CN111187309A - Preparation process and application of four components in cistanche - Google Patents
Preparation process and application of four components in cistanche Download PDFInfo
- Publication number
- CN111187309A CN111187309A CN202010111714.5A CN202010111714A CN111187309A CN 111187309 A CN111187309 A CN 111187309A CN 202010111714 A CN202010111714 A CN 202010111714A CN 111187309 A CN111187309 A CN 111187309A
- Authority
- CN
- China
- Prior art keywords
- cistanche
- water
- echinacoside
- ethanol
- elution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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- 238000002360 preparation method Methods 0.000 title claims abstract description 33
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- NJYVDFDTLLZVMG-UHFFFAOYSA-N echinacoside Natural products CC1OC(OC2C(O)C(OCCc3ccc(O)c(O)c3)OC(COC4OC(CO)C(O)C(O)C4O)C2OC(=O)C=Cc5cc(O)cc(O)c5)C(O)C(O)C1O NJYVDFDTLLZVMG-UHFFFAOYSA-N 0.000 claims abstract description 46
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
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- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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- A—HUMAN NECESSITIES
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Abstract
The invention particularly discloses a method for extracting, purifying and simultaneously preparing four high-purity components including echinacoside, verbascoside, isoverbascoside and tubular flower glycoside A from cistanche, and application of four single components and compositions with different proportions thereof in nerve protection and bone protection. The method comprises the following steps: refining by combining the steps of extracting medicinal materials, precipitating an extracting solution with ethanol, purifying macroporous resin and an HPLC-DAD/MS component automatic collection system, namely extracting the cistanche medicinal materials by adopting a hot reflux method; then carrying out primary purification by adopting an alcohol precipitation mode; then adopting a macroporous resin adsorption method to further purify the four components to obtain needed phenylethanoid glycoside parts (the content is more than or equal to 50 percent and meets the requirement of the traditional Chinese medicine parts of the new medicine 5) of the four components; finally, the HPLC-DAD/MS component automatic collection system is utilized to realize the high-efficiency and high-purity preparation of the four-component monomers, and the purity is higher than 98%; simultaneously, the four single components and the compositions with different proportions thereof are researched for the neuroprotective effect and the bone protective effect.
Description
Technical Field
The invention belongs to the field of natural products, relates to medical application of natural products, and particularly relates to application of four component monomers in cistanche and compositions with different proportions thereof in nerve protection and bone protection.
Background
Cistanche deserticola (Cistanche Deserticola Ma) is a plant of the genus Cistanche of the family Orobanchaceae, parasitic on the roots of plants of the genus Salix, such as Haloxylon tatum, Salix purpurea, Nitri leucotrichum, Calligonum mongolicum and Artemisia annua, and is mainly produced in Takrama desert area of Xinjiang and in the county near the area. The part of cistanche deserticola used as medicine is dry fleshy stem with scaly leaves. The cistanche deserticola is recorded to be divided into four varieties, namely cistanche deserticola, cistanche tubulosa, cistanche deserticola, cistanche salsa and cistanche baihua saline, and the varieties contained in the pharmacopoeia are cistanche and cistanche tubulosa. Since ancient books of physicians, Shen nong Ben Cao Jing, records cistanche, which has the effects of tonifying kidney yang, benefiting essence and blood, loosening bowel to relieve constipation, etc., modern medical research shows that cistanche has the functions of resisting aging, resisting oxidation, protecting nerves and regulating immunity.
The phenylethanoid glycosides are the main effective components of cistanche, and are also relatively widely researched components of cistanche, the mother nucleus of which is usually β -glucose, wherein phenethyl and hydroxyl are substituted by hydroxyl or methoxy, cinnamoyl is substituted by methoxy, and natural glycoside compounds are formed through ester bonds or glycosidic bonds.
In view of the pharmacological action of phenylethanoid glycosides, the methods for extracting and purifying phenylethanoid glycosides are widely studied.
The extraction method of phenylethanoid glycosides mainly comprises high shear homogenization extraction, solvent extraction, ultrasonic-assisted extraction, microwave-assisted extraction and supercritical fluid extraction. The high-shear homogenizing extraction method is based on the super-shear principle and realizes the emulsification of solid phase micro-particles and liquid phase. The combination of high shear, impact, pressure and other forces can quickly achieve solubility balance between the solid sample and the solvent, and has the advantages of short time, small solvent amount, high yield and the like. Solvent extraction methods can be divided into three categories: cold dipping, hot dipping and heavy dipping, wherein the cold dipping refers to dipping extraction carried out at normal temperature and is suitable for extracting components which are unstable at high temperature; hot dipping refers to extraction carried out in a water bath or steam heating, suitable for high temperature stable components, with extraction efficiency higher than cold dipping; the re-impregnation refers to a method of impregnating a plurality of times to improve extraction efficiency. The ultrasonic-assisted extraction method is characterized in that ultrasonic energy is used for vibrating, heating and cavitating medicinal particles, and accelerating a solvent to enter the medicinal particles, so that rapid extraction is realized. The microwave-assisted extraction method is characterized in that microwave energy is transmitted to cause molecular motion through ion conduction and dipole rotation, the continuous high temperature breaks cells, and effective substances in the cells flow out, so that the extraction of effective components is realized. The method needs strict control of extraction time to prevent target product from decomposing under strong action of microwave. The supercritical fluid extraction method is an excellent solvent for extracting and separating substances from materials by using high density, low viscosity and high diffusion coefficient of a supercritical fluid, and most studies on the supercritical fluid use CO2As the supercritical fluid, it is inexpensive and difficult to useCombustion, safety, environmental protection, recoverability and the like, and is the first choice for supercritical fluid extraction.
The purification method of the phenylethanoid glycosides mainly comprises a mesoporous carbon adsorption method, a molecular imprinting method, a solvent extraction method, a membrane separation method, a column chromatography method, a macroporous resin adsorption method, a high-efficiency counter-current chromatography method and a preparative high-efficiency liquid chromatography method. The mesoporous carbon adsorption method utilizes the adsorption and desorption performance of mesoporous carbon, the inner diameter of the mesoporous carbon is 2-50nm, and the molecular size of the phenylethanoid glycoside is about 2nm, so that the mesoporous carbon can be used for purifying and separating the phenylethanoid glycoside. The molecular imprinting method is divided into covalent molecular imprinting and non-covalent molecular imprinting, and the separation and purification of natural products usually selects non-covalent molecular imprinting because it is easier to elute than covalent molecular imprinting. The solvent extraction method utilizes the solubility of different components in different solvents to further realize purification, and the common purification solvents comprise petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol. The membrane separation method is a separation method using a difference in molecular weight. The column chromatography is mainly divided into silica gel column chromatography, polyamide column chromatography and gel column chromatography, wherein the silica gel column chromatography and the polyamide column chromatography are both separation realized according to the adsorption and desorption performance of separated substances on fillers, and the gel column chromatography is separation realized according to the size and the shape of molecules. The macroporous resin adsorption method is a common means for separating natural products, and substances with different polarities can be separated and purified by macroporous resins with different types. The high-efficiency counter-current chromatography is a separation and purification method based on liquid-liquid partition chromatography, and has the advantages of high recovery rate, high efficiency and the like. The preparative high performance liquid chromatography is used for preparing the phenylethanoid glycoside in a short time by utilizing the advantages of high sample loading and high separation of the preparative high performance liquid chromatography.
