Method for preparing ephedra root phenolic compounds from ephedra root and application thereof

Abstract

The application relates to a method for preparing ephedra root phenolic compounds from ephedra roots and the application of the ephedra root phenolic compounds, which comprises the following steps: taking dried ephedra roots and rhizomes as raw materials, adding water, decocting and extracting, concentrating to obtain an extract, dispersing the extract in water, washing and eluting, stirring, separating, identifying, loading on a chromatographic column, collecting flow fractions, concentrating and drying, obtaining ephedra root phenol K and ephedra root phenol M; heating and refluxing the ephedra roots to extract, concentrating to obtain an extract, dispersing the extract in water, separating, washing and eluting, identifying and combining, loading on a chromatographic column, collecting flow fractions, concentrating and drying, separating and purifying, obtaining ephedra root phenol I; further separating, loading on a chromatographic column, collecting flow fractions, concentrating and drying, collecting dark spot color bands, eluting, drying and obtaining ephedra root phenol J. The method is easy to operate, the product has high purity, has an anti-depression activity, can be effectively used for preparing medicines for treating depression, expands the medicinal value of the ephedra roots, and has huge economic and social benefits.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine, in particular, a method for preparing phenolic compounds from the roots of Ephedra sinica Stapf and the application thereof. BACKGROUND

[0002] Depression, as a mental illness, is the result of the combined effects of social, psychological and biological factors. Its clinical features mainly include emotional dysfunction, slow thinking, weakened will, impaired cognitive function and social dysfunction. It poses a serious threat to human life and quality of life. In the latest clinical studies, it was found that the severity of depression is closely related to the level of corticosterone. The plasma corticosterone level of patients with depression is generally high. For patients with major depressive disorder, anti-glucocorticoid therapy has shown significant therapeutic effect, and their plasma corticosterone levels have been reduced after treatment. Further studies have shown that the sustained increase in the level of corticosterone in plasma and cerebrospinal fluid may play a key role in the pathogenesis of depression. Therefore, finding natural products with potential antidepressant activity is an urgent technical problem for medical researchers.

[0003] The roots of Ephedra sinica Stapf, also known as bitter elm, are the dried roots and rhizomes of Ephedra sinica Stapf or Ephedra intermedia Schrenket C.A.Mey. of the Ephedraceae Dumortier family. In China, it is mainly distributed in Shanxi, Shaanxi, Sichuan and other places, and is a traditional Chinese medicine, which is recorded in the Chinese Pharmacopoeia. It was first mentioned in Shennong Bencao Jing, and has the effects of treating stroke, cold headache, warm malaria, sweating, removing evil heat, relieving cough and reversing qi, eliminating cold and heat, and breaking accumulation. With the rapid development of modern Chinese medicine and chemical technology, the research on Ephedra has become more and more comprehensive. However, the current research on Ephedra mainly focuses on the aboveground part of Ephedra, and the underground part of Ephedra is relatively less studied, and the literature records are relatively early. In order to further study the chemical components of the roots of Ephedra sinica Stapf, clarify the material basis of its efficacy, and promote the further development and utilization of the roots of Ephedra sinica Stapf, the chemical components thereof were isolated and identified, and two new glycosides and two new flavanols were isolated therefrom. The compounds were screened for activity by using a corticosterone-induced PC-12 cell damage model, which can significantly improve the corticosterone-induced PC-12 cell damage and exert potential antidepressant activity, and can be used in the preparation of drugs for treating depression. However, there has been no public report so far. SUMMARY

[0004] In view of the above, in order to overcome the deficiencies of the prior art, the purpose of the present application is to provide a method for preparing ephedra root phenolic compounds from ephedra root and its application, which can effectively solve the problem of preparing ephedra root phenolic compounds from ephedra root and realize the application in the preparation of drugs for treating depression.

[0005] The technical solution solved by the present application is: a method for preparing ephedra root phenolic compounds from ephedra root, the phenolic compounds are glycoside compounds ephedra root phenol I and ephedra root phenol J, flavanol compounds ephedra root phenol K and ephedra root phenol M, and their molecular structural formulas are respectively:

[0006]

[0007] Among them, ephedra root phenol I and ephedra root phenol J are a pair of the same molecular formula.

[0008] The preparation method is: taking the dried roots and rhizomes of ephedra vulgaris with impurities removed as raw materials, adding 15 times the amount of water based on the weight of the raw materials, wherein the weight of the solid is measured in kg and the volume of the liquid is measured in L, using decoction method to extract twice, each time for 2 hours, combining the two extraction solutions, and reducing pressure to concentrate to 0.100-0.102 times the weight of the raw materials, to obtain the first extract. The first extract is dispersed with water and passed through a Diaion HP-20 macroporous adsorption resin column, and then gradient eluted with water and ethanol to obtain the water part, the 10% ethanol part, the 20% ethanol part, the 30% ethanol part, the 40% ethanol part, the 50% ethanol part, the 70% ethanol part and the 95% ethanol part.

