A lignan extracted from the root of ephedra and its preparation method and application

Abstract

A lignan extracted from the root of Ephedra sinica Stapf and a preparation method and application thereof: the root of Ephedra sinica Stapf extracted by water decoction is extracted by ethanol reflux, concentrated to extract, dissolved by water dispersion, and then extracted by petroleum ether, dichloromethane, ethyl acetate and n-butanol to obtain corresponding parts; the dichloromethane part is separated by silica gel column chromatography, gradient eluted by petroleum ether-ethyl acetate and dichloromethane-methanol, chromatographically separated, gradient eluted by petroleum ether-ethyl acetate and dichloromethane, chromatographically separated, the retention time fractions are collected, concentrated and dried to obtain Ephedra sinica root phenol E; the ethyl acetate part is chromatographically separated, gradient eluted, reduced pressure concentrated, spray identified, the same fractions are combined, chromatographically separated, the retention time fractions are collected, concentrated and dried to obtain Ephedra sinica root phenol G; the method is easy to operate, the product has high purity, has an antidepressant activity, is effectively used for preparing a drug for treating depression, develops the medicinal value of the root of Ephedra sinica Stapf, and has huge economic and social benefits.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine, in particular to a lignan extracted from the roots of Ephedra sinica Stapf for treating depression and a preparation method and application thereof. BACKGROUND

[0002] Depression is a mental illness characterized by emotional dysfunction, slow thinking, decreased willpower, impaired cognitive function and social dysfunction. According to the WHO survey, depression is widespread worldwide. Only in China, the number of people with depression accounts for 4.2% of the total population. The survey shows that the proportion of female patients with depression is higher than that of male patients, and the proportion of the unemployed is higher than that of the employed. The main group of patients is adults. Studies have found that the sustained increase in the level of corticosterone in the plasma and cerebrospinal fluid may play an important role in the occurrence of depression. Therefore, it is an urgent technical problem for pharmaceutical researchers to screen for potential natural products with antidepressant activity by using a PC-12 cell damage model induced by corticosterone.

[0003] The root of Ephedra sinica Stapf, also known as bitter elm, is the dried root and rhizome of Ephedra sinica Stapf or Ephedra intermedia Schrenket C.A.Mey. of the Ephedraceae Dumortier family, which is recorded in the Chinese Pharmacopoeia. The modern pharmacological activities of the root of Ephedra sinica Stapf mainly include antihypertensive, antioxidant, anti-inflammatory and antitumor effects. The main chemical components are alkaloids, flavonoids and lignans. In China, Ephedra sinica Stapf is mainly produced in Hebei, Inner Mongolia, Sichuan and Shanxi. Due to the lack of chemical research on the root of Ephedra sinica Stapf, the activity research is relatively weak, which seriously hinders the development and utilization of the root of Ephedra sinica Stapf. Therefore, the chemical components of the root of Ephedra sinica Stapf were systematically extracted, separated and identified, and new lignan compounds were isolated. The activity of the compounds was screened by using a PC-12 cell damage model induced by corticosterone, which can significantly improve the PC-12 cell damage induced by corticosterone and has potential antidepressant activity, and can be used for preparing drugs for treating depression. However, there is no public report so far. SUMMARY

[0004] In view of the above problems, the present application aims to provide a lignan extracted from the roots of Ephedra sinica Stapf and a preparation method and application thereof, which can effectively solve the problem of developing drugs for treating depression.