Although the contents of the four phenylethanoid glycosides, namely echinacoside, verbascoside, isoverbascoside and tubular glycoside A, in the cistanche are high, the cistanche still contains other substances such as polysaccharide, iridoid, lignin and the like, so that the conventional procedures for extracting and purifying phenylethanoid glycosides from cistanche medicinal materials are complicated, high in cost and low in purity. Therefore, there is a need to optimize the process steps from extraction to purification to preparation, to ensure a higher transfer rate while reducing costs and pollution. Meanwhile, in the prior art, cistanche is used for neuroprotection to obtain a better curative effect, but the effective components in cistanche are not clear, and the microscopic analysis of drug effect substances is lacked. The separation and extraction of the effective components of the traditional Chinese medicine can improve the treatment effect, is a hotspot and trend of the international development of the traditional Chinese medicine research, and has important significance for developing new medicines with less components, controllable quality and definite mechanism.
Disclosure of Invention
The invention aims to simultaneously prepare four main components (echinacoside, verbascoside, isoverbascoside and tubular glycoside A) with high purity (HPLC purity is more than or equal to 98%) in cistanche by adopting a low-cost and high-efficiency method. The invention relates to a preparation process method for simultaneously preparing four components by refining medicinal material extraction, extract alcohol precipitation, macroporous resin purification and an HPLC-DAD/MS component automatic collection system, which simultaneously considers the neuroprotective effect and the bone-related disease prevention and/or treatment effect of the four component parts and prompts that the parts can be used for treating neurodegenerative diseases such as Mild Cognitive Impairment (MCI), Alzheimer Disease (AD), osteoporosis, fracture healing and the like.
The technical scheme of the invention is as follows:
the first purpose of the invention is to provide a method for simultaneously extracting four effective component monomers from cistanche, wherein the four effective component monomers are echinacoside, verbascoside, isoverbascoside and tubular glycoside A, and the extraction method comprises the following steps:
1) water extraction: cutting the cistanche medicinal materials into small pieces, soaking in water for 1-3 h, decocting and extracting with water for 2 times to obtain cistanche water extract;
2) alcohol precipitation: adjusting the cistanche water extract obtained in the step 1) to an alcohol precipitation solution with the density of 0.7-0.8 (0.7-0.8 g crude drug/mL), carrying out alcohol precipitation by adopting 95% ethanol to remove polysaccharide in the cistanche water extract obtained in the step 1) so that the final concentration of the alcohol precipitation solution is 50-70%, obtaining an alcohol precipitation supernatant, standing the alcohol precipitation supernatant at 4 ℃ for 12h, centrifuging to obtain the supernatant, recovering ethanol under reduced pressure, and carrying out freeze drying on water solution to obtain an alcohol precipitation supernatant solid matter;
3) and (3) purification: purifying the solid matters of the alcohol precipitation supernatant obtained in the step 2) by adopting macroporous resin, wherein the purification conditions are as follows: dissolving the solid matters of the alcohol precipitation supernatant obtained in the step 2) in water to prepare a uniform clear solution with the echinacoside concentration of 2.8mg/mL, wherein the sample loading amount is 5BV, the sample loading flow rate is 1BV/h, eluting with water, the impurity removal volume of the water is 10BV, and the elution flow rate is 2BV/h to obtain a water elution part; the elution volume of 25 percent ethanol is 15BV, and the elution flow rate is 4BV/h, thus obtaining 25 percent ethanol elution parts; the elution volume of 50 percent ethanol is 6BV, the elution flow rate is 2BV/h, and the diameter-height ratio is 1: 8; collecting 25% ethanol eluate, concentrating, and volatilizing ethanol to obtain purified extract;
4) an HPLC-DAD/MS component automatic collection system simultaneously extracts four effective component monomers: dissolving the purified extract obtained in the step 3) in water to prepare the extract with the maximum concentration capable of being completely dissolved, injecting 5mL of the extract into an HPLC-DAD/MS component automatic collection system, automatically collecting chromatographic peaks for 6min, 9min, 10min and 16min respectively, and volatilizing the solvent to obtain four effective component monomers of echinacoside, isoacteoside, verbascoside and tubular glycoside A respectively;
the chromatographic conditions of the HPLC-DAD/MS component automatic collection system are as follows:
a chromatographic column: shim-pack PRC-ODS 20X 25 cm;
mobile phase: methanol-formic acid aqueous solution (volume ratio is 45:55, wherein the volume fraction of formic acid in the formic acid aqueous solution is 0.1%)
Flow rate: 3 mL/min;
sample introduction amount: 5 mL;
detection wavelength: 330 nm.
TABLE 1 molecular Structure of the four component monomers
Further, in the step 1), the cistanche medicinal material is cut into smaller than 1cm3The water is added for soaking for 1 hour.
Further, the extraction time of 2 times in the step 1) is 1 hour respectively, and the material-liquid ratio is 1: 11-12; preferably, the feed-liquid ratio is 1: 11.85.
Further, the macroporous resin comprises one or a mixture of HPD-300, D-101-I, D-101 and HPD-100;
further preferably, the macroporous resin also comprises NKA-9, and the NKA-9 is mixed with one or more of HPD-300, D-101-I, D-101 and HPD-100;
even more preferably, selected from HPD-300 and NKA-9;
the purity of the four component monomers extracted by the method is higher than 98 percent (HPLC purity is more than or equal to 98 percent).
The second purpose of the invention is to provide the cistanche effective components extracted by the extraction method, wherein the cistanche effective components are one or more of the echinacoside, verbascoside, isoverbascoside and tubular glycoside A effective component monomers obtained in the step 4).
The third purpose of the invention is to provide the cistanche deserticola effective part extracted by the extraction method, wherein the effective part is the 25% ethanol elution part obtained in the step 3).
Furthermore, the total content of echinacoside, verbascoside, isoverbascoside and tubular flower glycoside A in the effective part is not less than 50%.