[0009] The 50% ethanol part is dissolved with methanol, then the sample:silica gel is 1:1.2, and then gradient eluted with dichloromethane-methanol system:50:1, 30:1, 20:1, 10:1, 5:1, 1:1, each elution part is 10 times the column volume, 2-3 days per gradient, and anisaldehyde-concentrated sulfuric acid spray is used for identification, every 50ml is identified once, and the same fractions are combined to obtain components Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6. The component Fr.3 is separated by silica gel column chromatography with a particle size of 100-200, gradient eluted with petroleum ether-ethyl acetate:10:1, 5:1, 3:1, TLC identification, and the same fractions are combined to obtain components Fr.3.1, Fr.3.2, Fr.3.3, Fr.3.4, Fr.3.5, Fr.3.6. The component Fr.3.4 is further separated by semi-preparative HPLC, and the specifications are:250×10mm, particle size 5μm, pore size 12nm, 5C 18-MS-II column, mobile phase: methanol: water = 54:46 (by volume), flow rate 3 ml / min, fractions with retention time t R = 16-18 min were collected, concentrated and dried to give compound ephedra root phenol K; fractions with retention time t R = 20-22 min were collected, concentrated and dried to give compound MHG-50-5-3-4-2; fractions with retention time t R = 35-37 min were collected, concentrated and dried to give compound MHG-50-5-3-4-3; compound MHG-50-5-3-4-2 was further separated by semi-preparative HPLC, column specifications: 250 x 10 mm, particle size 5 μm, pore size 12 nm, 5C 18 -MS-II column, mobile phase: acetonitrile: water = 27:73 (by volume), flow rate 3 ml / min, fractions with retention time t R = 40-42 min were collected, concentrated and dried to give compound ephedra root phenol M;

[0010] The ephedra root after water decoction extraction was extracted twice using 95% ethanol by volume by heating refluxing device, 1 kg of ephedra root was added to 4 L of ethanol each time, heated and refluxed for 2 h, the extraction liquid was combined and concentrated under reduced pressure to obtain a second extract with a weight volume of 1 / 4 of the raw material, which was dispersed and dissolved with water, then sequentially extracted with petroleum ether 5 times, each time using an amount of 0.7 times the weight volume of the extract, extracted with dichloromethane 10 times, each time using an amount of 1 times the weight volume of the extract, extracted with ethyl acetate 12 times, each time using an amount of 1 times the weight volume of the extract, and extracted with n-butanol 6 times, each time using an amount of 0.6 times the weight volume of the extract, the solvent was recovered to obtain petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, and n-butanol fraction;

[0011] The ethyl acetate fraction was subjected to silica gel column chromatography, gradient elution was performed using dichloromethane-methanol (50:1, 30:1, 20:1, 10:1, 5:1, 3:1) by volume ratio, 3-4 days per gradient, TLC detection and the same fraction was combined to obtain components E1, E2-E9. Component E9 was concentrated under reduced pressure, Toyopearl HW-40C gel column chromatography was used, gradient elution was performed using methanol with a volume concentration of 70%, 80%, and 85% in sequence, the amount of each elution fraction was 10 times the column volume, anisaldehyde-concentrated sulfuric acid spray detection was used, detection was performed every 10 ml, and the same fraction was combined to obtain components E9-1, E9-2, E9-3, E9-4-E9-8. Component E9-4 was concentrated under reduced pressure, Toyopearl HW-40C gel column chromatography was used, elution was performed using methanol with a volume concentration of 70%, the amount was 7 times the column volume, anisaldehyde-concentrated sulfuric acid spray detection was used, detection was performed every 5 ml, and the same fraction was combined to obtain component Er. Component Er was subjected to semi-preparative HPLC crude separation, a 5C 18 -MS-II column, the mobile phase was methanol:water = 42:58 by volume ratio, the flow rate was 3 ml / min, the fractions with retention times t R = 8-10 min were collected, concentrated and dried to obtain component Er-1. The fractions with retention times t R = 13-16 min were collected, concentrated and dried to obtain component Er-2. The fractions with retention times t R = 18-21 min were collected, concentrated and dried to obtain component Er-3. The fractions with retention times t R = 24-26 min were collected, concentrated and dried to obtain component Er-4. Er-3 was further separated and purified by semi-preparative HPLC, a 5C 18 -MS-II column, the mobile phase was methanol:water = 40:60 by volume ratio, the flow rate was 3 ml / min, the fractions with retention times t R = 17-19 min were collected, concentrated and dried to obtain compound Ephedra root phenol I. Er-2 was further separated and purified by semi-preparative HPLC, a 5C 18 -MS-II column, the mobile phase was methanol:water = 35:65 by volume ratio, the flow rate was 3 ml / min, the fractions with retention times t R = 28-30 min were collected, concentrated and dried to obtain component Er-2-3. Component Er-2-3 was separated by preparative thin layer chromatography, the developing agent was dichloromethane:methanol = 5:1 by volume ratio, the R fThe minimum dark spot color band, using a volume ratio of dichloromethane:methanol = 1:1 mixed solution 6ml elution, eluent concentration and drying, to obtain compound ephedra root phenol J.

[0012] The application of the glycoside compounds ephedra root phenol I and ephedra root phenol J, flavanol compounds ephedra root phenol K and ephedra root phenol M prepared by the method in the preparation of antidepressants.

[0013] The application has the advantages of rich raw materials, easy operation, good product quality, good extraction effect, high purity, the obtained compound has anti-depression activity, realizes the application in the preparation of drugs for treating depression, develops the medicinal value of ephedra root, and has great economic and social benefits. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The HR-ESI-MS spectrum (in MeOH) of ephedra root phenol I of the application is shown in the figure. 1 The H-NMR spectrum (in 500MHz, DMSO-d6) of ephedra root phenol I of the application is shown in the figure.

[0015] Figure 2 The C-NMR spectrum (in 125MHz, DMSO-d6) of ephedra root phenol I of the application is shown in the figure. 13 The C-NMR spectrum (in 125MHz, DMSO-d6) of ephedra root phenol I of the application is shown in the figure.

[0016] Figure 3 The HR-ESI-MS spectrum (in MeOH) of ephedra root phenol I of the application is shown in the figure.

[0017] Figure 4 The IR spectrum (in MeOH) of ephedra root phenol I of the application is shown in the figure.

[0018] Figure 5 The UV spectrum (in MeOH) of ephedra root phenol I of the application is shown in the figure.