[0005] The technical solution provided by the present invention is: a lignan extracted from the root of Ephedra, wherein the lignan is Ephedra root phenol E of the monoepoxy lignan class and Ephedra root phenol G of the benzofuran type lignan, and the molecular formulas thereof are: Ephedra root phenol E is C 24 H 28 O6; Ephedrine G is C 19 H 22 O8; molecular structures are:

[0006]

[0007] The preparation method is as follows: take the dried roots and rhizomes of Ephedra sinica, add 15 times the weight volume of water, extract twice by water decoction, each time for 2 hours, the weight volume refers to the solid in kg and the liquid in L, the Ephedra sinica root after water decoction extraction is then extracted twice by heating and reflux device with 95% volume concentration ethanol (abbreviated as ethanol, the same below), each time 1 kg of Ephedra sinica root is added with 4L ethanol, heated and refluxed for 2 hours, the extract is concentrated under reduced pressure to obtain an extract, the extract is dispersed and dissolved in water, and then extracted with petroleum ether 5 times in sequence, each time the amount is 0.7 times the weight volume of the extract, extracted with dichloromethane 10 times, each time the amount is 1 times the weight volume of the extract, extracted with ethyl acetate 12 times, each time the amount is 1 times the weight volume of the extract, extracted with n-butanol 6 times, each time the amount is 0.6 times the weight volume of the extract, and the solvent is recovered to obtain a petroleum ether fraction, a dichloromethane fraction, and a 10 times the weight volume of the extract. The dichloromethane fraction was separated by 100-200 mesh silica gel column chromatography to obtain the sample silica gel, which was then gradient eluted with petroleum ether-ethyl acetate in a volume ratio of 10:1, 5:1, 3:1, 1:1 and dichloromethane-methanol 20:1 to obtain the corresponding fractions M1, M2, M3, M4 and M5; the 3:1 fraction M3 was separated by 200-300 mesh silica gel column chromatography, and then gradient eluted with petroleum ether-ethyl acetate in a volume ratio of 30:1, 20:1, 10:1, 5:1 and dichloromethane to obtain the corresponding fractions M3-1, M3-2, M3-3, M3-4 and M3-5; the fraction M3-3 was separated by semi-preparative HPLC using a 5C HPLC with a specification of 250×10 mm, a particle size of 5 μm and a pore size of 12 nm. 18 -MS-Ⅱ chromatographic column, the mobile phase is a mixture of 0.45% triethylamine in water and methanol, the volume ratio of water and methanol is 15:84, the flow rate is 3ml / min, and the retention time t R = The fraction from 23 to 25 min was concentrated and dried to obtain ephedrine E;

[0008] The ethyl acetate part is separated by silica gel column chromatography with silica gel of 100-200 mesh, and gradient elution is performed with dichloromethane-methanol of 50:1, 30:1, 20:1, 10:1, 5:1 and 3:1 by volume ratio to obtain corresponding fractions E1, E2, E3-E9. Fraction E9 is separated by silica gel column chromatography with silica gel of 200-300 mesh, and gradient elution is performed with petroleum ether-ethyl acetate of 20:1, 10:1, 5:1, 5:1, 1:1 and 2:3 by volume ratio to obtain corresponding fractions E9-1, E9-2-E9-5. Fraction E9-2 is concentrated under reduced pressure, and Toyopearl HW-40C gel column chromatography is performed with methanol of 50% and 70% by volume concentration to obtain fractions E9-2-1, E9-2-2, E9-2-3, E-9-2-4, wherein the elution amount of each elution part is 10 times of the column volume, and anisaldehyde-concentrated sulfuric acid spraying detection is performed every 5 ml. The same fractions are combined to obtain fractions E9-2-3-1, E9-2-3-2, E-9-2-3-3. Fraction E-9-2-3-2 is separated by semi-preparative HPLC, and the column specification is 250*10mm, the particle size is 5um, and the pore size is 12nm. The mobile phase is methanol-water of 15:85 by volume ratio, the flow rate is 3ml / min, the fractions with retention time t 18 =26-28min are collected, concentrated and dried to obtain compound ephedra root phenol G. R =26-28min are collected, concentrated and dried to obtain compound ephedra root phenol G.

[0009] The lignans prepared by the method have anti-depression activity, and can be effectively used for preparing a medicine for treating depression, and the application in preparing the medicine for treating depression is realized.