The fourth purpose of the invention is to provide the application of the cistanche salsa effective components extracted by the extraction method or the cistanche salsa effective parts extracted by the extraction method in the preparation of preparations for preventing and/or treating neurodegenerative diseases.
Furthermore, the cistanche effective component is one or a combination of more of echinacoside, verbascoside, isoverbascoside and tubular flower glycoside A, and the total content of the four effective component monomers in the preparation for preventing and/or treating neurodegenerative diseases is not less than 50%.
The fifth purpose of the invention is to provide the application of the cistanche salsa effective components extracted by the extraction method or the cistanche salsa effective parts extracted by the extraction method in the preparation of preparations for preventing and/or treating bone-related diseases.
Preferably, the cistanche effective component is one or a combination of more of echinacoside, verbascoside, isoverbascoside and tubular flower glycoside A, and the total content of the four effective component monomers in the preparation for preventing and/or treating neurodegenerative diseases is not less than 50%.
The sixth purpose of the invention is to provide a method for simultaneously and quantitatively detecting the cistanche salsa effective part and/or four component monomers extracted by the extraction method, wherein the simultaneous and quantitative detection method is high performance liquid chromatography;
the preparation method of the reference stock solution of the simultaneous quantitative detection method comprises the following steps: precisely weighing 1-2mg each of echinacoside, acteoside, isoacteoside and tubular flower glycoside A, respectively placing in 1mL volumetric flask, adding 50% methanol to dissolve and fix volume, and respectively making into four reference substance stock solutions;
the preparation method of the test solution comprises the following steps: weighing 5mg of the 25% ethanol elution part obtained in the step 3), precisely weighing, adding 50% methanol for dissolving, fixing the volume to 1mL, completely mixing, and storing at 4 ℃ for later use; and/or the presence of a gas in the gas,
respectively weighing 2mg of each of the four effective component monomers obtained in the step 4), precisely weighing, adding 50% methanol for dissolving, fixing the volume to 1mL, completely mixing, and storing at 4 ℃ for later use;
the quantitative method comprises the following steps: the quantitative method is an external standard method for quantification, four reference substance stock solutions are sequentially and precisely diluted into concentration gradients of 1/2, 1/4, 1/8, 1/16 and 1/32 with the original concentration, 10 mu L (automatic sample injector) is respectively injected for analysis, the peak area is determined, the injection concentration (X) of the reference substance stock solution is taken as a horizontal coordinate, the corresponding peak area (Y) is taken as a vertical coordinate, and a standard curve is made;
the chromatographic conditions were as follows:
a chromatographic column: a chromatographic column: agilent ZORBAX extended-C18(250mm×4.6mm，1.8μm)；
Mobile phase: 1% formic acid in water (A) -acetonitrile (B) with a volume fraction of formic acid;
column temperature: 30 ℃;
detection wavelength: 330 nm;
flow rate: 1 mL/min;
sample volume of test solution: 10 μ L.
The elution procedure was:
the seventh purpose of the invention is to provide the application of phenylethanoid glycosides compounds in preparing preparations for preventing and/or treating neurodegenerative diseases, wherein the phenylethanoid glycosides compounds are one or a combination of more of echinacoside, verbascoside, isoverbascoside and tubular anthocyanin A.
Further, in the preparation for preventing and/or treating neurodegenerative diseases, the total content of the phenylethanoid glycosides compounds is not less than 50%.
The eighth purpose of the invention is to provide the application of phenylethanoid glycosides compounds in preparing preparations for preventing and/or treating bone-related diseases, wherein the phenylethanoid glycosides compounds are one or more of echinacoside, verbascoside, isoverbascoside and tubular anthocyanin A.
Furthermore, in the preparation for preventing and/or treating bone-related diseases, the total content of the phenylethanoid glycosides compounds is not less than 50%.
BV as used herein refers to the volume of the filled resin.
The technical scheme of the invention has the following beneficial effects:
the extraction method provided by the invention realizes the simultaneous extraction of four high-purity component monomers from the cistanche deserticola medicinal material: the echinacoside, the verbascoside, the isoverbascoside and the tubular glycoside A are obtained by investigating an extraction method, a purification method and a preparation method, the transfer rate of each link reaches a higher level, high efficiency and low cost are realized, the extraction method has strong repeatability and stable process quality, the higher transfer rate (72.38 percent) is ensured, and the cost and the pollution are reduced. Meanwhile, the purity of the prepared monomer is improved, and the purity of the four-component monomer obtained by extraction is higher than 98 percent (HPLC purity is more than or equal to 98 percent).
In addition, the invention provides a detection method for simultaneously and quantitatively detecting the four effective component monomers and/or quantitatively detecting the content of the four effective component monomers in the effective part of the invention, and the detection method has good precision and repeatability.
The invention proves the neuroprotective effect and the bone protective activity of the four effective component monomers and the effective parts determined by the invention, and provides a new choice for preparing preparations for preventing and/or treating neurodegenerative diseases and preparations for preventing and/or treating bone-related diseases.
Drawings
FIG. 1 comparison of echinacoside, verbascoside, coronariside A and isoverbascoside content in 25% ethanol purified fraction and unpurified fraction of example 2;
FIG. 2 finger print of the purified product of cistanche tubulosa
Peak 1 echinacoside; peak 2 tubular glycoside A; peak 3 verbascoside; peak 4 Isoverbascoside.
FIG. 3 Standard Curve of echinacoside
FIG. 4 Standard Curve of tubular flower glycoside A
FIG. 5 Standard Curve for verbascoside
FIG. 6 Standard Curve for Isoverbascoside
FIG. 7 is a comparison of the reference and test solutions
Peak 1: echinacoside; peak 2: tubuloside A; peak 3: verbascoside; peak 4: isoverbascoside
FIG. 8 Effect of different concentrations of each extract treated cells in example 5 on the viability of PC12 cells for 24 h.
P-value <0.05, p-value <0.01, p-value <0.001, compared to Ctrl group.
FIG. 9 Effect of different concentrations of each monomer-treated cell 24h on the viability of PC12 cells in example 5.
P-value <0.05, p-value <0.01 compared to Ctrl group.
FIG. 10 Effect of various concentrations of each extract in example 5 on cell viability following oxidative damage to PC12 after 24h treatment.
P-value <0.05, p-value <0.01, p-value <0.001, compared to Ctrl group.
FIG. 11 Effect of various concentrations of each monomer treatment for 24h in example 5 on cell viability following oxidative damage to PC 12.
P-value <0.01, p-value <0.001 compared to Ctrl group.
Figure 12 effect of different concentrations of each extract treatment for 24h in example 5 on LDH leakage after oxidative damage by PC 12. P-value <0.01, p-value <0.001 compared to Ctrl group.
