An arylnaphthalene phenolic acid compound extracted from Ephedra root, its preparation method and application

Abstract

The present invention relates to arylnaphthalene phenolic acid compounds extracted from Ephedrae Radix, their preparation methods and applications, which can effectively develop new medicinal values of Ephedra and realize the application in the preparation of antidepressant drugs. The technical solution for solving this problem is that an arylnaphthalene phenolic acid compound extracted from Ephedrae Radix includes Compound 1, Compound 2 and Compound 3, and their molecular formulas are C 14 H 12 O 5、 C 13 H 11 NO3 and C 13 H 10 O4. It is identified in the present invention that these are three arylnaphthalene phenolic acid compounds extracted from Ephedrae Radix. Their preparation methods are easy to operate, have strong directivity and high product purity. These compounds can be effectively used to prepare antidepressant drugs, opening up the medicinal value of Ephedrae Radix and bringing great economic and social benefits.

Description

1. Technical Field

[0001] The present invention relates to the field of medicine, and in particular to an arylnaphthalene phenolic acid compound extracted from Ephedra root, a preparation method thereof, and an application thereof. 2. Background Art

[0002] Depression is a mental disease mainly characterized by emotional dysfunction, slow thinking, decreased willpower, cognitive function impairment, and social function disorder. Depression has now become the leading cause of suicide, seriously affecting the quality of human life. Currently, clinical studies have shown that the level of corticosterone is related to the severity of depression. The plasma corticosterone level of depression patients increases, and anti-glucocorticoid treatment has obvious effects on severe depression patients, and the plasma corticosterone level decreases after treatment. The continuous increase in the levels of corticosterone in 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 the antidepressant activity of compounds using a corticosterone-induced PC-12 cell injury model and to find potential natural products with antidepressant activity.

[0003] Ephedra root, also known as bitter toona sinensis, is the dried root and rhizome of Ephedra sinica Stapf. or Ephedra intermedia Schrenket C.A.Mey. of the genus Ephedra (Ephedra Tournex Linn.) in the family Ephedraceae Dumortier. It is mainly distributed in Shanxi, Shaanxi, Sichuan and other places. It is a traditional Chinese medicine and is included in the Chinese Pharmacopoeia. It was first seen in the Shennong Ben Cao Jing and has the effects of treating stroke, typhoid headache, warm malaria, inducing sweating, removing pathogenic heat, relieving cough and counteracting adverse qi, dispelling cold and heat, and breaking up accumulations. Relevant research shows that the chemical components of Ephedra root are mainly alkaloids, flavonoids, phenolic acids, polysaccharides, etc. However, there is currently little further research on Ephedra root and stem. In order to make full use of China's rich Ephedra resources, further develop the new medicinal value of Ephedra, and find its new active ingredients, the present invention selects Ephedra root as the research object. However, so far, there has been no report on the preparation of arylnaphthalene phenolic acid compound 1 (Ephedra root phenolic acid B), compound 2 (Ephedra root phenolic acid C), and compound 3 (Ephedra root phenolic acid D) with potential antidepressant activity from Ephedra root, nor has there been any public report on how to extract the antidepressant-active Ephedra root phenolic acid components from Ephedra root. 3. Summary of the Invention

[0004] In view of the above situation, to solve the defects of the prior art, the purpose of the present invention is to provide an arylnaphthalene phenolic acid compound extracted from Ephedra root, a preparation method thereof, and an application thereof, which can effectively develop the new medicinal value of Ephedra and realize the application problem in the preparation of antidepressant-active drugs.

[0005] The technical solution to the problem of the present invention is an arylnaphthalene phenolic acid compound extracted from Ephedrae Radix, including compound 1, compound 2 and compound 3, whose molecular formulas are C 14 H 12 O5, C 13 H 11 NO3 and C 13 H 10 O4 respectively, and the structural formulas are as follows:

[0006]

[0007] The preparation method includes the following steps:

[0008] 1) Take the dried roots and rhizomes of Ephedra sinica, extract twice with 15 times the amount of water by water decoction method for 2 hours each time. After water decoction extraction, the Ephedra sinica roots are further extracted twice with 95% ethanol using a 10L heating reflux device. For each 1kg of Ephedra sinica roots, add 4L of ethanol and heat reflux for 2 hours. The extract is concentrated under reduced pressure to obtain the total extract, which is dispersed and dissolved in water, and successively extracted with petroleum ether (5 times × 7L), dichloromethane (10 times × 10L), ethyl acetate (12 times × 10L), and n-butanol (6 times × 6L). After recovering the solvents, the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, and n-butanol fraction are obtained;