[0010] The raw material is rich, the preparation method is easy to operate, the product quality is good, the extraction effect is good, the purity is high, the obtained compound has anti-depression activity, the application in preparing the medicine for treating depression is realized, the medicinal value of ephedra root is developed, and great economic and social benefits are obtained. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 The ephedra root phenol E of the application is 1 H-NMR spectrum (in 500MHz, CDCl3) chart.

[0012] Figure 2 The ephedra root phenol E of the application is 13 C-NMR spectrum (in 125MHz, CDCl3) chart.

[0013] Figure 3 The figure is the HR-ESI-MS spectrum (in MeOH) of ephedra root phenol E of the present invention.

[0014] Figure 4 The IR spectrum (in MeOH) of ephedrine E of the present invention is shown in FIG.

[0015] Figure 5 The UV spectrum (in MeOH) of ephedrine E of the present invention is shown.

[0016] Figure 6 For the ephedrine G of the present invention 1 H-NMR spectrum (in 500 MHz, CD3OD) diagram.

[0017] Figure 7 For the ephedrine G of the present invention 13 C-NMR spectrum (in 125 MHz, CD3OD) diagram.

[0018] Figure 8 The figure is the HR-ESI-MS spectrum (in MeOH) of ephedrine G of the present invention.

[0019] Figure 9 The IR spectrum (in MeOH) of ephedrine G of the present invention is shown.

[0020] Figure 10 The UV spectrum (in MeOH) of ephedrine G of the present invention is shown.

[0021] Figure 11 The diagram shows the molecular structural formula of ephedrine E and ephedrine G of the present invention.

[0022] Figure 12 1H-1H COSY correlation and HMBC correlation diagram of ephedrine E and G.

[0023] Figure 13 The effect of ephedra root phenol E (1) and G (2) on the cell survival rate in the corticosterone-induced PC-12 cell injury model ( n=4. ## P<0.01vs CORT; *P<0.05, **P<0.01vs CORT). DETAILED DESCRIPTION

[0024] The specific implementation methods of the present invention are described in detail below with reference to examples and specific situations.

[0025] The present invention can be specifically implemented by the following examples:

[0026] The invention is a lignan extracted from the roots of Ephedra sinica Stapf, which is a monocyclic epoxy lignan, Ephedra sinica root phenol E, and a benzofuran-type lignan, Ephedra sinica root phenol G, with molecular formulas of C 24 H 28 O6; and C 19 H 22 O8, respectively, and molecular structural formulas of:

[0027]

[0028] The preparation method is as follows:

[0029] Take 40 kg of dried roots and rhizomes of Ephedra sinica Stapf, and extract twice using 600 L of water by decoction, 2 hours each time. The roots of Ephedra sinica Stapf after water decoction extraction are extracted twice using 95% ethanol by heating reflux device, 4 L of ethanol per kg of Ephedra sinica Stapf roots, heating reflux for 2 hours each time. The extract is concentrated under reduced pressure to obtain 10.0 kg of extract, which is dissolved by dispersion with water, and then extracted with petroleum ether 5 times, 7 L each time, with dichloromethane 10 times, 10 L each time, with ethyl acetate 12 times, 10 L each time, and with n-butanol 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 are obtained. The dichloromethane part is separated by silica gel column chromatography with 2000 g of silica gel of 100-200 mesh, to obtain 270 g of mixed silica gel, which is eluted by gradient elution with petroleum ether-ethyl acetate in a volume ratio of 10:1, 5:1, 3:1, 1:1, and dichloromethane-methanol in a volume ratio of 20:1, to obtain corresponding fractions M1, M2, M3-M5. The fraction M3 is separated by silica gel column chromatography with 200-300 mesh, and eluted by gradient elution with petroleum ether-ethyl acetate in a volume ratio of 30:1, 20:1, 10:1, 5:1, and dichloromethane, to obtain corresponding fractions M3-1, M3-2, M3-3-M3-5. The fraction M3-3 is separated by semi-preparative HPLC with a column of 250x10 mm, particle size of 5 μm, and pore size of 12 nm, to obtain 6.12 mg of Ephedra sinica root phenol E. 18 -MS-Ⅱ column, mobile phase is a mixture of 0.45% triethylamine aqueous solution and methanol, volume ratio of the aqueous solution to methanol is 15:84, flow rate is 3 ml / min, fractions with retention time t R =23-25 min are collected, concentrated and dried to obtain Ephedra sinica root phenol E (6.12 mg);