FIG. 13. Effect of different concentrations of each monomer treatment for 24h in example 5 on LDH leakage after oxidative damage to PC 12.
P-value <0.01, p-value <0.001 compared to Ctrl group.
FIG. 14 Effect of different concentrations of each extract treated cells for 24h on MC3T3-E1 cell viability in example 6.
P-value <0.05, p-value <0.01, p-value <0.001, compared to Ctrl group.
FIG. 15 Effect of different concentrations of each monomer-treated cell in example 6 on the viability of MC3T3-E1 cells over 24 h.
P-value <0.01, p-value <0.001 compared to Ctrl group.
FIG. 16 Effect of different concentrations of each extract treatment 5d on ALP activity of MC3T3-E1 cells in example 6.
P-value <0.05, p-value <0.001 compared to Ctrl group.
FIG. 17 Effect of different concentrations of each monomer treatment 5d on ALP activity in MC3T3-E1 cells in example 6.
P-value <0.05, p-value <0.001 compared to Ctrl group.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Example 1 cistanche extraction Process investigation
Adopting single factor investigation method to optimize and investigate soaking time, extraction method, extraction solvent, extraction time, extraction times, material-liquid ratio and extraction particle diameter, and cutting the cistanche medicinal material into pieces smaller than 1cm3Soaking the small blocks in water for 1h, and decocting and extracting with water for 2 times; orthogonal design experiment investigation items are extraction solvent, extraction time and feed-liquid ratio, then response surface design is adopted to carry out further investigation on the extraction solvent, the extraction time and the feed-liquid ratio, and the final investigation result is as follows: water is used as an extraction solvent, the extraction time is 1h, and the material-liquid ratio is 1: 11.85. The inspection basis is the extract yield, and the content determination method is according to the chromatographic method in the 2015 pharmacopoeia. A small experimental confirmation was carried out according to the above conditions.
The experimental results are as follows:
response surface optimization is performed by using 50% weight (%) as a target index Y, and response surface test data analysis by a reflux extraction method is performed by using Design Expert 8.05 software. The specific experimental arrangement and results are shown in table 1.
Table 150% weight response surface design and results
The results in the table are subjected to linear regression through software design, and a quadratic polynomial equation of the extract yield Y to three factors is obtained through fitting:
Y＝53.60+27.59A+1.14B+1.79C-1.10AB+0.18AC-2.10BC--9.73A2+0.21B2-5.49C2
R2＝0.9811
to verify the validity of this equation, analysis of variance was performed, the specific results are shown in table 2:
TABLE 2 analysis of variance results with extract yield as a single factor
R20.9811, corrected R2＝0.9567
As can be seen from the data in table 1, F is 40.32 and p <0.05, indicating that the quadratic equation obtained by regression is significant. The magnitude of the effect of each variable is: the concentration of ethanol is more than the ratio of material to liquid and the extraction time is more than the extraction time.
Software analysis shows that the ethanol concentration is 1 (pure water) when the weight of 50% is maximum, the extraction time is 1h, and the material-liquid ratio is 1: 11.85.
Example 2 macroporous resin purification Process Studies of Cistanchis herba extract
Removing polysaccharide in the cistanche water extract by adopting an alcohol precipitation method, and optimizing an alcohol precipitation process from the ethanol concentration and the density respectively, wherein the optimization result is as follows: adjusting the cistanche water extract obtained in the step 1) to an alcohol precipitation solution with the density of 0.7-0.8, carrying out alcohol precipitation by adopting 95% ethanol to remove polysaccharide in the cistanche water extract obtained in the step 1) so that the final concentration of the alcohol precipitation solution is 50-70%, obtaining an alcohol precipitation supernatant, standing the alcohol precipitation supernatant at 4 ℃ for 12h, centrifuging to obtain the supernatant, recovering the ethanol under reduced pressure, and carrying out freeze drying on water solution to obtain an alcohol precipitation supernatant solid matter.
The optimization indexes are the transfer rate of echinacoside and verbascoside and the amount of generated filter residue; under the alcohol precipitation process, the transfer rate of echinacoside and verbascoside and the amount of generated filter residue are as follows: the transfer rate of alcohol precipitation was 98.8%, and the amount of generated residue was 2.6g/500g crude drug.
Then adopting macroporous resin to further purify the filtrate after alcohol precipitation, firstly selecting 10 kinds of macroporous resin (respectively: HPD-100, HPD-300, AB-8, D101, ADS-7, ADS-5, G2, DM130, AB-8 and D101-I), and selecting the type of the macroporous resin with better adsorption and desorption capacities through static adsorption and dynamic adsorption investigation, wherein the investigation result is as follows: HPD-300> D-101-I > HPD-100> D-101; furthermore, NKA-9 adsorbs only poorly to the target substance (i.e., the four active ingredient monomers of the present invention), but adsorbs one or more other impurities efficiently, and is difficult to elute after adsorption, so that NKA-9 can be mixed with any one or more of HPD-300, D-101-I, HPD-100 and D-101 to obtain a better purification result.
And (3) mixing HPD-300 and NKA-9, further carrying out dynamic adsorption and desorption investigation, and investigating the sample loading amount, the sample loading flow rate, the elution ethanol concentration, the elution amount and the diameter-height ratio.
The experimental results are as follows: the sample loading amount is 5BV, the sample loading flow rate is 1BV/h, the water impurity removal volume is 10BV, the elution flow rate is 2BV/h, the 25 percent ethanol elution volume is 15BV, the elution flow rate is 4BV/h, the 50 percent ethanol elution volume is 6BV, the elution flow rate is 2BV/h, and the diameter-height ratio is 1: 8.
The results of the water elution fraction, 25% ethanol elution fraction, and 50% ethanol elution fraction elution, purity, and content experiments obtained under the purification conditions are shown in table 3:
TABLE 3
The comparison of the content of echinacoside, verbascoside, coronarin A and isoverbascoside in the 25% ethanol-purified fraction and the unpurified fraction is shown in FIG. 1. from Table 3 and FIG. 1, it can be seen that echinacoside and verbascoside are substantially completely in the 25% ethanol-eluted fraction, and the content of echinacoside, verbascoside, coronarin A and isoverbascoside in the 25% ethanol-purified fraction is significantly increased compared with the unpurified fraction, and the content of four components in the 25% ethanol-purified fraction is: echinacoside 47.29%, tubular flower glycoside A1.64%, verbascoside 4.20%, isoverbascoside 8.15%. Indicating successful purification.