[0019] Figure 6 The HR-ESI-MS spectrum (in MeOH) of ephedra root phenol J of the application is shown in the figure. 1 The H-NMR spectrum (in 500MHz, DMSO-d6) of ephedra root phenol J of the application is shown in the figure.

[0020] Figure 7 The C-NMR spectrum (in 125MHz, DMSO-d6) of ephedra root phenol J of the application is shown in the figure. 13 The C-NMR spectrum (in 125MHz, DMSO-d6) of ephedra root phenol J of the application is shown in the figure.

[0021] Figure 8 The HR-ESI-MS spectrum (in MeOH) of ephedra root phenol J of the application is shown in the figure.

[0022] Figure 9 The IR spectrum (in MeOH) of ephedra root phenol J of the application is shown in the figure.

[0023] Figure 10Figure for UV spectrum (in MeOH) of ephedrine phenol J of the present application.

[0024] Figure 11 Figure for UV spectrum (in MeOH) of ephedrine phenol K of the present application. 1 Figure for H-NMR spectrum (in 500MHz, CD3OD) of ephedrine phenol J of the present application.

[0025] Figure 12 Figure for H-NMR spectrum (in 500MHz, CD3OD) of ephedrine phenol K of the present application. 13 Figure for C-NMR spectrum (in 125MHz, CD3OD) of ephedrine phenol J of the present application.

[0026] Figure 13 Figure for HR-ESI-MS spectrum (in MeOH) of ephedrine phenol K of the present application.

[0027] Figure 14 Figure for UV spectrum (in MeOH) of ephedrine phenol K of the present application.

[0028] Figure 15 Figure for IR spectrum (in MeOH) of ephedrine phenol K of the present application.

[0029] Figure 16 Figure for UV spectrum (in MeOH) of ephedrine phenol M of the present application. 1 Figure for H-NMR spectrum (in 500MHz, CD3OD) of ephedrine phenol M of the present application.

[0030] Figure 17 Figure for H-NMR spectrum (in 500MHz, CD3OD) of ephedrine phenol M of the present application. 13 Figure for C-NMR spectrum (in 125MHz, CD3OD) of ephedrine phenol M of the present application.

[0031] Figure 18 Figure for HR-ESI-MS spectrum (in MeOH) of ephedrine phenol M of the present application.

[0032] Figure 19 Figure for UV spectrum (in MeOH) of ephedrine phenol M of the present application.

[0033] Figure 20 Figure for IR spectrum (in MeOH) of ephedrine phenol M of the present application.

[0034] Figure 21 Figure for molecular structure formula of ephedrine phenol I, ephedrine phenol J, ephedrine phenol K, ephedrine phenol M of the present application.

[0035] Figure 22 Figure for effect of compound 1 (ephedrine phenol I) and compound 2 (ephedrine phenol J) on cell survival rate in PC-12 cell damage model induced by corticosterone. n = 4. ## P < 0.01 vs CORT; *P < 0.05, **P < 0.01 vs CORT.

[0036] Figure 23 Effects of compound 3 (ephedradol K) and compound 4 (ephedradol M) of the present application on cell survival rate in a PC-12 cell damage model induced by corticosterone. n = 4. ## P<0.01 vs CORT; *P<0.05, **P<0.01 vs CORT graph. DETAILED DESCRIPTION

[0037] The specific embodiments of the present application will be described in detail below in conjunction with examples and specific cases.

[0038] The present application can be given in the following embodiments in specific implementation:

[0039] A preparation method of ephedradol compounds from ephedra roots, the ephedradol compounds being glycoside compounds ephedradol I and ephedradol J, flavanol compounds ephedradol K and ephedradol M, and the molecular formulae being: ephedradol I: C 20 H 20 O9, ephedradol J: C 20 H 20 O9, ephedradol K: C 22 H 18 O7, ephedradol M: C 24 H 18 O7, and the molecular structural formulae being respectively:

[0040]

[0041] The preparation method is: taking 40 kg of impurity-removed dry ephedra roots and rhizomes as raw materials, adding 600 L of water each time to decoct and extract 2 times, each time for 2 hours, combining the two extraction liquids, and reducing pressure to concentrate to obtain first extract 4.07 kg; the first extract is dispersed with water, and gradient elution is performed with Diaion HP-20 macroporous adsorption resin column, water and different volume concentrations of ethanol in sequence, to obtain water part 3.06 kg, volume concentration 10% ethanol part 191.82 g, volume concentration 20% ethanol part 180.05 g, volume concentration 30% ethanol part 120.12 g, volume concentration 40% ethanol part 147.45 g, volume concentration 50% ethanol part 97.80 g, volume concentration 70% ethanol part 53.40 g, and volume concentration 95% ethanol part 8.73 g;