[0009] 2) The dichloromethane fraction obtained in step 1) is separated by silica gel column chromatography (100 - 200 mesh). The ratio of sample-mixed silica gel to blank silica gel is 1:7 - 8, and gradient elution is carried out using petroleum ether - ethyl acetate at a volume ratio of 10:1, 5:1, 3:1, 1:1 and dichloromethane - methanol at a volume ratio of 20:1 to obtain 15 corresponding fractions M1 - M15;

[0010] 3) The fraction M12 obtained in step 2) is separated by silica gel column chromatography (200 - 300 mesh), and gradient elution is carried out using petroleum ether - ethyl acetate at a volume ratio of 30:1, 20:1, 10:1, 5:1 and dichloromethane - methanol at a volume ratio of 30:1, 20:1, 10:1 to obtain 6 corresponding fractions M12 - 1 - M12 - 6;

[0011] 4) The fraction M12 - 5 obtained in step 3) is concentrated under reduced pressure, and column chromatography on MCI gel CHP 20P is carried out, successively eluted with 70%, 80%, 90%, and 100% methanol. The dosage of each elution part is 10 times the column volume, the flow rate is 3ml / min, and detection is carried out by spraying with anisaldehyde - concentrated sulfuric acid. Detection is carried out once every 100ml, and the same fractions are combined to obtain 8 components M12 - 5 - 1 - M12 - 5 - 8;

[0012] 5) The fraction M12-5-4 obtained in step 4) was separated by semi-preparative HPLC using a 5C-MS-Ⅱ chromatographic column with the specification model of 250×10 mm, particle size of 5 μm, and pore size of 12 nm. The mobile phase was methanol: water = 47:53, the flow rate was 3 ml / min, and the fraction with a retention time t = 27 - 29 min was collected, concentrated and dried to obtain compound ephedra root phenolic acid B; 18 -MS-Ⅱ chromatographic column, the mobile phase was methanol: water = 47:53, the flow rate was 3 ml / min, and the fraction with a retention time t R = 27 - 29 min was collected, concentrated and dried to obtain compound ephedra root phenolic acid B;

[0013] 6) The fraction M14 obtained in step 2) was separated by silica gel column chromatography (200 - 300 mesh), and gradient elution was carried out with petroleum ether - ethyl acetate at a volume ratio of 50:1, 30:1, 15:1, 5:1, and 3:1 to obtain the corresponding 5 fractions M14-1 to M14-5;

[0014] 7) The fraction M14-2 obtained in step 6) was concentrated under reduced pressure and chromatographed on an MCI gel CHP 20P column. It was eluted successively with 70%, 80%, 90%, and 100% methanol. The amount of each elution part was 10 times the column volume, the flow rate was 3 ml / min, and it was detected by spraying with anisaldehyde - concentrated sulfuric acid, once every 100 ml. The same fractions were combined to obtain 5 components M14-2-1 to M14-2-5;

[0015] 8) The fraction M14-2-4 obtained in step 7) was separated by semi-preparative HPLC using a 5C-MS-Ⅱ chromatographic column with the specification model of 250×10 mm, particle size of 5 μm, and pore size of 12 nm. The mobile phase was methanol: water = 42:58, the flow rate was 3 ml / min, and the fraction with a retention time t 18 -MS-Ⅱ chromatographic column, the mobile phase was methanol: water = 42:58, the flow rate was 3 ml / min, and the fraction with a retention time t R = 52 - 54 min was collected, concentrated and dried to obtain compound ephedra root phenolic acid C;

[0016] 9) The fraction M11 obtained in step 2) was concentrated under reduced pressure and chromatographed on an MCI gel CHP 20P column. It was eluted successively with 50%, 70%, 80%, 90%, and 100% methanol. The amount of each elution part was 10 times the column volume, the flow rate was 3 ml / min, and it was detected by spraying with anisaldehyde - concentrated sulfuric acid, once every 100 ml. The same fractions were combined to obtain 9 components M11-1 to M11-8;

[0017] 10) The fraction M11-8 obtained in step 9) was separated by semi-preparative HPLC using a 5C-MS-Ⅱ chromatographic column with the specification model of 250×10 mm, particle size of 5 μm, and pore size of 12 nm. The mobile phase was methanol: water = 60:40, the flow rate was 3 ml / min, and the fraction with a retention time t 18 -MS-Ⅱ chromatographic column, the mobile phase was methanol: water = 60:40, the flow rate was 3 ml / min, and the fraction with a retention time t R = 7 - 9 min was collected, concentrated and dried to obtain compound ephedra root phenolic acid D.