Example 3 preparation of four high purity chemical Components Using an HPLC-DAD/MS component AutoCollection System
Taking Cistanchis herba (particle diameter less than 1 cm)2)250g, placing the mixture into a 10L round bottom flask, adding 11.85 times of water (2750mL), soaking for 1h, extracting twice, heating and decocting for 1h for two times, filtering, and combining filtrates. Steaming, concentrating to 357mL (0.7g crude drug/mL), adding773mL 95% ethanol, standing at 4 deg.C for 12h, centrifuging to get supernatant, steaming, recovering ethanol under reduced pressure, and freeze drying to get 130g solid of supernatant.
Taking solid matters of alcohol precipitation supernatant after alcohol precipitation, dissolving the solid matters in water to prepare a uniform clarified solution with echinacoside concentration of 2.8mg/mL, taking processed 32g HPD-300 and 32g NKA-9 (wet weight), carrying out wet column packing, loading into a glass column with the inner diameter of 4cm and the height of 32cm, loading the prepared sample solution to be tested into a sample with the volume of 2500mL and the flow rate of 5BV/h, eluting with water, 25% ethanol and 50% ethanol in sequence, wherein the elution volumes are respectively 5L for water impurity removal, 10L for 25% ethanol elution and 3L for 50% ethanol elution, and the elution flow rates are respectively 2BV/h for water impurity removal, 4BV/h for 25% ethanol elution and 2BV/h for 50% ethanol elution, collecting 25% ethanol eluent, concentrating, volatilizing ethanol, and obtaining 16.3g of purified extract.
Dissolving the purified extract in water to obtain the maximum concentration capable of being completely dissolved, injecting 5mL into HPLC-DAD/MS component automatic collection system, automatically collecting 6min, 9min, 10min and 16min chromatographic peaks, and volatilizing solvent to obtain echinacoside (6min), isoacteoside (9min), verbascoside (10min) and tubuloside A (16min) compounds. The chromatographic conditions were as follows:
a chromatographic column: shim-pack PRC-ODS 20X 25 cm;
mobile phase: methanol-formic acid water (volume ratio 45:55, volume fraction of formic acid in formic acid water solution is 0.1%)
Flow rate: 3 mL/min;
sample introduction amount: 5 mL;
detection wavelength: 330nm
And (3) blowing the solvent from the prepared solution by nitrogen to obtain 4 monomers respectively, wherein the purity of the 4 monomers is higher than 98% by determination.
Example 4 method for simultaneously quantifying four ingredients in cistanche tubulosa purified product and its methodological verification
Optimizing chromatographic conditions by investigating mobile phase, flow rate, sample amount, column temperature and detection wavelength to obtain a fingerprint spectrum as shown in figure 2, wherein peak 1 in figure 2 is echinacoside; peak 2 is tubular flower glycoside A; peak 3 is verbascoside; peak 4 is isoverbascoside.
The simultaneous quantitative detection method is high performance liquid chromatography, and the chromatographic conditions are as follows:
a chromatographic column: a chromatographic column: AgilentZORBAXExtend-C18(250mm×4.6mm，1.8μm)；
Mobile phase: 0.1% formic acid (a) -acetonitrile (B);
column temperature: 30 ℃;
detection wavelength: 330 nm;
flow rate: 1 mL/min.
Elution procedure: see table 4;
TABLE 4 elution procedure
Methodology investigation
1 investigation of Linear relationship
Taking echinacoside, verbascoside, isoverbascoside and tubular flower glycoside A reference substances, respectively 1-2mg, precisely weighing, placing in a 1mL volumetric flask, adding 50% methanol to dissolve and fix volume, and making into reference substance stock solution; the stock solution was sequentially diluted to concentration gradients of 1/2, 1/4, 1/8, 1/16, and 1/32 at the original concentration, and 10 μ L of each sample (autosampler) was added for analysis, and a standard curve was prepared by measuring the peak area with the sample concentration (X) as the abscissa and the peak area (Y) as the ordinate (see fig. 3 to 6).
2 detection limit and quantification limit
Injecting a reference substance solution with known concentration, and diluting the reference substance solution to a concentration (S/N ≈ 3) about 3 times of signal-to-noise ratio according to a corresponding peak height value, namely a detection Limit (LOD); the concentration of about 10 times the signal-to-noise ratio (S/N ≈ 10) is the limit of quantitation (LOQ), and the relative standard deviation of the peak area is calculated by repeating the sample injection 6 times at the limit of quantitation, and the results are shown in Table 5.
TABLE 5 examination of detection limits and quantitation limits
3 precision test
The preparation method of the test solution comprises the following steps: precisely weighing the same batch of purified cistanche tubulosa (Desertliving cistanche) Bunge, namely 5mg of 25% ethanol elution part, precisely weighing, adding 50% methanol for dissolving, fixing the volume to 1mL, mixing completely, and storing at 4 ℃ for later use;
precisely sucking 10 μ L of test solution (with concentration of echinacoside and verbascoside of 0.3025mg/mL), continuously feeding sample for 6 times according to the above chromatographic conditions, and measuring peak area, wherein RSD of four components is less than 3%, which indicates that the used liquid phase has good precision.
4 repeatability test
Precisely weighing 6 parts of the purified cistanche tubulosa (Desertliving cistanche) Bunge of the same batch, namely 6 parts of 25% ethanol elution part, each 5mg, precisely weighing, adding 50% methanol for dissolving, fixing the volume to 1mL, and completely mixing. Treating according to the preparation method of the test solution, and performing sample injection measurement according to the chromatographic conditions. The result shows that the RSD of the four components does not exceed 3 percent, which indicates that the method has better repeatability.
5 stability test
Taking a sample solution under the item of 'repeatability test', respectively injecting samples at 0, 2, 4, 8, 12 and 24 hours, recording the peak area of the cistanche purified substance in the sample solution, and calculating the RSD value of the measurement result.
The results show that the four components have better stability within 12 h. (Echinacoside RSD 0.68%, tubular flower glycoside RSD 1.70%, verbascoside RSD 1.74%, isoverbascoside RSD 0.77%)
6 sample recovery test
Precisely measuring 6 parts of the same batch of sample solution with known content, precisely adding appropriate amount of echinacoside, verbascoside, isoverbascoside and tubuloside A, respectively, processing according to the preparation method of the test solution, and performing sample injection measurement according to the above chromatographic conditions.
Percent recovery [% ]
TABLE 6 sample recovery test results of cistanche tubulosa purified material
TABLE 7 sample recovery results
The result shows that the average recovery rate of the four components is in the required range, the RSD is less than 3 percent, and the quality standard requirement is met.
7 determination in samples
Taking about 5mg of each batch of cistanche tubulosa purified substances (namely 25% ethanol elution parts), precisely weighing, weighing 2 parts in parallel, preparing according to a method for preparing a test solution, and carrying out sample injection 2 times on each part for determination. The 3 batches of cistanche tubulosa purified material were tested.