[0042] 97.80 g of the 50% ethanol fraction was dissolved in methanol and subjected to silica gel column chromatography with a sample-to-silica gel ratio of 1:1.2 (v / v). The sample was then eluted using a dichloromethane-methanol system at ratios of 50:1, 30:1, 20:1, 10:1, 5:1, and 1:1 (v / v), with 3.5 L used for each elution fraction. Elutions were performed every 50 ml using anisaldehyde-sulfuric acid spray chromatography. Fractions of the same type were combined to obtain fractions Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, and Fr.6. Fraction Fr.3... 0.10 g was separated by silica gel column chromatography using a gradient elution of petroleum ether-ethyl acetate at ratios of 10:1, 5:1, and 3:1 (v / v), with 0.5 L of each gradient and 3-4 h per gradient. TLC detection was performed, and fractions of the same type were combined to obtain components Fr.3.1, Fr.3.2, Fr.3.3, Fr.3.4, Fr.3.5, and Fr.3.6. Component Fr.3.46 (2.03 mg) was further separated by semi-preparative HPLC. The sample was obtained using a 5C filter with a particle size of 250 × 10 mm, a particle size of 5 μm, and a pore size of 12 nm. 18 MS-II column, mobile phase: methanol:water = 54:46 (v / v), flow rate: 3 mL / min, collection retention time t R The fraction was collected for 16-18 min, concentrated and dried to give 3.06 mg of ephedrine K; the retention time was t. R The fraction was concentrated and dried over a period of 20-22 min to obtain compound MHG-50-5-3-4-2; the retention time was collected. R The fraction was concentrated and dried over 35-37 min to obtain compound MHG-50-5-3-4-3. Compound MHG-50-5-3-4-3 (62.03 mg) was further separated using semi-preparative HPLC. The sample was obtained from a 5C HPLC system with a particle size of 250 × 10 mm, a particle size of 5 μm, and a pore size of 12 nm. 18 MS-II column, mobile phase acetonitrile:water = 27:73 (v / v), flow rate 3 mL / min, collection retention time t R The fraction was concentrated and dried over a period of 40-42 minutes to obtain 1.61 mg of ephedrine M.

[0043] The roots of E. herba after water decocting extraction were extracted with 95% ethanol using 10 L heating reflux device for 2 times, 1 kg of the roots of E. herba and 4 L of ethanol were used for each time, and heating reflux was performed for 2 h. The extract was concentrated under reduced pressure to obtain 10.0 kg of total extract, which was dissolved by water dispersion, and was extracted with petroleum ether for 5 times, 7 L each time, with dichloromethane for 10 times, 10 L each time, with ethyl acetate for 12 times, 10 L each time, and with n-butanol for 6 times, 6 L each time. After recovery of the solvents, 211.6 g of petroleum ether part, 245.1 g of dichloromethane part, 335.5 g of ethyl acetate part, and 62.5 g of n-butanol part were obtained.

[0044] The ethyl acetate part 335.5 g was subjected to silica gel column chromatography separation, and gradient elution was performed using a dichloromethane-methanol system with a volume ratio of 50:1, 30:1, 20:1, 10:1, 5:1, and 3:1, 6.3 L for each gradient, 3-4 days for each gradient, TLC detection and combination of the same fractions to obtain components E1, E2-E9, and E9 50.0 g was concentrated under reduced pressure to 20 ml. Toyopearl HW-40C gel column chromatography was used, and 70%, 80%, and 85% methanol was used for elution in sequence, and the amount of each elution part was 10 times the column volume. Anisaldehyde-concentrated sulfuric acid spray detection was used, and detection was performed every 10 ml. The same fractions were combined to obtain components E9-1, E9-2, E9-3, E9-4-E9-8. Component E9-4 1.25 g was concentrated under reduced pressure to 20 ml, Toyopearl HW-40C gel column chromatography was used, and 70% methanol was used for elution, and the amount was 7 times the column volume. Anisaldehyde-concentrated sulfuric acid spray detection was used, and detection was performed every 5 ml. The same fractions were combined to obtain component Er. Component Er 223.08 mg was subjected to semi-preparative HPLC crude separation, and the column specification was 250*10 mm, the particle size was 5 μm, and the pore size was 12 nm. The mobile phase was a mixture of methanol and water with a volume ratio of 40:60, the flow rate was 3 ml / min, and the fractions with retention times t 18 =8-10 min were collected, concentrated and dried to obtain component Er-1. The fractions with retention times t R =13-16 min were collected, concentrated and dried to obtain component Er-2. The fractions with retention times t R =18-21 min were collected, concentrated and dried to obtain component Er-3. The fractions with retention times t R =24-26 min were collected, concentrated and dried to obtain component Er-4. Er-3 10.59 mg was further separated and purified by semi-preparative HPLC, and the column specification was 250*10 mm, the particle size was 5 μm, and the pore size was 12 nm. The mobile phase was a mixture of methanol and water with a volume ratio of 40:60, the flow rate was 3 ml / min, and the fractions with retention times t R =8-10 min were collected, concentrated and dried to obtain component Er-1. The fractions with retention times t 18 =13-16 min were collected, concentrated and dried to obtain component Er-2. The fractions with retention times t =18-21 min were collected, concentrated and dried to obtain component Er-3. The fractions with retention times t =24-26 min were collected, concentrated and dried to obtain component Er-4.R = 17-19 min, concentrated and dried to obtain compound ephedraol I 5.58 mg; component Er-2 28.47 mg was further separated by semi-preparative HPLC, the column was 250 x 10 mm, particle size 5 μm, pore size 12 nm, 5C 18 MS-II column, mobile phase was methanol: water = 35:65 by volume, flow rate 3 ml / min, collection of retention time t R = 28-30 min, concentrated and dried to obtain component Er-2-3, Er-2-3 10.01 mg was separated by preparative thin layer chromatography, developing agent was dichloromethane: methanol = 5:1 by volume, collection of R f The smallest dark spot color band, using dichloromethane: methanol = 1:1 by volume 6 ml elution 1 h, the eluent was concentrated and dried to obtain compound ephedraol J 3.52 mg.

[0045] The composition given in the above examples can be prepared in any amount of traditional Chinese medicine as needed, the examples given are only used to illustrate the specific embodiments of the present application, and are not used to limit the scope of protection of the present application, the technical core of the present application is the traditional Chinese medicine composition, which can be prepared into any amount of glycoside compounds ephedraol I and ephedraol J, flavanol compounds ephedraol K and ephedraol M according to actual needs.