[0018] Use of phenolic acid compounds ephedra root phenolic acid B, ephedra root phenolic acid C, and ephedra root phenolic acid D according to the present invention in the preparation of antidepressant drugs.

[0019] Identified by the present invention, they are three arylnaphthalene phenolic acid compounds extracted from ephedra root. Their preparation method is easy to operate, has strong directivity, and high product purity. These compounds can be effectively used in the preparation of drugs with antidepressant activity, opening up the medicinal value of ephedra root and bringing great economic and social benefits. IV. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Chemical structure diagrams of compounds ephedra root phenolic acid 1, 2, and 3 according to the present invention.

[0021] Figure 2 Effects of compounds 1, 2, and 3 according to the present invention on cell viability in the corticosterone-induced PC-12 cell damage model, where x — ±s, n = 4. ## P < 0.01 vs CORT; *P < 0.05, **P < 0.01 vs CORT.

[0022] Figure 3 1H-NMR spectrum (in DMSO-d6) of compound ephedra root phenolic acid B according to the present invention. 1 1H-NMR spectrum (in DMSO-d6).

[0023] Figure 4 13C-NMR spectrum (in DMSO-d6) of compound ephedra root phenolic acid B according to the present invention. 13 13C-NMR spectrum (in DMSO-d6).

[0024] Figure 5 DEPT 135 spectrum (in DMSO-d6) of compound ephedra root phenolic acid B according to the present invention.

[0025] Figure 6 1H- 1 H COSY spectrum (in DMSO-d6) of compound ephedra root phenolic acid B according to the present invention. 1 1H-

[0026] Figure 7 HSQC spectrum (in DMSO-d6) of compound ephedra root phenolic acid B according to the present invention.

[0027] Figure 8 HMBC spectrum (in DMSO-d6) of compound ephedra root phenolic acid B according to the present invention.

[0028] Figure 9 HR-ESI-MS spectrum (in MeOH) of compound ephedra root phenolic acid B according to the present invention.

[0029] Figure 10 IR spectrum (in MeOH) of ephedra root phenolic acid B, a compound of the present invention.

[0030] Figure 11 UV spectrum (in MeOH) of ephedra root phenolic acid B, a compound of the present invention.

[0031] Figure 12 For 1 1H-NMR spectrum (in DMSO-d6) of ephedra root phenolic acid C, a compound of the present invention.

[0032] Figure 13 For 13 13C-NMR spectrum (in DMSO-d6) of ephedra root phenolic acid C, a compound of the present invention.

[0033] Figure 14 DEPT 135 spectrum (in DMSO-d6) of ephedra root phenolic acid C, a compound of the present invention.

[0034] Figure 15 For 1 H- 1 1H-1H COSY spectrum (in DMSO-d6) of ephedra root phenolic acid C, a compound of the present invention.

[0035] Figure 16 HSQC spectrum (in DMSO-d6) of ephedra root phenolic acid C, a compound of the present invention.

[0036] Figure 17 HMBC spectrum (in DMSO-d6) of ephedra root phenolic acid C, a compound of the present invention.

[0037] Figure 18 HR-ESI-MS spectrum (in MeOH) of ephedra root phenolic acid C, a compound of the present invention.

[0038] Figure 19 IR spectrum (in MeOH) of ephedra root phenolic acid C, a compound of the present invention.

[0039] Figure 20 UV spectrum (in MeOH) of ephedra root phenolic acid C, a compound of the present invention.

[0040] Figure 21 For 1 1H-NMR spectrum (in CD3OD) of ephedra root phenolic acid D, a compound of the present invention.

[0041] Figure 22 For 13 13C-NMR spectrum (in CD3OD) of ephedra root phenolic acid D, a compound of the present invention.

