method disclosed in the Chinese patent application CN1294132A "Preparation process of total flavonoid glycosides of Epimedium" is as follows: the coarse powder of Epimedium is boiled and extracted with an aqueous solution containing antioxidants, and the extract is absorbed by a macroporous resin column. The method disclosed in another Chinese patent application CN1283627A "A Preparation Method for Extracting Icariin from Epimedium" is: soak the aerial part of Epimedium in water, decoct, concentrate the water, and pass through the large pores Resin adsorption, ethanol elution, and the eluent is heated to reflux with activated carbon to decolorize. Although the methods of the above two patent applications are extracted with water, there are many impurities in the water extraction, so a macroporous resin column is used for separation. In the patent application CN1294132A, antioxidants are added during hydrothermal extraction because the total flavonoids of Epimedium are easily oxidized during high-temperature extraction and processing, see page 2 of the patent application publication for this invention. The oxidation of total flavonoids in Epimedium will inevitably lead to darker color, increased impurities, and reduced yield, which makes the separation of total flavonoids in the effective part more difficult, and the total flavonoids content in the final product is low, thereby affecting the therapeutic effect. The anti-osteoporotic effect of flavonoids is dose-related. At the same time, the reduction in the yield of total flavonoids also affects the cost and price of medicines, especially when patients need to be treated for a longer period of time and with higher doses, this effect is more obvious.

Contents of the invention

Aiming at the deficiencies in the extraction and separation process of total flavonoids of Epimedium, the present invention will solve the following technical problems: research and development of a method for the industrial production of total flavonoids of Epimedium, which can use water as the extraction solvent and does not use macroporous resins, avoiding the The adsorption of the resin and its possible harmful residues affect the further promotion and application in medicine; at the same time, in order to ensure the therapeutic effect, the total flavonoid content of the product should be high, because the anti-osteoporosis effect of the total flavonoids of Epimedium is the same as that of the total In addition, the transfer rate of total flavonoids should be high in order to reduce production costs. Only with low production costs can the price of drugs be low, because anti-osteoporosis drugs often need to be taken for a long time, and the patient's economic affordability is a social benefit. One aspect of comprehensive consideration.

In order to solve the above technical problems, the present invention develops the following technical solutions.

A method for extracting and separating total flavonoids of Epimedium: take Epimedium medicinal material, extract with water, concentrate the water extract to a density of 1.05-1.11, and then extract with n-butanol.

Epimedium can be crushed in advance, passed through a 20-mesh sieve, and soaked in water for 1-3 hours or 1.5 hours.

Water for soaking can be tap water, deionized water, or other clean water suitable for extracting medicinal materials; 23-26 times as well.

After the Epimedium medicinal material is soaked, it is extracted by heating. It can be heated to boiling, and then kept for 0.3-2 hours, preferably 0.5-1 hour. The medicinal material can be repeatedly extracted with water for 2-4 times, preferably 3 times.

The combined water extracts are concentrated in vacuo at 65-55°C, preferably at 60°C. After the water extracts are concentrated to a density of 1.05-1.11, they are extracted with n-butanol. The density of the concentrated water extract has a great influence on the efficiency of n-butanol extraction, and its density is preferably 1.06-1.10, and the density of 1.08-1.09 is more ideal.

During n-butanol extraction, the consumption of n-butanol is 1-4 times (volume/volume) of concentrated solution volume, is better with 2-3 times of amount, 2.5-2.8 times is also good. The n-butanol extraction is more complete for multiple extractions, generally 3-8 times, 4-7 times are also acceptable, and 5-6 times are better.

The n-butanol extract was recovered in vacuum to obtain the total flavonoids of Epimedium. The extract has obvious anti-osteoporosis effect, so the total flavonoids of Epimedium can be mixed with pharmaceutically applicable carriers to prepare various anti-osteoporosis preparations.

The invention provides a method for the industrial preparation of total flavonoids of Epimedium, which not only uses water as the extraction solvent, but also avoids the use of macroporous resins, and will not affect the further development of medicine due to the adsorption of the resins and its possible harmful residues. promotional application.

The preparation method provided by the invention not only simplifies the operation steps, but also simplifies the operations in the steps, and does not need macroporous resin column treatment and activated carbon decolorization.