[0030] The ethyl acetate part 335.5 g was separated by silica gel column chromatography with 100-200 mesh silica gel 6000 g, and 500 g of silica gel was obtained, and gradient elution was carried out with dichloromethane-methanol in a volume ratio of 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, to obtain corresponding fractions E1-E9, and fraction E9 was separated by 200-300 mesh silica gel column chromatography, and gradient elution was carried out with petroleum ether-ethyl acetate in a volume ratio of 20:1, 10:1, 5:1, 5:1, 1:1 and 2:3, to obtain corresponding fractions E9-1, E9-2-E9-5, fraction E9-2 was concentrated under reduced pressure, Toyopearl HW-40C gel column chromatography was used, and elution was carried out with methanol in a volume concentration of 50%, 70% in turn, each elution part was 10 times the column volume, anisaldehyde-concentrated sulfuric acid spray detection was used, and detection was carried out every 5 ml, and the same fractions were combined to obtain components E9-2-1, E9-2-2, E-9-2-4, component E-9-2-3 was concentrated to 15 mL under reduced pressure, Sephadex LH-20 gel column chromatography was used, elution was carried out with 70% methanol, anisaldehyde-concentrated sulfuric acid spray detection was used, and detection was carried out every 3 ml, and the same fractions were combined to obtain components E9-2-3-1, E9-2-3-2, E-9-2-3-3, component E-9-2-3-2 was separated by semi-preparative HPLC, and the specifications of the column were: 250*10 mm, particle size 5 μm, pore size 12 nm, 5C 18 -MS-Ⅱ column, the mobile phase was methanol:water=15:85 by volume, the flow rate was 3 ml / min, the fractions with retention time t R =26-28 min were collected, concentrated and dried to obtain compound ephedra root phenol G (3.86 mg).

[0031] According to the composition given in the above examples, any amount of traditional Chinese medicine can be prepared as needed, and the examples given are only used to illustrate the specific embodiments of the present application, but not to limit the protection scope of the present application, and the technical core protected by the present application is the traditional Chinese medicine composition, which can also be prepared into any amount of ephedra root phenol E and ephedra root phenol G as needed.

[0032] The present application has the advantages of rich raw materials, easy operation, high yield, good quality, high purity, and the prepared ephedra root phenol E and ephedra root phenol G have anti-depression activity, can be effectively used for treating depression, and can be applied to 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:

[0033] I. Experimental instruments and reagents.

[0034] 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).

[0035] 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.

[0036] 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);

[0037] The grass ephedra root selected in the present subject was purchased from Bozhou medicinal material market in May 2021, and was identified by Professor Dong Chengming of the College of Pharmacy as the dried root and rhizome of Ephedra sinica Stapf. of the Ephedraceae family, and the plant specimen (NO. 20210517) was preserved in the Traditional Chinese Medicine Chemical Extraction and Separation Laboratory of Henan University of Chinese Medicine.

[0038] II. Structure identification.