The average content of the purified cistanche tubulosa in 3 batches is 61.28%, wherein the minimum content is 60.10%, and the maximum content is 61.73%. The content limit of the total content of the four components in the cistanche tubulosa is recommended to be not less than 50%. (FIG. 7)
Weighing 2mg of each of the four separated effective component monomers respectively, precisely weighing, weighing 2 parts in parallel, preparing according to a method for preparing a test solution, and carrying out sample injection 2 times on each sample for determination.
Example 5 study of neuroprotective Effect of purified cistanche deserticola and its four ingredients
To investigate the possible protective effect of the four extracts and their four monomeric components (echinacoside, verbascoside, isoverbascoside and angioside A) on rat adrenomedullin chromaffin tumor differentiated cell line PC12 at 200. mu.M H2O2Cells were treated for 24h to undergo oxidative damage.
The specific treatment method comprises the following steps:
CCK-8 method for detecting cytotoxicity of different extracts on PC12
PC12 cells in the logarithmic growth phase are taken and inoculated to a 96-well plate at an appropriate density, and are placed in an incubator for 24 hours. After 24h, the culture solution containing 1% FBS, 500, 250, 125, 0 mu g/mL of different extracts and 80, 40 and 20 mu mol/L of different monomers is replaced, each group has no less than 3 more wells, and the culture solution is put into an incubator for further culture for 24 h. After 24h, the supernatant was discarded, and the cell viability was measured by the CCK-8 method (FIGS. 8 to 9).
Method for detecting survival rate of PC12 cells by CCK-8 method
PC12 cells in logarithmic growth phase are inoculated to a 96-well plate at an appropriate density and cultured in an incubator. After the cells are cultured for 24 hours, the cells are treated differently according to the following components, and each group has not less than 3 multiple holes: control group of Normal cells (Ctrl), H2O2Model group (200. mu.M), VC (25. mu.M) positive control group, H2O2+ different extract administration groups (500, 250, 125. mu.g/mL), H2O2+ different monomer administration groups (80, 40, 20. mu. mol/L). After 24h of treatment, the supernatant is discarded, the cell survival rate is detected by adopting a CCK-8 method, and the treatment results of the extracts with different concentrations and the monomers with different concentrations show that:
as a result, it was found that H was compared with the control group of normal cells2O2The cell survival rate after 24h of the treated cells is only 68.82% (compared with the normal cell control group, P<0.001), description H2O2Causing significant damage to the cells.
After the four extracts at different doses are treated, the cell survival rate is remarkably improved to different degrees.
After the water elution parts prepared in the purification step are regulated to 250 mu g/mL and 125 mu g/mL for treatment, the cell survival rate is increased to 91.58 percent and 103.05 percent; after the 50% ethanol elution part prepared in the purification step is regulated to 250 mu g/mL and 125 mu g/mL, the cell survival rate is increased to 86.28% and 86.97%; after the 25% ethanol elution part prepared in the purification step is subjected to dosage adjustment to 500 mu g/mL, 250 mu g/mL and 125 mu g/mL, the cell survival rate is increased to 102.89%, 90.58% and 84.02%, and certain dosage dependence is presented; after the water extraction and alcohol precipitation liquid prepared in the alcohol precipitation step is treated by adjusting the dosage to 500 mug/mL, 250 mug/mL and 125 mug/mL, the cell survival rates are respectively increased to 89.27%, 89.58% and 96.03%.
The results show that the four extracts can antagonize H to a certain extent2O2The survival rate of the cells is reduced, the water elution part, the 25% ethanol elution part and the water extraction and alcohol precipitation liquid have relatively good effects, and the 25% ethanol elution part comprises four components of echinacoside, verbascoside, isoverbascoside and tubular anthocyanin A, so that the 25% part has better drug effect than other parts, and the preparation for preventing and/or treating neurodegenerative diseases is suggested to be prepared (figure 10).
After the four monomers with different dosages are treated, the cell survival rate is remarkably improved to different degrees.
After the echinacoside is regulated to 80, 40 and 20 mu mol/L, the cell survival rate is increased to 84.97%, 84.08% and 79.31%; after the verbascoside is regulated to 80, 40 and 20 mu mol/L for treatment, the cell survival rate is increased to 96.03%, 89.58% and 86.97%; after the isoverbascoside is regulated to 80, 40 and 20 mu mol/L, the cell survival rate is increased to 102.90%, 92.98% and 84.03%; after the pipefish glycoside A is adjusted to 80, 40 and 20 mu mol/L, the cell survival rate is respectively increased to 88.78%, 89.27% and 80.93%.
The results show that four monomers can antagonize H to a certain extent2O2The resulting decrease in cell viability suggests that the monomeric compounds and their various combinations can be used to prepare agents for the prevention and or treatment of neurodegenerative diseases (fig. 11).
3. Method for detecting leakage of Lactate Dehydrogenase (LDH) of PC12 cells by microplate method
To further investigate the protective effect of the four extracts on PC12 cells, 200. mu.M H was added2O2And (3) damaging the cells for 24 hours, and detecting the leakage amount of LDH in cell supernatant by adopting a microplate method: PC12 cells in logarithmic growth phase are inoculated to a 96-well plate at an appropriate density and cultured in an incubator. After 24h of cell culture, the cells were treated differently according to the following protocolAnd (3) arranging each group with at least two holes: control group of Normal cells (Ctrl), H2O2Model group (200. mu.M), VC (25. mu.M) positive control group, H2O2+ different extract administration groups (500, 250, 125. mu.g/mL), H2O2+ different monomer administration groups (80, 40, 2. mu. mol/L). After 24h of treatment, the supernatant was aspirated and transferred to a clean 96-well plate, and Lactate Dehydrogenase (LDH) activity in the supernatant was determined according to the instructions of Nanjing's kit for the establishment of lactate dehydrogenase detection.
The results show that H2O2The LDH content in the cell supernatant rose to 194.53% 24h after the treatment of the cells (P compared to Ctrl group<0.001), description H2O2Causing significant damage to the cells.
The cellular supernatants showed different decreases in LDH levels after treatment with the four extracts at different doses. As shown in fig. 12, the LDH content of the water elution sites of 500, 250 and 125 μ g/mL was reduced to 134.98%, 116.36% and 119.56%; 250. the LDH content in the cell supernatant after the treatment of the 50% ethanol elution part with 125 mu g/mL is reduced to 152.07% and 134.05%; 500. after the treatment of 25% ethanol elution sites of 250 and 125 mu g/mL, the LDH content is reduced to 102.89%, 90.58% and 84.02%, and obvious dose dependence is presented; 500. the LDH content of the treated water extraction and alcohol precipitation solution of 250 and 125 mu g/mL is respectively reduced to 123.97%, 121.76% and 108.81%, and certain dose dependence is also shown. The above results show that the four extracts can antagonize H to some extent2O2The resulting leakage of LDH was somewhat protective of PC12 cells, with the 25% ethanol elution site acting relatively best.