The above preparation method of the present invention improves the transfer rate of total flavones and the content of total flavones in the product. The content of total flavonoids is determined by the method of "Pharmacopoeia of the People's Republic of China" (2000 edition), and the calculation of icariin is 55-65%; the transfer rate of total flavonoids reaches 75%-80%.

A high transfer rate of total flavonoids can reduce production costs, and low production costs can lead to low drug prices, because anti-osteoporosis drugs often need to be taken for a long time, and the patient's economic affordability is an aspect of comprehensive consideration of social benefits. The high content of total flavonoids in the product provides a guarantee for achieving a good therapeutic effect, because the anti-osteoporosis effect of total flavonoids of Epimedium is related to the content of total flavonoids. Pharmacodynamic tests showed that there were significant differences in curative effect between low doses of total flavonoids of Epimedium and medium and high doses; moreover, total flavonoids of Epimedium had no estrogen-like effect. Pharmacodynamic tests have proved that in order to achieve a good anti-osteoporosis effect, medium and high doses of total flavonoids of Epimedium should be used for treatment, and the preparation method of total flavonoids of Epimedium disclosed in the present invention is to prepare Epimedium with higher flavonoid content. The total flavonoids preparation of sheep gourd provides a method for industrial production.

1. Effect of total flavonoids of Epimedium on osteoblasts cultured in vitro

Animals: newborn 1-3 day old SD rats, provided by the Experimental Animal Center of our school.

Medicine: total flavonoids of Epimedium (content: 62.5%, batch number: G011003) prepared according to the process. Prepare a solution with a concentration of 1 mg/ml with DMEM culture solution, adjust the pH to 7.0, and then filter to sterilize.

Main reagents: DMEM and type II collagenase are products of Gibco; trypsin is products of Merck; newborn bovine serum is products of Shanghai Yangsheng Biological Products Co., Ltd.; vitamin C and β-sodium glycerophosphate are products of Sigma; sodium fluoride (NaF, Analytical pure) is the product of Shanghai Reagent No. 3 Factory affiliated to Shanghai Chemical Reagent General Factory; Alizarin Red (ARS) is the product of Linhai Huifeng Chemical Factory. The rest of the reagents were commercially available analytically pure.

Methods: Newborn 1-3d SD rats were cultured with osteoblasts, and the calvaria was aseptically removed, and the adhered periosteum, blood vessels and connective tissues were carefully removed, cut into pieces, digested with 0.25% trypsin at 37°C for 15 minutes, and shaken every 3 minutes Once, discard the digestion solution, then digest with 0.1% type II collagenase at 37°C and magnetic stirring (50 rpm) for 60 min, centrifuge the digestion solution at 1000 rpm for 5 min, collect the cells, and inoculate them in a medium containing 10% newborn bovine serum Place in a culture bottle of DMEM culture medium, culture in an incubator at 37°C and 5% CO ₂ , change the medium every 24 hours, and then every 3 days. When the cells cover the culture bottle, digest with 0.25% trypsin and subculture , the experimental cells used are the 3rd passage cells. Osteoblasts were triangular, polygonal, and irregular in shape, with larger bodies, larger nuclei, and darker staining.

Cell proliferation detection Cells were inoculated in 96-well plates at a density of 2.5×10 ⁴ cells/ml, and 24 hours later, total epimedium flavonoids were added at concentrations of 1, 10, and 100 μg/ml, and a positive control group was set with NaF 1×10 ^-7 , 1×10 ^-5 M and the blank control group, each concentration was repeated for 6 wells. 68 hours after adding the test drug, add 20 μl MTT to each well, continue to cultivate for 4 hours, then discard the culture medium, then add 150 μl DMSO (analytical pure) to each well, measure the absorbance value of each well at a wavelength of 490 nm on a microplate reader, and use OD ₄₉₀ indicated.

Determination of mineralized nodule formation Cells were inoculated on a 24-well plate at a density of 2.0×10 ⁴ cells/cm ² . After 24 hours, the DMEM medium containing 10% newborn bovine serum was changed, and at the same time, kinesin at a concentration of 1, 10, and 100 μg/ml was added. Total flavonoids of goatskin, positive control group NaFl×10 ^-7 , 1×10 ^-5 M and blank control group, each concentration repeated 3 wells. Change the medium every 3 days, fix in situ with 95% ethanol on the 15th day, stain with 0.1% alizarin red for 30 minutes, take orange-red nodules, clear boundaries, and >200 μm as the standard, and use low-power light microscope as mineralized nodules count.