[0039] The ephedra root phenol E is white amorphous powder (CH2Cl2), and the purity is (98.2%); HR-ESI-MS gives a quasi-molecular ion peak m / z: 435.1795 [M+Na] + (Calcd. 435.1778), and the molecular formula is determined as C 24 H 28 O6; UV (MeOH) λ max (logε): 202 (2.47), 233 (1.63), 386 (1.55) nm; IR v max : 3404, 2935, 1490, 1443, 1248 cm -1 ; 1 HNMR (CDCl3, 500MHz) and 13 C NMR (CDCl3, 125MHz) data are shown in Table 1;

[0040] The ephedra root phenol G is white amorphous powder (CD3OD), (99.1%); HR-ESI-MS quasi-molecular ion peak m / z: 401.1197 [M+Na] +( Calcd. 401.1206), and the molecular formula is determined as C 19 H 22 O8; UV (MeOH) λ max (logε): 208 (2.68), 281 (1.40) nm; IR v max : 3400, 2938, 1277, 1029 cm -1 ; 1 H NMR (CD3OD, 500MHz) and 13 C NMR (CD3OD, 125MHz) data are shown in Table 1, and the 1H-1H COSY correlation and HMBC correlation of ephedra root phenol E and G are shown in Figure 12The molecular structures of ephedrine E and ephedrine G are as follows:

[0041]

[0042] Table 1 NMR spectrum data of ephedra root phenol E and G

[0043]

[0044] a Recorded in CDCl3,b Recorded in CD3OD

[0045] 3. Activity detection

[0046] PC-12 cells were placed in a 5% CO2, 37°C constant temperature incubator and cultured in RPMI1640 medium containing 10% fetal bovine serum. Cells in the best growth phase were selected and digested with 0.25% trypsin. After digestion was terminated, the cells were prepared with RPMI1640 medium containing 10% fetal bovine serum to a cell concentration of 1×10 4 , seeded on a 96-well plate, 100 μL of cell suspension per well, placed in an incubator, and after the cells adhered, the culture medium was replaced with serum-free medium. After starvation for 24 h, PC-12 cells were divided into 4 groups: normal group NC (control, cultured in RPMI1640 medium), model group CORT (model, corticosterone), positive control group FXT (fluoxetine, 0.4 μmol·L -1 ), drug-treated group (1-2, 2 μmol·L -1 , i.e., ephedrin E and ephedrin G of the present invention). Each group had 6 replicate wells. After culturing for 24 h, new culture medium was replaced, and 10 μL of CCK8 solution was added to each well. After incubation at 37° C. for 1.5 h, the absorbance OD value was measured at 450 nm using a microplate reader.

[0047] Cell survival rate = (OD value of drug administration group - OD value of blank group) / (OD value of normal control group - OD value of blank group) × 100%.

[0048] The CCK8 method was used to detect the cell survival rate of ephedra root phenol E and ephedra root phenol G in the corticosterone-induced PC-12 cell injury model. Figure 13 As shown in Table 2, compared with the normal control group, the cell viability of the model group was significantly decreased (P<0.05); compared with the model group, ephedra root phenol E and ephedra root phenol G could significantly improve cell viability (P<0.01) and improve the damage of PC-12 cells induced by corticosterone, indicating that ephedra root phenol E and ephedra root phenol G have good potential antidepressant activity.

[0049] Table 2 Effect of compounds on cell survival rate in the PC-12 cell damage model induced by corticosterone

[0050] Group Dose (pmol L -1 ) Cell survival rate (%) Normal group (NC) 0 100.00±4.66 Model group (CORT) 800 59.83±4.75 Positive control group (FXT) 0.4 76.40±13.17 Dosing group 1 (ephedra root phenol E) 2 130.51±21.45 Dosing group 2 (ephedra root phenol G) 2 129.34±17.03

[0051] From the above, it can be clearly seen that the raw material of the present application is abundant, the preparation method is easy to operate, the product quality is good, the extraction effect is good, the yield is high, the purity is as high as 98.5%, the obtained compound has anti-depression activity, realizes the application in the preparation of drugs for treating depression, is an innovation in treatment drugs, has actual clinical application value, opens up a new use of Ephedrae Radix, and has huge economic and social benefits.