[0046] The present application has abundant raw materials, easy-to-operate preparation method, high yield, good quality and high purity, the prepared glycoside compounds ephedraol I and ephedraol J, flavanol compounds ephedraol K and ephedraol M have anti-depression activity, can be effectively used for treating depression, and are applied in the preparation of drugs for treating depression, and very good beneficial technical effects have been achieved through experiments, and the relevant data are as follows:

[0047] I. Experimental instruments and reagents.

[0048] NMR was performed on a Bruker AVANCE III 500 NMR spectrometer (TMS internal standard) (Bruker), IR spectra were recorded on a Nicolet is 10 Microscope Spectrometer (Thermo Scientific, USA), high resolution mass spectra were recorded on a Bruker maxis HD mass spectrometer, UV spectra were recorded on a Shimadzu UV-2401PC apparatus, high performance liquid chromatography was performed on a Waters Alliance series 2695 high performance liquid system equipped with a 2998 diode array detector, Empower3 chromatographic data workstation, LC50 high pressure preparative liquid chromatograph, UV200 ultraviolet detector [Spectrum Labs (Beijing) Technology Co., Ltd.], YMC-Pack ODS-A chromatographic column (250 x 10 mm.D.S-5 mm, 12 mm) (YMC Co., Ltd.), and the rest were N-1100 rotary evaporator (Shanghai Ailang Instrument Co., Ltd.), A-1000S water flow air extractor (Shanghai Ailang Instrument Co., Ltd.), N-1111 refrigerated water circulating device (Shanghai Ailang Instrument Co., Ltd.), FDU-2110 freeze dryer (Shanghai Ailang Instrument Co., Ltd.), DFZ-60508 vacuum drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.), AB204-N one-thousandth precision analytical balance (METTLER TOLEDO), iMARK enzyme marker (BIO-RAD, USA), carbon dioxide incubator (Shanghai STIK), super clean bench (Sujing Group).

[0049] Rat adrenal pheochromocytoma cells PC-12 were purchased from the Shanghai Cell Library of the Chinese Academy of Sciences; Corticosterone (China Shanghai Aladdin Biochemical B2302817); Fluoxetine (USA Med Chem Express Company 251273); CCK8 (USA GLPBIO Company 43); 5-HT7 antibody (China Wuhan Sanyue Biological Technology Co., Ltd. 00058665); Tubulin antibody (GR3398636-5); DAPI staining agent (USA ABCAM Company GR3445296-5); Column chromatography filler Diaion HP-20, MCIGel CHP-20P (Japan Mitsubishi Chemical Corporation), silica gel H (100-200 mesh) used for column chromatography was produced by Qingdao Marine Chemical Plant, and chromatographically pure reagents were produced by Tianjin Sifang Fine Chemicals Co., Ltd., and analytically pure reagents were produced by Beijing Chemical Plant and Tianjin Third Chemical Reagent Factory.

[0050] Goat anti-rabbit IgG H&L (Alexa 488) pre-adsorbed secondary antibody (GR3449096-1); goat anti-mouse IgG H&L (Alexa 594)Pre-adsorbed secondary antibody (American ABCAM Company GR3413419-1); RPMI1640 medium (American Invitrogen Company 2315381); Fetal bovine serum (South American ExCell Bio Company 12A218);

[0051] The roots of E. sinica Stapf. used in this study were purchased from Bozhou Medicinal Material Market in May 2021 and identified by Professor Dong Chengming of the College of Pharmacy as the dried roots and rhizomes of Ephedra sinica Stapf. (Ephedraceae), and the plant specimen (NO. 20210517) was preserved in the Traditional Chinese Medicine Chemical Extraction and Separation Laboratory of Henan University of Chinese Medicine.

[0052] II. Structure identification.

[0053] Ephedrine I yellow oil (DMSO), HR-ESI-MS gave the quasi-molecular ion peak m / z: 427.0992 [M+Na] + (Calcd. For 427.0999), the molecular formula was determined as C 20 H 20 O9; UV (MeOH) λ max (logε): 208 (2.74), 249 (2.65), 311 (2.06) nm; IRν max : 3410, 1632, 1507, 1109, 1023 cm -1 ; 1 H NMR (DMSO-d6, 500MHz) and 13 C NMR (DMSO-d6, 125MHz) data are shown in Table 1.

[0054] Ephedrine J red amorphous powder (DMSO), HR-ESI-MS quasi-molecular ion peak m / z: 427.1006 [M+Na] + (Calcd. For 427.0999), the molecular formula was determined as C 20 H 20 O9; UV (MeOH) λ max (logε): 204 (2.98), 314 (3.20) nm; IRν max : 3302, 1615, 1506, 1264, 1079 cm -1 ; 1 H NMR (DMSO-d6, 500MHz) and 13 C NMR (DMSO-d6, 125MHz) data are shown in Table 1.

[0055] Mahuangrenphenol K yellow amorphous powder (MeOH), HR-ESI-MS quasi-molecular ion peak m / z: 417.0959 [M+H]+ (Calcd. For 417.0944), molecular formula is C 22 H 18 O7; UV (MeOH) λ max (log ε): 204 (4.58), 262 (4.12) nm; IR v max : 3389, 2923, 1705 cm -1 ; 1 H NMR (CD3OD, 500 MHz) and 13 C NMR (CD3OD, 125 MHz) data are shown in Table 2.

[0056] Mahuangrenphenol M yellow amorphous powder (MeOH), HR-ESI-MS quasi-molecular ion peak m / z: 441.0958 [M+Na] + (calcd for C 24 H 18 O7Na, 441.0944), suggesting that the molecular formula of the compound is C 24 H 18 O7; UV (MeOH) λ max (log ε): 202 (4.45), 359 (4.08) nm; IR v max : 3242, 2922, 1697 cm -1 ; 1 H NMR (CD3OD, 500 MHz) and 13 C NMR (CD3OD, 125 MHz) data are shown in Table 2.