Effects on cell proliferation: Epimedium total flavonoids significantly promoted osteoblast proliferation at a concentration of 1-10 μg/ml, compared with the control group (P<0.001, P<0.001), see Table 1.

Effects on mineralized nodules: Osteoblasts were cultured for 15 days and mineralized nodules could be seen. Alizarin red staining showed orange-red nodules of different sizes and shapes. The number of mineralized nodules in the Epimedium total flavonoids 10 μg/ml group was significantly higher than that in the control group, P<0.05 compared with the control group, see Table 1.

Table 1. The effect of total flavonoids of Epimedium on promoting osteoblast proliferation and mineralization nodules ( x±SD)

Group Concentration OD490 Number of Mineralized Nodules

(n=6) (n=3)

Control group ---- 0.203±0.012 85.8±7.9

010409 1μg/ml 0.252±0.015*** 88.0±18.0

10 0.260±0.025*** 113.0±16.1*

100 0.212±0.003 77.3±6.7

NaF 1×10 ^-7 M 0.227±0.015* 100.3±4.2*

1×10 ^-5 0.221±0.008** 114.0±8.2**

*P<0.05, **P<0.01, ***P<0.001vs control group

2. Effect of total flavonoids of Epimedium on osteoporosis model of ovariectomized rats

Animals: Sprague-Dawley clean grade rats, female, 6 months old, purchased from Shanghai Xipuer-Bikay Experimental Animal Co., Ltd., animal quality certificate number: Yidongzi No. 02-49-3 (Shanghai Medical Experimental Animals Co., Ltd. Issued by the Management Committee).

Medicines and reagents: Epimedium total flavonoids prepared by the method of the present invention (content is 62.5%, batch number: G011003); Nylestriol (Nylestriol, batch number: 010801, Shanghai Hualian Pharmaceutical Co., Ltd.); Gushukang (batch number : 000402, Donggang Kangchen Pharmaceutical Co., Ltd.); osteocalcin radioimmunoassay kit (BGP, batch number: March 2002, China Institute of Atomic Energy); serum alkaline phosphatase, serum anti-tartrate acid phosphatase, serum calcium , Phosphorus kits are all products of Nanjing Jiancheng Biotechnology Co., Ltd.

Methods: Grouping and drug administration Rats were anesthetized with 20% urethane 1g/kg intraperitoneally, fixed in the prone position, incisions were made on both sides of the back spine, and bilateral ovaries were removed under aseptic conditions as osteoporosis model rats. In the operation group, a small piece of fat was removed bilaterally as a control. After 5 days, 56 surviving healthy rats were randomly divided into 7 groups, 8 rats in each group, and given the following drugs orally for 3 consecutive months.

Sham operation group (Sham) 0.5% CMC-Na solution 10ml/kg/d

Pathological model group (model group) 0.5% CMC-Na solution 10ml/kg/d

Positive control group Nylestriol: 1 mg/kg (herein referred to as E ₃ , administered twice a week)

 Gushukang: 4g/kg/d (herein referred to as GSK)

The test drug group Epimedium total flavonoids low dose: 75mg/kg/d (hereinafter referred to as L-EPF)

The dose of Epimedium total flavonoids: 150mg/kg/d (hereinafter referred to as M-EPF)

High dose of total flavonoids of Epimedium: 300mg/kg/d (herein referred to as H-EPF)

Bone Densitometry Two days before the end of the experiment, dual-energy X-ray (DEXA) bone density was detected on the lumbar spine and femur under ether anesthesia.

Determination of serum biochemical indicators One day before the end of the experiment, blood was collected from the orbital venous plexus to measure serum osteocalcin (BGP), alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), calcium (Ca), phosphorus (P) content.

Determination of Bone Biomechanical Indexes The next day after the last administration, the rats were killed by dislocation of the cervical spine, the right femur was quickly peeled off, and its length, long diameter and short diameter were measured with a vernier caliper, and then placed on a 2000N spring pressure testing machine (span: 20mm) Perform a three-point bending test, and then measure the wall thickness of the broken bone. The force (load) and deflection (the maximum distance that the midline of the sample deviates from the original position when the sample is bent) is picked up by the microcomputer-sensor system. Using Grapher software, the deflection is taken as the abscissa and the load is taken as the ordinate to draw a graph. From the graph, the maximum load (the maximum force the femur can bear) and the maximum deflection (the displacement that occurs when the femur bears the maximum force) can be obtained.