Claims

1. A method for preparing lignans extracted from Ephedra root, characterized in that: The lignans are ephedrine E of the monoepoxy lignan class and ephedrine G of the benzofuran-type lignan class, and their molecular structural formulas are: ； The preparation method comprises the following steps: taking dried roots and rhizomes of Ephedra sinica from which impurities have been removed as raw materials, adding water 15 times the weight volume of the raw materials, extracting twice by water decoction, each time for 2 hours, where the weight volume refers to the solid in kg and the liquid in L, extracting the Ephedra sinica roots extracted by water decoction twice by using a 95% volume concentration ethanol heating reflux device, adding 4 L of ethanol to 1 kg of the roots each time, heating and reflux for 2 hours, concentrating the extract under reduced pressure to obtain 10 kg of extract, dispersing and dissolving the extract in water, extracting five times by using petroleum ether, each time using 0.7 times the weight volume of the extract, extracting ten times by using dichloromethane, each time using 1 times the weight volume of the extract, extracting twelve times by using ethyl acetate, each time using 1 times the weight volume of the extract, extracting six times by using n-butanol, each time using 0.6 times the weight volume of the first extract, and recovering the solvent. The petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, and n-butanol fraction were obtained; the dichloromethane fraction was separated by 100-200 mesh silica gel column chromatography to obtain sample-mixed silica gel, which was then gradient eluted with petroleum ether-ethyl acetate in a volume ratio of 10:1, 5:1, 3:1, 1:1, and dichloromethane-methanol 20:1, to obtain corresponding fractions M1, M2, M3, M4, and M5; the 3:1 fraction M3 was separated by 200-300 mesh silica gel column chromatography, and then gradient eluted with petroleum ether-ethyl acetate in a volume ratio of 30:1, 20:1, 10:1, 5:1, and dichloromethane to obtain corresponding fractions M3-1, M3-2, M3-3, M3-4, and M3-5; fraction M3-3 was separated by semi-preparative HPLC using a 5C HPLC with a specification of 250×10 mm, a particle size of 5 μm, and a pore size of 12 nm. 18 -MS-Ⅱ chromatographic column, the mobile phase is a mixture of 0.45% triethylamine in water and methanol, the volume ratio of water to methanol is 15:84, the flow rate is 3ml / min, and the retention time t R = 23-25 ​​min fraction, concentrated and dried to obtain ephedrine E; The ethyl acetate fraction was separated by silica gel chromatography on a 100-200 mesh silica gel column to obtain the sample silica gel, which was then eluted with a gradient of dichloromethane-methanol in a volume ratio of 50:1, 30:1, 20:1, 10:1, 5:1, and 3:1 to obtain the corresponding fractions E1, E2, E3-E9. Fraction E9 was separated by silica gel chromatography on a 200-300 mesh silica gel column and eluted with a gradient of petroleum ether-ethyl acetate in a volume ratio of 20:1, 10:1, 5:1, 1:1, and 2:3 to obtain the corresponding fractions E9-1, E9-2-E9-5. Fraction E9-2 was concentrated under reduced pressure and eluted using a Toyopearl HW-40C gel column chromatography with 50% and 70% methanol, respectively. The amount used for each elution fraction was 10 times the column volume. Anisaldehyde-concentrated sulfuric acid spray was used for identification. The fractions were separated by HPLC. The fractions were eluted with 70% methanol and sprayed with anisaldehyde-concentrated sulfuric acid. The fractions were separated by HPLC. The fractions were separated by HPLC. The fractions were separated by HPLC. The fractions were eluted with 70% methanol and sprayed with anisaldehyde-concentrated sulfuric acid. The fractions were eluted with 3 ml of the mixture. The fractions were fused to obtain the components E9-2-1, E9-2-2, and E9-2-3. The fraction E-9-2-3-2 was separated by HPLC. The size of the column was 250×10 mm, particle size 5 μm, pore size 12 nm. 18 -MS-Ⅱ chromatographic column, mobile phase is methanol: water = 15:85 by volume, flow rate 3ml / min, collection retention time t R = 26-28 min fraction was concentrated to dryness to obtain compound ephedrine G.