[0042] Figure 23DEPT 135 spectrum of ephedra root phenolic acid D, a compound of the present invention (in CD3OD).

[0043] Figure 24 For ephedra root phenolic acid D, a compound of the present invention 1 H- 1 H COSY spectrum of ephedra root phenolic acid D, a compound of the present invention (in CD3OD).

[0044] Figure 25 HSQC spectrum of ephedra root phenolic acid D, a compound of the present invention (in CD3OD).

[0045] Figure 26 HMBC spectrum of ephedra root phenolic acid D, a compound of the present invention (in CD3OD).

[0046] Figure 27 HR-ESI-MS spectrum of ephedra root phenolic acid D, a compound of the present invention (in MeOH).

[0047] Figure 28 IR spectrum of ephedra root phenolic acid D, a compound of the present invention (in MeOH).

[0048] Figure 29 UV spectrum of ephedra root phenolic acid D, a compound of the present invention (in MeOH). V. Specific Embodiments

[0049] The following further elaborates on the specific embodiments of the present invention in conjunction with the accompanying drawings and examples.

[0050] Example 1

[0051] When the present invention is specifically implemented, it includes the following steps:

[0052] 1) Take 40 kg of the dried roots and rhizomes of Ephedra sinica, extract twice with 15 times the amount of water by water decoction method for 2 hours each time. After water decoction extraction, the Ephedra sinica roots are further extracted twice with 95% ethanol using a 10 L heating reflux device, with 4 L of ethanol added per 1 kg of Ephedra sinica roots, and heated under reflux for 2 hours. The extract is concentrated under reduced pressure to obtain 10.0 kg of total extract, which is dispersed and dissolved in water, and successively extracted with petroleum ether (5 times × 7 L), dichloromethane (10 times × 10 L), ethyl acetate (12 times × 10 L), and n-butanol (6 times × 6 L). After recovering the solvents, the petroleum ether fraction (211.6 g), dichloromethane fraction (245.1 g), ethyl acetate fraction (335.5 g), and n-butanol fraction (62.5 g) are obtained;

[0053] 2) The dichloromethane fraction (170.0 g) obtained in step 1) was separated by silica gel column chromatography (100 - 200 mesh) using blank silica gel (2000 g) and sample-mixed silica gel (270 g). Gradient elution was carried out using petroleum ether - ethyl acetate at a volume ratio of 10:1, 5:1, 3:1, 1:1 and dichloromethane - methanol at a volume ratio of 20:1 to obtain 15 corresponding fractions M1 - M15;

[0054] 3) The fraction M12 (13.31 g) obtained in step 2) was separated by silica gel column chromatography (200 - 300 mesh). Gradient elution was carried out using petroleum ether - ethyl acetate at a volume ratio of 30:1, 20:1, 10:1, 5:1 and dichloromethane - methanol at a volume ratio of 30:1, 20:1, 10:1 to obtain 6 corresponding fractions M12 - 1 - M12 - 6;

[0055] 4) The fraction M12 - 5 (6.3 g) obtained in step 3) was concentrated under reduced pressure (to about 20 ml), and then separated by MCI gel CHP 20P column chromatography. It was eluted successively with 70%, 80%, 90%, 100% methanol. The amount of each elution fraction was 10 times the column volume, the flow rate was 3 ml / min. Detection was carried out using anisaldehyde - concentrated sulfuric acid spray, once every 100 ml. The same fractions were combined to obtain 8 components M12 - 5 - 1 - M12 - 5 - 8;

[0056] 5) The fraction M12 - 5 - 4 (101.76 mg) obtained in step 4) was separated by semi - preparative HPLC using a 5C 18 -MS - Ⅱ chromatographic column with a specification model of 250×10 mm, particle size 5 μm, pore size 12 nm. The mobile phase was methanol:water = 47:53, the flow rate was 3 ml / min. The fraction with a retention time t R = 28.4 min was collected, concentrated and dried to obtain compound ephedra root phenolic acid B;

[0057] 6) The fraction M14 (10.23 g) obtained in step 2) was separated by silica gel column chromatography (200 - 300 mesh). Gradient elution was carried out using petroleum ether - ethyl acetate at a volume ratio of 50:1, 30:1, 15:1, 5:1, 3:1 to obtain 5 corresponding fractions M14 - 1 - M14 - 5;