Bone Index Measurement The left femur was stripped and dried in an oven at 110°C for 1 hour, and the bone length (L), short diameter of bone (s-D), long diameter of bone (l-D) and bone weight (W) were measured. The dried femur was charred and placed in a muffle furnace for 8 hours at 600°C for ashing. After taking it out, the ashes were dissolved in 6N HCl solution, and the bone calcium and phosphorus contents were determined.

Bone Morphometric Measurement The broken head was fixed in 2% glutaraldehyde, and the femoral head was sawed sagittal with a dental diamond saw (grinding wheel). One piece was taken, cleaned, soaked in 10% sodium hypochlorite solution for 6 hours, and ultrasonically cleaned for 15 hours. Minutes, ethanol gradient dehydration, ether immersion, and then air-dried, ion sputtering coating. Observed under a scanning electron microscope, the accelerating voltage was 20 kV. The photos taken by SEM were combined with the CMIAS-98A image analyzer made by Beihang University to measure the surface percentage of trabecular bone.

Statistical analysis: t test was used to compare the means between groups.

Effects on rat body weight and uterine coefficient: After ovariectomized rats, the body weight increased significantly, and the uterine coefficient decreased significantly, indicating that the modeling was successful. After taking the total flavonoids of Epimedium, the weight gain tended to slow down, but it was not significant, and the uterine coefficient was not different from that of the model group, indicating that the total flavonoids of Epimedium had no estrogen-like effect, see Table 2.

Table 2. Effects of Epimedium total flavonoids on body weight and uterine coefficient of ovariectomized rats (n=8, x±SD)

Group Body Weight(g) Uterus Index(g/kg)

Sham 296±34** 1.486±0.552***

Model group 356±24 0.220±0.067

E ₃ 281±19*** 0.925±0.207***

GSK 329±27 0.271±0.106

L-EPF 336±31 0.256±0.073

M-EPF 341±34 0.206±0.060

H-EPF 345±17 0.223±0.034

**P<0.01, ***P<0.001 vs model group

Effects on serological indicators: Compared with the Sham group, the s-BGP, s-ALP, and s-TRAP in the model group were significantly increased, while s-Ca and sP had no significant changes. After 3 months of administration, compared with the model group, the s-BGP and s-TRAP in the E ₃ group were significantly reduced, and the s-ALP also had a tendency to decrease. However, s-BGP and s-ALP in each EPF group remained at relatively high levels, but s-TRAP showed a tendency to decrease, especially in L-EPF and M-EPF groups (P<0.01, P<0.05), see Table 3 .

Table 3. Effects of Epimedium total flavonoids on serological indicators in ovariectomized rats (n=8, x±SD)

s-BGP s-ALP s-TRAP s-Ca s-P

Group

(ng/ml) (u) (U/L) (mmol/L) (mmol/L)

Sham 1.71±0.38** 15.00±3.18*** 15.39±5.80* 1.70±0.16 1.91±0.54

Model group 3.29±1.23 28.65±8.06 21.54±3.74 1.77±0.26 1.87±0.19

E ₃ 1.37±0.33** 21.27±6.28 13.72±6.56* 1.59±0.18 1.70±0.14

GSK 3.29±0.99 27.37±5.23 15.91±7.45 1.37±0.11** 1.75±0.32

L-EPF 3.76±0.87 33.09±5.50 16.09±2.55** 1.64±0.09 1.75±0.25

M-EPF 3.60±0.86 35.73±10.41 16.24±4.44* 1.85±0.15 2.35±0.32**

H-EPF 4.64±1.54 32.94±9.05 18.01±2.65 1.39±0.27* 2.12±0.69

*P＜0.05, **P＜0.01, ***P＜0.001 vs model group

Effects on bone index and bone mineral content: Continuous administration of EPF for 3 months has no significant effect on femoral length and diameter; it has no effect on femoral dry weight, but low, medium and high dose groups all have the effect of increasing femoral coefficient . Compared with Sham, the model group W _ash /W _femur decreased significantly. Compared with the model group, the _Wash /W _femur in the EPF medium and high dose groups increased significantly (P<0.01, P<0.01), and the _Wash /L also tended to increase. All EPF groups could increase the femoral calcium content and significantly increase the bone phosphorus content (P<0.01, P<0.01, P<0.05), see Table 4.