2. The method for preparing lignans extracted from the root of Ephedra according to claim 1, characterized in that: 40 kg of dried roots and rhizomes of Ephedra sinica after impurities removal were taken as raw materials, and 600 L of water was added for decoction and extraction twice, each time for 2 hours. The roots of Ephedra sinica after water decoction and extraction were then extracted twice with 95% ethanol using a heating reflux device, adding 4 L of ethanol per kg of Ephedra sinica root each time, heating and reflux for 2 hours, and the extract was concentrated under reduced pressure to obtain 10.0 kg of extract, which was dispersed and dissolved in water, and extracted with petroleum ether 5 times, 7 L each time, with dichloromethane 10 times, 10 L each time, with ethyl acetate 12 times, 10 L each time, and with n-butanol 6 times, 6 L each time. After recovering the solvent, 211.6 g of petroleum ether fraction, 245.1 g of dichloromethane fraction, 335.5 g of ethyl acetate fraction, and 62.5 g of n-butanol fraction were obtained. The dichloromethane fraction was separated by 100-200 mesh silica gel column chromatography 2000 g of silica gel to obtain 270 g of mixed silica gel. Petroleum ether-acetic acid was used in a volume ratio of Ethyl ester: 10:1, 5:1, 3:1, 1:1 and dichloromethane-methanol 20:1 were used for gradient elution to obtain the corresponding fractions M1, M2, M3~M5. Fraction M3 was separated by 200~300 mesh silica gel column chromatography and petroleum ether-ethyl acetate: 30:1, 20:1, 10:1, 5:1 by volume and dichloromethane were used for gradient elution to obtain the corresponding fractions M3-1, M3-2, M3-3~M3-5. Fraction M3-3 was separated by semi-preparative HPLC with a 5C column with specifications of 250×10 mm, particle size of 5 μm, pore size of 12 nm. 18 -MS-Ⅱ chromatographic column, the mobile phase is a mixture of 0.45% triethylamine in water and methanol, the volume ratio of water to methanol is 15:84, the flow rate is 3ml / min, and the retention time t R = 23-25 ​​min fraction was concentrated to dryness to obtain 6.12 mg of ephedrine E; 335.5 g of the ethyl acetate fraction was separated by 100-200 mesh silica gel column chromatography with 6000 g of silica gel to obtain 500 g of sample silica gel. Gradient elution was performed using dichloromethane-methanol in a volume ratio of 50:1, 30:1, 20:1, 10:1, 5:1, and 3:1 to obtain the corresponding fractions E1-E9. Fraction E9 was separated by 200-300 mesh silica gel column chromatography and gradient elution was performed using petroleum ether-ethyl acetate in a volume ratio of 20:1, 10:1, 5:1, 1:1, and 2:3 to obtain the corresponding fractions E9-1, E9-2-E9-5. Fraction E9-2 was concentrated under reduced pressure and eluted using a Toyopearl HW-40C gel column chromatography with 50% and 70% methanol by volume, respectively. The amount used for each elution fraction was 10 times the column volume. Anisaldehyde-concentrated sulfuric acid spray detection was performed. Every 5 The fractions were detected once every 3 ml, and the same fractions were combined to obtain components E9-2-1, E9-2-2, E9-2-13, and E-9-2-4. Component E-9-2-3 was concentrated to 15 mL under reduced pressure and chromatographed on a Sephadex LH-20 gel column, eluted with 70% methanol, and spray-detected with anisaldehyde-concentrated sulfuric acid. The fractions were detected once every 3 ml, and the same fractions were combined to obtain components E9-2-3-1, E9-2-3-2, and E-9-2-3-3. Component E-9-2-3-2 was separated by semi-preparative HPLC using a 5C column with specifications of 250×10 mm, a particle size of 5 μm, and a pore size of 12 nm. 18 -MS-Ⅱ column, mobile phase is methanol: water = 15:85 by volume, flow rate 3ml / min, collection retention time t R = 26-28 min fraction was concentrated to dryness to obtain 3.86 mg of compound ephedrine G.

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