[0058] 7) The fraction M14 - 2 (3.3 g) obtained in step 6) was concentrated under reduced pressure (to about 20 ml), and then separated by MCI gel CHP 20P column chromatography. It was eluted successively with 70%, 80%, 90%, 100% methanol. The amount of each elution fraction was 10 times the column volume, the flow rate was 3 ml / min. Detection was carried out using anisaldehyde - concentrated sulfuric acid spray, once every 100 ml. The same fractions were combined to obtain 5 components M14 - 2 - 1 - M14 - 2 - 5;

[0059] 8) The fraction M14-2-4 (67.71 mg) obtained in step 7) was separated by semi-preparative HPLC using a 5C 18 -MS-II chromatographic column with a specification model of 250×10 mm, particle size of 5 μm, and pore size of 12 nm. The mobile phase was methanol:water = 42:58, the flow rate was 3 ml / min, and the fraction with a retention time t R = 53.3 min was collected, concentrated and dried to obtain compound ephedra root phenolic acid C;

[0060] 9) The fraction M11 (4.8 g) obtained in step 2) was concentrated under reduced pressure (to about 20 ml), and then subjected to column chromatography on MCI gel CHP 20P. It was eluted successively with 50%, 70%, 80%, 90%, and 100% methanol, with the amount of each elution part being 10 times the column volume, and the flow rate was 3 ml / min. Detection was carried out using anisaldehyde-sulfuric acid spray, once every 100 ml. The same fractions were combined to obtain 9 components M11-1 to M11-8;

[0061] 10) The fraction M11-8 (78.46 mg) obtained in step 9) was separated by semi-preparative HPLC using a 5C 18 -MS-II chromatographic column with a specification model of 250×10 mm, particle size of 5 μm, and pore size of 12 nm. The mobile phase was methanol:water = 60:40, the flow rate was 3 ml / min, and the fraction with a retention time t R = 8.6 min was collected, concentrated and dried to obtain compound ephedra root phenolic acid D.

[0062] In the present invention, ephedra root was selected as the research object, and its chemical components were systematically separated and identified. Three new arylnaphthalene phenolic acid compounds, namely ephedra root phenolic acids B-D, were isolated therefrom. The compound activity screening was carried out using a corticosterone-induced PC-12 cell injury model. The results showed that compounds 1-3 could significantly improve corticosterone-induced PC-12 cell injury and had potential antidepressant activity. The relevant experimental data are as follows:

[0063] I. Instruments and reagents

[0064] Bruker AVANCEⅢ500 nuclear magnetic resonance spectrometer for nuclear magnetic resonance (with TMS internal standard) (Bruker), Nicolet is 10 Microscope Spectrometer for infrared spectroscopy (Thermo Scientific, USA), Bruker maxis HD mass spectrometer for high-resolution mass spectrometry, Shimadzu UV-2401PC apparatus for ultraviolet spectroscopy, Waters Alliance series 2695 high-performance liquid chromatography system for high-performance liquid chromatography, equipped with 2998 type diode array detector, Empower3 chromatographic data workstation, LC50 type high-pressure preparative liquid chromatography, UV200 type ultraviolet detector [Sepurest (Beijing) Technology Co., Ltd.], YMC-Pack ODS-A chromatographic column (250×10mm.D.S-5mm, 12mm) (YMC Co., Ltd.), and the rest include N-1100 type rotary evaporator (Shanghai Ailang Instrument Co., Ltd.), A-1000S type water aspirator (Shanghai Ailang Instrument Co., Ltd.), N-1111 type chilled water circulation device (Shanghai Ailang Instrument Co., Ltd.), FDU-2110 type freeze dryer (Shanghai Ailang Instrument Co., Ltd.), DFZ-60508 type vacuum drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.), AB204-N one-ten-thousandth precision analytical balance (METTLER TOLEDO), iMARK type microplate reader (BIO-RAD, USA), carbon dioxide incubator (Shanghai STIK), laminar flow hood (Sujing Group).