Table 4, Epimedium total flavonoids on the impact of ovariectomized rats on bone index and bone mineral content (n=8, x±SD)

Group W _ash (g) W _ash /W _femur (g/g) W _ash /L(g/cm) Ca(g/g) P(g/g)

Sham 0.392±0.019 0.621±0.014*** 0.105±0.004 0.222±0.016* 0.150±0.008*

Model group 0.394±0.038 0.586±0.009 0.104±0.008 0.199±0.017 0.141±0.007

E ₃ 0.391±0.026 0.625±0.013*** 0.107±0.006 0.208±0.013 0.149±0.012

GSK 0.401±0.025 0.612±0.071 0.107±0.008 0.216±0.009* 0.156±0.011**

L-EPF 0.389±0.026 0.593±0.007 0.103±0.006 0.207±0.012 0.153±0.009**

M-EPF 0.403±0.030 0.606±0.014** 0.105±0.007 0.206±0.010 0.157±0.009**

H-EPF 0.401±0.027 0.609±0.014** 0.106±0.005 0.206±0.020 0.155±0.012*

**P<0.01, ***P<0.001 vs model group

Effects on bone density: DEXA test analysis results showed that the femur bone density of rats in the model group decreased significantly, and the lumbar bone density also showed a downward trend. The bone mineral density of femur and lumbar spine in EPF medium and high dose groups both increased.

Effects on femoral bone biomechanics: the maximum load and maximum bending stress of the model group were lower than those of the sham operation group, indicating that the bending resistance and tensile capacity of the rat femur in the model group were lower than those of the sham operation group, and the drug was administered continuously for three months Afterwards, the maximum load, flexural elastic modulus and flexural rigidity of the low, medium and high dose groups of total flavonoids of Epimedium were all higher than those of the model group, indicating that the flexural capacity, Impact resistance and fatigue resistance have been improved.

Effects on bone morphology: The trabecular bone of rats in the ovariectomized model group had many cavities and large areas; the arch bridge structure was obviously thinner, with defects or even fractures; the bone surface was rough and not smooth. In the middle and high dose groups of total flavonoids of Epimedium, the cavity of bone trabecula was reduced and the area was also reduced; the structure of the arch bridge was complete and the gap was thicker; the surface of the bone became smooth and even.

As can be seen from Table 5, the average width of the bone trabeculae in the model group rats was significantly reduced (P<0.001), and the MTT was significantly increased after giving Epimedium total flavonoids compared with the model group, and the middle and high dose groups were extremely significant (P<0.001, P<0.001, P <0.001). The area percentage of trabecular bone TS% decreased significantly (P<0.001), and the middle and high dose groups of total flavonoids of Epimedium can significantly increase the reduced TS% (P<0.001, P<0.001).

Table 5. Effects of Epimedium total flavonoids on bone mineral density and bone morphology in ovariectomized rats (n=6, x±SD)

Group MTT(μm) TS%

Sham 105.00±13.78*** 86.178±0.029***

Model group 35.00±6.78 63.664±0.032

E ₃ 100.33±5.13*** 84.701±0.044***

GSK 108.33±8.43*** 85.679±0.022***

L-EPF 53.67±17.36* 65.464±0.061

M-EPF 96.33±6.25*** 82.264±0.043***

H-EPF 104.33±8.98*** 83.336±0.056***

*P＜0.05, ***P＜0.001 vs model group

Detailed ways

The medicinal material is selected from one of the following 5 Epimedium medicinal materials recorded in "Pharmacopoeia of the People's Republic of China" (2000 edition), namely: Epimedium brevicornum Maxim., Epimedium brevicornum Maxim., E. sagittatum (Sieb.et Zucc.) Maxim., Epimedium vellus E.pubescens Maxim., Epimedium Wushanense E.wushanense T.S.Ying, E.koreanum Nakai, the medicinal part is the dry aerial part. The properties, physical and chemical identification characteristics and content determination of the medicinal material conform to the content under the item "Epimedium" in "Pharmacopoeia of the People's Republic of China" (2000 Edition Part One).