[0057] Molecular structural formula of mahuangrenphenol I, mahuangrenphenol J, mahuangrenphenol K, mahuangrenphenol M are as follows:

[0058]

[0059] Table 1 NMR spectral data of mahuangrenphenol I-J

[0060]

[0061] Table 2 NMR spectral data of mahuangrenphenol K and M

[0062]

[0063] a Recorded in DMSO-d6, b Recorded in CD3OD

[0064] III. Activity detection.

[0065] PC-12 cells were placed in a 5% CO2, 37°C constant temperature incubator, while using RPMI1640 culture medium containing 10% fetal bovine serum to culture, and the best growth period of the cells was selected, 0.25% trypsin was used for digestion, and after the digestion was terminated, RPMI1640 containing 10% fetal bovine serum was configured into a cell concentration of 1×10 4 , 100 μL of cell suspension was inoculated in each well of a 96-well plate, and it was placed in a culture box. After the cells adhered, the culture medium was replaced with a serum-free culture medium. After 24 hours of starvation, the PC-12 cells were divided into four groups: a normal group NC (control, cultured in RPMI1640 medium), a model group CORT (corticosterone), a positive control group FXT (fluoxetine, 0.4 μmol·L -1 ), and a drug group (ephedra root phenol I, ephedra root phenol J, ephedra root phenol K, ephedra root phenol M, 2 μmol·L -1 ). Each group had 6 replicate wells, and after 24 hours of culture, new culture medium was replaced, 10 μL of CCK8 solution was added to each well, and after 1.5 hours of incubation at 37°C, the absorbance OD value was detected at 450 nm by an enzyme marker.

[0066] Cell survival rate = (drug group OD value - blank group OD value) / (normal control group OD value - blank group OD value) x 100%

[0067] The CCK8 method was used to detect the cell survival rate of the PC-12 cell damage model induced by corticosterone, and the results are shown in Tables 3 and 4. Compared with the normal control group, the cell viability of the model group decreased significantly (P<0.05); compared with the model group, ephedra root phenol I, ephedra root phenol J, ephedra root phenol K, and ephedra root phenol M can significantly improve the cell viability (P<0.01) and improve the damage of corticosterone to PC-12 cells.

[0068] Table 3 Effect of ephedra root phenol I and ephedra root phenol J on cell survival rate in PC-12 cell damage model induced by corticosterone n = 4.

[0069] Group ]]> ​ Dose (pmol L-1) ]]> ​ Cell survival rate (%) ]]> ​ Normal group ]]> ​ 0 ]]> ​ 124.25±21.02 ]]> ​ Model group ]]> ​ 800 ]]> ​ 60.39±7.73 ]]> ​ Positive control group ]]> ​ 0.4 ]]> ​ 82.92±6.81 ]]> ​ Mahuanggenkol I ]]> ​ 2 ]]> ​ 92.81±4.46 ]]> ​ Mahuanggenkol J ]]> ​ 2 ]]> ​ 87.69±7.53 ]]> ​

[0070] Table 4 Effect of ephedra root phenol K and ephedra root phenol M on cell survival rate in PC-12 cell damage model induced by corticosterone n = 4.

[0071] Group ]]> ​ Dose (pmol L -1 ) ]]> ​ Cell survival rate (%) ]]> ​ Normal group ]]> ​ 0 ]]> ​ 100±4.66 ]]> ​ Model group ]]> ​ 800 ]]> ​ 59.83±4.75 ]]> ​ Positive control group ]]> ​ 0.4 ]]> ​ 74.99±16.48 ]]> ​ Mahuanggenkol K ]]> ​ 2 ]]> ​ 115.14±8.37 ]]> ​ Mahuanggenkol M ]]> ​ 2 ]]> ​ ]]>

[0072] ​

[0072] From the above, it can be clearly seen that the present application has abundant raw materials, easy-to-operate preparation method, good product quality, good extraction effect, high yield, and high purity of up to 98.5%, the obtained compound has anti-depression activity, realizes the application in the preparation of drugs for treating depression, is an innovation in therapeutic drugs, has actual clinical application value, opens up a new use of Ephedrae Radix, and has huge economic and social benefits.

Claims

1. A phenolic compound prepared from the roots of Ephedra sinica Stapf, the phenolic compound being glycoside phenolic compound Ephedra sinica phenol I and Ephedra sinica phenol J, flavanol compound Ephedra sinica phenol K and Ephedra sinica phenol M, the molecular structural formulae of which being: Ephedra sinica phenol I and Ephedra sinica phenol J are a pair of compounds with the same molecular formula. ， wherein The dried roots and rhizomes of E. sinica free of impurities are taken as raw materials, 15 times the volume of water based on the weight of the raw materials is added, the weight of the solid is measured in kg and the volume of the liquid is measured in L, decoction extraction is used twice, each time for 2 hours, the two extraction solutions are combined, and concentrated under reduced pressure to 0.100-0.102 times the volume based on the weight of the raw materials to obtain a first extract, the first extract is dispersed with water, and passed through a Diaion HP-20 macroporous adsorption resin column, and gradient elution is performed with water and ethanol in sequence to obtain the water fraction, the 10% ethanol fraction, the 20% ethanol fraction, the 30% ethanol fraction, the 40% ethanol fraction, the 50% ethanol fraction, the 70% ethanol fraction and the 95% ethanol fraction; 2. The method of claim 1, wherein the ephedra root phenolic compound is prepared by the steps of: The roots of E. sinica free of impurities after water decoction extraction are extracted twice with 95% ethanol using a heating reflux device, 4 L of ethanol is added for each kg of the roots, and heating reflux is performed for 2 hours, the extraction solutions are combined, and concentrated under reduced pressure to obtain a second extract with a volume of 1 / 4 based on the weight of the raw materials, the second extract is dispersed and dissolved with water, and then extracted with petroleum ether 5 times, each time using 0.7 times the volume based on the weight of the extract, extracted with dichloromethane 10 times, each time using 1 times the volume based on the weight of the extract, extracted with ethyl acetate 12 times, each time using 1 times the volume based on the weight of the extract, and extracted with n-butanol 6 times, each time using 0.6 times the volume based on the weight of the extract, and the solvent is recovered to obtain the petroleum ether fraction, the dichloromethane fraction, the ethyl acetate fraction and the n-butanol fraction; ​ The 50% ethanol extract was dissolved in methanol, and then column chromatography was performed on silica gel with a sample: silica gel ratio of 1: 1.2, followed by gradient elution with a dichloromethane-methanol system (50:1, 30:1, 20:1, 10:1, 5:1, 1:1) at a volume ratio, and each elution fraction was 10 times the column volume, 2-3 days per gradient, and anisaldehyde-sulfuric acid spray detection was performed every 50 ml, and the same fractions were combined to obtain components Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, and Fr.