To further investigate the protective effect of the four monomers on PC12 cells, LDH levels in cell supernatants showed different decreases after treatment with different doses of the four monomers. As shown in FIG. 13, LDH content decreased to 137.54%, 119.02%, 118.30% after 80, 40, 20. mu. mol/L echinacoside treatment; 80. LDH content in cell supernatant was reduced to 115.81%, 149.96%, 137.14% after 40, 20. mu. mol/L verbascoside treatment; 80. 40, 20. mu. mol/L Isoverbascoside treatmentThen, the LDH content is reduced to 119.77%, 117.09% and 155.78%; 80. LDH content after 40 and 20 mu mol/L verbascoside A treatment is respectively reduced to 126.83%, 148.43% and 171.81%. The above results show that four monomers can antagonize H to some extent2O2The leakage of LDH is caused, and PC12 cells are protected to a certain extent.
Example 6 study of bone protection Effect of Cistanchis herba purified product and its four components
In order to examine the possible protective effect of four extracts and four monomer components (echinacoside, verbascoside, isoverbascoside and tubular glycoside A) on mouse embryonic osteoblast precursor cells MC3T3-E1, the effect on cell proliferation and intracellular ALP activity was examined. The specific treatment method comprises the following steps:
CCK-8 method for detecting cytotoxicity of different extracts and four components thereof on MC3T3-E1
Cells in the logarithmic growth phase are taken and inoculated to a 96-well plate with proper density, and the 96-well plate is placed in an incubator to be cultured for 24 hours. After 24h, the extract containing 1% FBS and different extracts of 500, 250, 125 and 0 mu g/mL are replaced; 80. 40 and 20 mu mol/L culture solution of different monomers, each group has no less than 3 more holes, and the culture solution is put into an incubator to be continuously cultured for 24 hours. And (3) discarding the supernatant after 24h, and detecting the cell viability by adopting a CCK-8 method, wherein the treatment results of the extracts with different concentrations and the monomers with different concentrations are as follows:
to examine the effect of different extracts on the viability of MC3T3-E1, cells were treated with four extracts at 500, 250, 125, and 0 μ g/mL for 24h, respectively, and the results are shown in FIG. 16. 500. 250 and 125 mu g/mL water elution parts, 50% ethanol elution parts and water extraction and alcohol precipitation liquid, wherein the 250 and 125 mu g/mL 25% ethanol elution parts have obvious promotion effect on the activity of MC3T3-E1 cells. And the 25% ethanol elution part of 500 mu g/mL has obvious inhibition effect on the cell viability, which indicates that the cell is cytotoxic at the concentration. Therefore, the subsequent experiments selected 250, 125, 62.5 μ g/mL of 25% ethanol elution sites as the active concentration, and the active concentrations of the remaining 3 extracts were set to 500, 250, 125 μ g/mL (FIG. 14).
To examine the effect of different monomers on the viability of MC3T3-E1 cells were treated with 80, 40, 20, 0. mu. mol/L of each of the four monomers for 24h, and the results are shown in FIG. 2. After 80 mu mol/L echinacoside treatment, the cell activity is increased to 120.33%; 80. after 40 mu mol/L verbascoside treatment, the cell activity is respectively increased to 123.05 percent and 129.03 percent; after 80 mu mol/L isoverbascoside treatment, the cell activity is increased to 115.55%; after 80. mu. mol/L treatment with tubular flower glycoside A, the cell viability rose to 121.38%. The results show that 80 mu mol/L echinacoside, 80 mu mol/L verbascoside, 40 mu mol/L isoverbascoside and 80 mu mol/L angioside A have obvious promotion effect on the activity of MC3T3-E1 cells, and the rest concentration has no obvious influence on the cell activity. The results suggest that four monomers may have an effect of promoting osteoblast proliferation (fig. 15).
2. Microplate assay for detecting the activity of various extracts and four components thereof on MC3T3-E1 cell alkaline phosphatase (ALP)
Cells in the logarithmic growth phase are taken and inoculated to a 24-well plate with proper density, and the plate is placed in an incubator to be cultured for 24 hours. And after 24h, replacing the culture solution containing 10% FBS and extracts with different concentrations, placing each group into an incubator for continuously culturing for 5d, and replacing the culture solution once every 1 d. After washing the cells for 2 times with PBS, the cells were collected with a cell scraper, and a suitable amount of RIPA inhibitor-free potent lysate was added to the cells to lyse the cells sufficiently on ice. The activity of ALP in the lysates was determined as per the instructions.
To further examine the bone protective effects of the four extracts, ALP activity of the four extracts after culturing MC3T3-E1 cells 5d, respectively, was measured as shown in FIG. 16. As a result, the ALP activity was found to be significantly reduced after the treatment of the 250/125. mu.g/mL water elution sites compared with the Ctrl group, while the 500/250/125. mu.g/mL 50% ethanol elution sites had no significant effect on the ALP activity, indicating that there may be no significant promotion effect on the differentiation of MC3T3-E1 cells. 250. After the treatment of the 25% ethanol elution part with 125 mug/mL, compared with the Ctrl group, the ALP activity is respectively increased to 134.61% and 128.67%, and the significant difference exists; the ALP activity after the treatment of 500 mug/mL water extraction and alcohol precipitation liquid is 199.06%. The results show that the 25% ethanol elution part and the water extraction and alcohol precipitation solution have certain promotion effect on the differentiation of MC3T3-E1 cells into osteoblasts, and the bone protection activity of the MC3T3-E1 cells is prompted.
To further investigate the protective effects of the four monomeric bones, ALP activity of the four extracts after culturing MC3T3-E1 cells 5d, respectively, was determined as shown in FIG. 17. As a result, it was found that the intracellular ALP activity was increased to 125.48% after 80. mu. mol/L treatment with echinacoside; after 80 mu mol/L verbascoside treatment, the intracellular ALP activity is increased to 134.61% and 129.03%; after 80 μmol/L isoverbascoside treatment, intracellular ALP activity was increased to 127.87%; 80. after 40. mu. mol/L treatment with tubular flower glycoside A, intracellular ALP activity was increased to 125.72% and 118.28%, respectively. The results show that the four monomers have certain promotion effect on the differentiation of MC3T3-E1 cells into osteoblasts, and the bone protection activity of the monomers is prompted.