Example 1

Weigh 20 kg of Epimedium medicinal material, pulverize, pass through a 20-mesh sieve, add 15 times the amount of ion-exchanged water, soak for 1 hour, heat to boiling, keep for 0.5 hour, and turn off the heat source. The extract was filtered while hot. Extracted 3 times, combined the 3 extracts, concentrated in vacuo at 65°C to a density of 1.064. The content of total flavonoids is determined by the method of "Pharmacopoeia of the People's Republic of China" (2000 edition), and the transfer rate is greater than 70%.

Extract 6 times with n-butanol, the dosage of n-butanol is 2.5 times of the extract concentrate, combine the n-butanol extracts, recover n-butanol under reduced pressure, until there is no n-butanol smell, vacuum dry to obtain the total flavonoid extract 1536g. The total flavonoid content of the extract is determined according to the method of "Pharmacopoeia of the People's Republic of China" (2000 edition), and is 62.5% based on icariin. The transfer rate of total flavonoids reaches 80%.

Example 2

Weigh 50 kg of Epimedium medicinal material, pulverize, pass through a 20-mesh sieve, add 20 times the amount of ion-exchanged water, soak for 1.5 hours, heat to boiling, keep for 1 hour, and turn off the heat source. The extract was filtered while hot. Extract twice, combine the two extracts, and concentrate in vacuo at 60°C to a density of 1.108.

Extract 6 times with n-butanol, each dosage of n-butanol is 1.5 times of the extract concentrate, combine the n-butanol extracts, recover n-butanol under reduced pressure until there is no n-butanol smell (if necessary, add 95% 7-alcohol Drive away n-butanol), and vacuum drying to obtain 3718g of total flavonoid extract. The total flavonoid content of the extract was determined to be 61.5% based on icariin. The transfer rate of total flavonoids reaches 80%.

Example 3

Weigh 50 kg of Epimedium medicinal material, pulverize, pass through a 20-mesh sieve, add 25 times the amount of ion-exchanged water, soak for 2 hours, heat to boiling, keep for 1.5 hours, and turn off the heat source. The extract was filtered while hot. Extracted 3 times, combined the 3 extracts, concentrated in vacuo at 55°C to a density of 1.08. The content of total flavonoids is measured, and the transfer rate is greater than 70%.

Extract 6 times with n-butanol, each dosage of n-butanol is 2 times of the extract concentrate, combine the n-butanol extracts, recover n-butanol under reduced pressure until there is no n-butanol smell (if necessary, add 95% ethanol to drive n-butanol), vacuum-dried to obtain 3746g of total flavonoid extract. The total flavonoid content of the extract is 60.4% calculated as icariin. The transfer rate of total flavonoids reaches 80%.

Claims (6)

1. The method for extracting and separating total flavonoids of Epimedium, which is characterized in that: take Epimedium medicinal material, extract with water, concentrate the water extract to a density of 1.05-1.11, and then extract with n-butanol. 2. The extraction and separation method according to claim 1, characterized in that: heating and extracting with water, concentrating the water extract to a density of 1.06-1.10, and then extracting with n-butanol. 3. The extraction and separation method according to claim 2, characterized in that: extract by heating with water, concentrate the water extract to a density of 1.08-1.09, extract with n-butanol, recover n-butanol, and obtain total flavonoids of Epimedium. 4. The extraction and separation method according to claim 1, 2 or 3, characterized in that: heat to boiling with water for extraction, then keep for 0.3-2 hours, extract the medicinal materials with water for 2-4 times, combine the water extracts, and extract at 65-55 ℃ for vacuum concentration, and extract 3-8 times with n-butanol which is 1-4 times the volume of the concentrated solution. 5. The extraction and separation method according to claim 4, characterized in that: heat to boiling with water for extraction, then keep for 0.5-1 hour, extract the medicinal materials with water for 3 times, combine the water extracts, and conduct vacuum concentration at 60°C for concentration Extract 4-7 times with 2-3 times the amount of n-butanol. 6. The extraction and separation method according to claim 5, characterized in that: extract with n-butanol 2.5-2.8 times the volume of the concentrated solution for 5-6 times.