6. Component Fr.3 was separated by column chromatography on silica gel with a 100-200 mesh size, gradient elution with a petroleum ether-ethyl acetate system (10:1, 5:1, 3:1) at a volume ratio, TLC detection, and the same fractions were combined to obtain components Fr.3.1, Fr.3.2, Fr.3.3, Fr.3.4, Fr.3.5, and Fr.3.

6. Component Fr.3.4 was further separated by semi-preparative HPLC, and the column specifications were: 250x10 mm, 5C 18 -MS-II column, mobile phase: methanol: water = 54:46 at a volume ratio, flow rate: 3 ml / min, and fractions with retention times t R =16-18 min were collected, concentrated and dried to obtain compound Ephedra Root Phenol K; fractions with retention times t R =20-22 min were collected, concentrated and dried to obtain compound M HG-50-5-3-4-2; Retention time t R Fractions with retention time = 35-37 min were concentrated and dried to give compound MHG-50-5-3-4-3; compound MHG-50-5-3-4-2 was further separated by semi-preparative HPLC on a 5C18 18 -MS-II column, mobile phase: acetonitrile: water = 27:73 by volume, flow rate 3 ml / min, retention time t R Fractions with retention time = 40-42 min were concentrated and dried to give compound ephedrine M; 40 kg of the dried roots and rhizomes of E. sinica free of impurities are taken as raw materials, and extracted twice with 600 L of water each time for 2 hours, the two extraction solutions are combined, and concentrated under reduced pressure to obtain a first extract 4.07 kg, the first extract is dispersed with water, and passed through a Diaion HP-20 macroporous adsorption resin column, and gradient elution is performed with water and ethanol with different concentrations in sequence to obtain the water fraction 3.06 kg, the 10% ethanol fraction 191.82 g, the 20% ethanol fraction 180.05 g, the 30% ethanol fraction 120.12 g, the 40% ethanol fraction 147.45 g, the 50% ethanol fraction 97.80 g, the 70% ethanol fraction 53.40 g and the 95% ethanol fraction 8.73 g; The ethyl acetate fraction was subjected to silica gel column chromatography, gradient elution was performed using dichloromethane-methanol at a volume ratio of 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 3-4 days per gradient, TLC detection and the same fraction was combined to obtain components E1, E2-E9. Component E9 was concentrated under reduced pressure, Toyopearl HW-40C gel column chromatography was used, gradient elution was performed using methanol at a volume concentration of 70%, 80%, 85% in sequence, the amount of each elution fraction was 10 times the column volume, anisaldehyde-concentrated sulfuric acid spray detection was used, detection was performed every 10 ml, the same fraction was combined to obtain components E9-1, E9-2, E9-3, E9-4-E9-8; component E9-4 was concentrated under reduced pressure, Toyopearl HW-40C gel column chromatography was used, elution was performed using methanol at a volume concentration of 70%, the amount was 7 times the column volume, anisaldehyde-concentrated sulfuric acid spray detection was used, detection was performed every 5 ml, the same fraction was combined to obtain component Er; component Er was subjected to semi-preparative HPLC crude separation, a 5C 18 -MS-II column, the mobile phase was methanol:water = 42:58 by volume, the flow rate was 3 ml / min, the fractions with retention times t R =8-10 min were collected, concentrated and dried to obtain component Er-1; the fractions with retention times t R =13-16 min were collected, concentrated and dried to obtain component Er-2; the fractions with retention times t R =18-21 min were collected, concentrated and dried to obtain component Er-3; the fractions with retention times t R =24-26 min were collected, concentrated and dried to obtain component Er-4; Er-3 was further separated and purified by semi-preparative HPLC, a 5C 18 -MS-II column, the mobile phase was methanol:water = 40:60 by volume, the flow rate was 3 ml / min, the fractions with retention times t R =17-19 min were collected, concentrated and dried to obtain compound Ephedra root phenol I; Er-2 was further separated and purified by semi-preparative HPLC, a 5C 18 -MS-II column, the mobile phase was methanol:water = 35:65 by volume, the flow rate was 3 ml / min, the fractions with retention times t t R =28-30 min were collected, concentrated and dried to obtain component Er-2-3; component Er-2-3 was separated by preparative thin layer chromatography, the developing agent was dichloromethane:methanol = 5:1 by volume, the fractions with retention times t R f The minimum dark spot color band, using a volume ratio of dichloromethane: methanol = 1:1 mixture of 6 ml elution, eluent concentrated and dried to obtain compound ephedra root phenol J.