Claims (10)
1. A method for simultaneously extracting four effective component monomers from cistanche is characterized in that the four effective component monomers are echinacoside, verbascoside, isoverbascoside and tubular glycoside A, and the extraction method comprises the following steps:
1) water extraction: cutting the cistanche medicinal materials into small pieces, soaking in water for 1-3 h, decocting and extracting with water for 2 times to obtain cistanche water extract;
2) alcohol precipitation: adjusting the cistanche water extract obtained in the step 1) to an alcohol precipitation solution with the density of 0.7-0.8, carrying out alcohol precipitation by adopting 95% ethanol to remove polysaccharide in the cistanche water extract obtained in the step 1) so that the final concentration of the alcohol precipitation solution is 50-70%, obtaining an alcohol precipitation supernatant, standing the alcohol precipitation supernatant at 4 ℃ for 12h, centrifuging to obtain a supernatant, recovering the ethanol under reduced pressure, and carrying out freeze drying on water solution to obtain an alcohol precipitation supernatant solid matter;
3) and (3) purification: purifying the solid matters of the alcohol precipitation supernatant obtained in the step 2) by adopting macroporous resin, wherein the purification conditions are as follows: dissolving the solid matters of the alcohol precipitation supernatant obtained in the step 2) in water to prepare a uniform clear solution with the echinacoside concentration of 2.8mg/mL, wherein the sample loading amount is 5BV, the sample loading flow rate is 1BV/h, eluting with water, the impurity removal volume of the water is 10BV, and the elution flow rate is 2BV/h to obtain a water elution part; the elution volume of 25 percent ethanol is 15BV, and the elution flow rate is 4BV/h, thus obtaining 25 percent ethanol elution parts; the elution volume of 50 percent ethanol is 6BV, the elution flow rate is 2BV/h, and the diameter-height ratio is 1: 8; collecting 25% ethanol eluate, concentrating, and volatilizing ethanol to obtain purified extract;
preferably, the macroporous resin comprises one or more of HPD-300, D-101-I, D-101 and HPD-100;
further preferably, the macroporous resin also comprises NKA-9, and the NKA-9 is mixed with one or more of HPD-300, D-101-I, D-101 and HPD-100;
even more preferably, selected from HPD-300 and NKA-9;
4) an HPLC-DAD/MS component automatic collection system simultaneously extracts four effective component monomers: dissolving the purified extract obtained in step 3) in water to obtain the maximum concentration capable of being completely dissolved, injecting 5mL into an HPLC-DAD/MS component automatic collection system, automatically collecting chromatographic peaks for 6min, 9min, 10min and 16min respectively, and volatilizing the solvent to obtain echinacoside, isoacteoside, verbascoside and tubular glycoside A respectively;
the chromatographic conditions of the HPLC-DAD/MS component automatic collection system are as follows:
a chromatographic column: shim-pack PRC-ODS 20X 25 cm;
mobile phase: methanol-formic acid water (volume ratio is 45:55, the volume fraction of formic acid in the formic acid water solution is 0.1%)
Flow rate: 3 mL/min;
sample introduction amount: 5 mL;
detection wavelength: 330 nm.
2. The method for simultaneously extracting four effective component monomers from cistanche as claimed in claim 1, wherein the cistanche is cut into less than 1cm in step 1)3The water is added for soaking for 1 hour.
3. The method for simultaneously extracting four effective component monomers from cistanche as claimed in claim 1, wherein the 2 times of extraction in step 1) are each 1h, and the ratio of material to liquid is 1: 11-12; preferably, the feed-liquid ratio is 1: 11.85.
4. The cistanche salsa effective component extracted by the extraction method of claim 1, wherein the cistanche salsa effective component is one or more of echinacoside, verbascoside, isoverbascoside and tubular glycoside A obtained in step 4).
5. The cistanche deserticola effective part extracted by the extraction method of claim 1, wherein the effective part is the 25% ethanol elution part obtained in the step 3).
6. Use of the cistanche salsa effective component of claim 4 or the cistanche salsa effective fraction of claim 5 in the preparation of a preparation for preventing and/or treating neurodegenerative diseases.
7. Use of the cistanche salsa effective component of claim 4 or the cistanche salsa effective fraction of claim 5 in the preparation of a preparation for preventing and/or treating bone-related diseases.
8. A method for simultaneously and quantitatively detecting the effective parts and/or four effective component monomers of cistanche deserticola extracted by the extraction method of claim 1, which is characterized in that the method for simultaneously and quantitatively detecting is a high performance liquid chromatography,
the preparation method of the reference stock solution comprises the following steps: precisely weighing 1-2mg each of echinacoside, acteoside, isoacteoside and tubular flower glycoside A, respectively placing in 1mL volumetric flask, adding 50% methanol to dissolve and fix volume, and respectively making into four reference substance stock solutions;
the preparation method of the test solution comprises the following steps: weighing 5mg of the 25% ethanol elution part obtained in the step 3), precisely weighing, adding 50% methanol for dissolving, fixing the volume to 1mL, completely mixing, and storing at 4 ℃ for later use; and/or the presence of a gas in the gas,
respectively weighing 2mg of each of the four effective component monomers obtained in the step 4), precisely weighing, adding 50% methanol for dissolving, fixing the volume to 1mL, completely mixing, and storing at 4 ℃ for later use;
the quantitative method comprises the following steps: the quantitative method is an external standard method for quantification, four reference substance stock solutions are sequentially and precisely diluted into concentration gradients of 1/2, 1/4, 1/8, 1/16 and 1/32 with the original concentration, 10 mu L of the concentration gradients are respectively injected for analysis, the peak area is determined, the injection concentration (X) of the reference substance stock solutions is used as a horizontal coordinate, the corresponding peak area (Y) is used as a vertical coordinate, and a standard curve is drawn;
the chromatographic conditions were as follows:
a chromatographic column: a chromatographic column: AgilentZORBAXExtend-C18(250mm×4.6mm，1.8μm)；
Mobile phase: volume fraction 0.1% formic acid water (a) -acetonitrile (B);
column temperature: 30 ℃;
detection wavelength: 330 nm;
flow rate: 1 mL/min;
sample introduction amount: 10 mu L of the solution;
the elution procedure was:
9. the application of the phenylethanoid glycosides compounds in preparing preparations for preventing and/or treating neurodegenerative diseases is characterized in that the phenylethanoid glycosides compounds are one or a combination of more of echinacoside, verbascoside, isoverbascoside and tubular glycoside A.
10. The application of the phenylethanoid glycosides compounds in preparing preparations for preventing and/or treating bone related diseases is characterized in that the phenylethanoid glycosides compounds are one or a combination of more of echinacoside, verbascoside, isoverbascoside and tubular glycoside A.
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