[0065] Rat adrenal pheochromocytoma cells PC-12, purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences; corticosterone (Aladdin Biochemical B2302817, Shanghai, China); fluoxetine (Med Chem Express, USA, 251273); CCK8 (GLPBIO, USA, 43); 5-HT7 antibody (Wuhan Sanying Biotechnology Co., Ltd., China, 00058665); Tubulin antibody (GR3398636-5); DAPI staining agent (ABCAM, USA, GR3445296-5); column chromatography packing Diaion HP-20, MCIGel CHP-20P (Mitsubishi Chemical Corporation, Japan), silica gel H (100-200 mesh) used for column chromatography is produced by Qingdao Ocean Chemical Factory, chromatographically pure reagents used are produced by Tianjin Siyou Fine Chemicals Co., Ltd., and analytical pure reagents used are produced by Beijing Chemical Factory and Tianjin Third Chemical Reagent Factory.

[0066] 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 (GR3413419-1 from ABCAM, USA); RPMI 1640 medium (2315381 from Invitrogen, USA); fetal bovine serum (12A218 from ExCell Bio, South America);

[0067] The Ephedra root selected in this invention was purchased from Bozhou Chinese herbal medicine market in May 2021. It was identified by Professor Dong Chengming of the School of Pharmacy as the dried root and rhizome of Ephedra sinica Stapf. (Ephedraceae), and the plant specimen (NO. 20210517) is stored in the Laboratory of Chinese Medicine Chemical Extraction and Isolation, Henan University of Chinese Medicine.

[0068] Secondary structure identification

[0069] Ephedradic acid B is a white amorphous powder (CH2Cl2), and HR-ESI-MS gives the quasi-molecular ion peak m / z: 261.0757 [M+H] + (Calcd.For 261.0751), and the molecular formula is determined to be C 14 H 12 O5; UV (MeOH) λ max (logε): 249 (1.38), 256 (1.38), 290 (0.77) nm; IRν max : 3369, 1639, 1462, 1255 cm -1 ; 1 1H NMR (DMSO-d6, 500 MHz) and 13 13C NMR (DMSO-d6, 125 MHz) data are shown in Table 1.

[0070] Ephedradic acid C is a white amorphous powder (CH2Cl2), and the HR-ESI-MS quasi-molecular ion peak is m / z: 230.0810 [M+H] + (Calcd.For 230.0811), and the molecular formula is C 13 H 11 NO3; UV (MeOH) λ max (logε): 203 (1.38), 247 (1.55), 288 (0.89) nm; IRν max : 3441, 1679, 1466, 1241, 1209 cm -1 ; 1 1H NMR (CD3OD, 500 MHz) and 13 13CNMR (CD3OD, 125 MHz) data are shown in Table 1.

[0071] Ephedra root phenolic acid D is a white amorphous powder (CH2Cl2), and the HR-ESI-MS quasi-molecular ion peak is m / z: 253.0460 [M+H] + (Calcd. For 253.0471), and the molecular formula is C 13 H 10 O4; UV (MeOH) λ max (logε): 210 (0.87), 244 (1.25), 251 (1.24), 285 (0.55) nm; IR ν max : 3405, 1618, 1574, 1397, 1241 cm -1 ; 1 1H NMR (CD3OD, 500 MHz) and 13 13C NMR (CD3OD, 125 MHz) data are shown in Table 1.

[0072] Table 1 NMR spectroscopic data of ephedra root phenolic acids B, C, and D

[0073]

[0074] b Recorded in DMSO-d6, c Recorded in CD3OD, d Recorded in CD3OD

[0075] Three activity detections

[0076] PC-12 cells were placed in a 5% CO2, 37 °C constant temperature incubator and cultured with RPMI1640 medium containing 10% fetal bovine serum. The cells in the best growth phase were selected, digested with 0.25% trypsin, and after terminating the digestion, they were configured into a cell concentration of 1×10 4 , inoculated on a 96-well plate, with 100 μL of cell suspension in each well, placed in the incubator. After the cells adhered, the medium was changed to a serum-free medium. After starving for 24 h, the PC-12 cells were divided into 6 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 administration groups (1 - 3, 2 μmol·L -1 ), and experimental groups. There were 6 replicate wells in each group. After culturing for 24 h in each group, after changing to a new medium, 10 μL of CCK8 solution was added to each well. After incubating at 37 °C for 1.5 hours, the absorbance OD value was detected at 450 nm with an enzyme-labeled instrument, and the cell survival rate was calculated according to the following formula:

[0077] Cell viability = (OD value of the drug-administered group - OD value of the blank group) / (OD value of the normal control group - OD value of the blank group) × 100%.