3. The method for preparing ephedrine compounds according to claim 2, characterized in that, ​ 50% ethanol fraction 97.80 g was dissolved in methanol, and column chromatography was performed using silica gel with sample: silica gel = 1: 1.2 by volume ratio, followed by gradient elution with dichloromethane-methanol system: 50:1, 30:1, 20:1, 10:1, 5:1, 1:1 by volume ratio, and each elution fraction was 3.5 L, 2-3 days per gradient, and anisaldehyde-concentrated sulfuric acid spray detection was performed every 50 ml, and the same fractions were combined to obtain components Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.

6. Component Fr.3 0.10 g was separated by silica gel column chromatography with petroleum ether-ethyl acetate = 10:1, 5:1, 3:1 by volume ratio gradient elution, and each gradient was 0.5 L, 3-4 h per gradient, and TLC detection was performed, and the same fractions were combined to obtain components Fr.3.1, Fr.3.2, Fr.3.3, Fr.3.4, Fr.3.5, Fr.3.

6. Component Fr.3.4 62.03 mg was further separated by semi-preparative HPLC with a column specification of 250x10 mm, particle size 5 μm, and pore size 12 nm, 5C 18 -MS-Ⅱ chromatographic column, and the mobile phase was methanol: water = 54:46 by volume ratio, and the flow rate was 3 ml / min, and the fractions with retention times t R =16-18 min were collected, concentrated and dried to obtain compound ephedra root phenol K 3.06 mg; the fractions with retention times t R =20-22 min were collected, concentrated and dried to obtain compound M HG-50-5-3-4-2; Retention time t R Fractions with retention time = 35-37 min were concentrated and dried to give compound MHG-50-5-3-4-3-4; compound MHG-50-5-3-4-3-4 62.03 mg was further separated by semi-preparative HPLC with column size: 250 x 10 mm, particle size 5 μm, pore size 12 nm, 5C 18 - MS-II column, mobile phase: acetonitrile: water = 27:73 by volume, flow rate 3 ml / min, retention time t R Fractions with retention time = 40-42 min were concentrated and dried to give compound ephedrine M 1.61 mg; The grass ephedra root after decocting extraction again uses 95% ethanol to use 10L's heating reflux device to extract 2 times, every time 1 kg ephedra root adds 4L ethanol, heating reflux 2h, and the extraction liquid is concentrated under reduced pressure to obtain total extract 10.0 kg, is dissolved with water dispersion, is extracted with petroleum ether 5 times in turn, every time 7L, dichloromethane is extracted 10 times, every time 10L, ethyl acetate is extracted 12 times, every time 10L, n-butanol is extracted 6 times, every time 6L, after recovering solvent, obtain petroleum ether part 211.6g, dichloromethane part 245.1g, ethyl acetate part 335.5g, n-butanol part 62.5g; The ethyl acetate fraction 335.5 g was subjected to silica gel column chromatography, gradient elution was performed using dichloromethane-methanol system with volume ratio: 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 6.3 L for each gradient, 3-4 days for each gradient, TLC detection and the same fractions were combined to obtain components E1, E2-E9, E9 50.0 g was concentrated to 20 ml under reduced pressure, Toyopearl HW-40C gel column chromatography was used, 70%, 80%, 85% methanol was used for elution in turn, the amount of each elution part was 10 times the column volume, anisaldehyde-concentrated sulfuric acid spray detection was used, detection was performed every 10 ml, the same fractions were combined to obtain components E9-1, E9-2, E9-3, E9-4-E9-8, component E9-4 1.25 g was concentrated to 20 ml under reduced pressure, Toyopearl HW-40C gel column chromatography was used, 70% methanol was used for elution, the amount was 7 times the column volume, anisaldehyde-concentrated sulfuric acid spray detection was used, detection was performed every 5 ml, the same fractions were combined to obtain component Er; component Er 223.08 mg was subjected to semi-preparative HPLC crude separation, the column specifications were: 250x10 mm, particle size 5 μm, pore size 12 nm, 5C 18 -MS-II column, the mobile phase was methanol:water = 42:58 by volume, the flow rate was 3 ml / min, the fractions with retention time t R =8-10 min were collected, concentrated and dried to obtain component Er-1, the fractions with retention time t R =13-16 min were collected, concentrated and dried to obtain component Er-2, the fractions with retention time t R =18-21 min were collected, concentrated and dried to obtain component Er-3, the fractions with retention time t R =24-26 min were collected, concentrated and dried to obtain component Er-4; Er-3 10.59 mg was further separated and purified by semi-preparative HPLC, the column specifications were: 250x10 mm, particle size 5 μm, pore size 12 nm, 5C 18 -MS-II column, the mobile phase was methanol:water = 40:60 by volume, the flow rate was 3 ml / min, the fractions with retention time t R =17-19 min were collected, concentrated and dried to obtain compound Ephedra root phenol I 5.58 mg; component Er-2 28.47 mg was further separated by semi-preparative HPLC, the column specifications were: 250x10 mm, particle size 5 μm, pore size 12 nm, 5C 18 -MS-II column, the mobile phase was methanol:water = 35:65 by volume, the flow rate was 3 ml / min, the fractions with retention time t R Fractions 28-30 min, concentrated and dried to give component Er-2-3, 10.01 mg of which was separated by preparative thin layer chromatography, developed with dichloromethane:methanol = 5:1 by volume, and the fractions collected R f The least dark band, eluted with 6 ml of a mixture of dichloromethane:methanol = 1:1 by volume for 1 h, and the eluate concentrated and dried to give compound Ephedrae Radix Phenol J 3.52 mg.

4. The use of the glycosides of ephedra root phenol I and ephedra root phenol J, flavanol compounds of ephedra root phenol K and ephedra root phenol M prepared by the method of claim 2 or 3 in the preparation of antidepressant drugs.

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