[0078] Four activity results

[0079] The cell viability of compounds 1-3 in the corticosterone-induced PC-12 cell injury model was detected by using the CCK8 method, and the results are as Figure 2 shown. Compared with the normal control group, the cell viability in the model group decreased significantly (P<0.05); compared with the model group, compounds 1-3 could significantly increase the cell viability (P<0.01), improve the injury of corticosterone to PC-12 cells, indicating that compounds 1-3 have good antidepressant activity.

[0080] Five conclusions

[0081] Three new arylnaphthalene phenolic acid compounds were isolated from Ephedrae Radix in the present invention, and were identified as ephedradic acid B, C, and D. The corticosterone-induced PC-12 cell injury model was used for the activity screening of the compounds. The results showed that ephedradic acid B, C, and D could significantly improve the corticosterone-induced PC-12 cell injury, have potential antidepressant activity, can be effectively used for preparing drugs for treating depression, have great development prospects, and have great economic and social benefits.

Claims

1. An arylnaphthalene phenolic acid compound extracted from Ephedrae Radix, characterized in that, Selected from Compound 1, Compound 2 or Compound 3, whose molecular formulas are C 14 H 12 O5, C 13 H 11 NO3 and C 13 H 10 O4, and the structural formulas are respectively:

2. The preparation method of the arylnaphthalene phenolic acid compound extracted from Ephedra root according to claim 1, characterized in that, It includes the following steps: 1) Take the dried roots and rhizomes of Ephedra sinica Stapf, extract them twice with 15 times the amount of water by water decoction method for 2 hours each time. After water decoction extraction, the Ephedra sinica Stapf roots are further extracted twice with 95% ethanol using a 10L heating reflux device. For each 1kg of Ephedra sinica Stapf roots, add 4L of ethanol and heat reflux for 2 hours. The extract is concentrated under reduced pressure to obtain the total extract, which is dispersed and dissolved in water, and then extracted successively with petroleum ether, dichloromethane, ethyl acetate, and n-butanol. After recovering the solvents, the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, and n-butanol fraction are obtained; 2) The dichloromethane fraction obtained in step 1) is separated by silica gel column chromatography. The ratio of the sample-mixed silica gel to the blank silica gel is 1:7 - 8, and gradient elution is carried out using petroleum ether - ethyl acetate at a volume ratio of 10:1, 5:1, 3:1, 1:1 and dichloromethane - methanol at a volume ratio of 20:1 to obtain 15 corresponding fractions M1 - M15; 3) The fraction M12 obtained in step 2) is separated by silica gel column chromatography, and gradient elution is carried out using petroleum ether - ethyl acetate at a volume ratio of 30:1, 20:1, 10:1, 5:1 and dichloromethane - methanol at a volume ratio of 30:1, 20:1, 10:1 to obtain 6 corresponding fractions M12 - 1 - M12 - 6; 4) The fraction M12 - 5 obtained in step 3) is concentrated under reduced pressure, and then separated by MCI gel CHP 20P column chromatography. It is eluted successively with 70%, 80%, 90%, and 100% methanol. The amount of each elution part is 10 times the column volume, and the flow rate is 3ml / min. It is detected by spraying with anisaldehyde - concentrated sulfuric acid, once every 100ml. The same fractions are combined to obtain 8 components M12 - 5 - 1 - M12 - 5 - 8; 5) Separate the fraction M12-5-4 obtained in step 4) by semi-preparative HPLC using a 5C 18 18 -MS-II chromatographic column with the specification model: 250×10 mm, particle size 5 μm, pore size 12 nm. The mobile phase is methanol: water = 47:53, the flow rate is 3 ml / min, and collect the fraction with the retention time t R R = 27 - 29 min, concentrate and dry to obtain compound 1 ephedra root phenolic acid B; 6) The fraction M14 obtained in step 2) is separated by silica gel column chromatography, and gradient elution is carried out using petroleum ether - ethyl acetate at a volume ratio of 50:1, 30:1, 15:1, 5:1, 3:1 to obtain 5 corresponding fractions M14 - 1 - M14 - 5; 7) The fraction M14 - 2 obtained in step 6) is concentrated under reduced pressure, and then separated by MCI gel CHP 20P column chromatography. It is eluted successively with 70%, 80%, 90%, and 100% methanol. The amount of each elution part is 10 times the column volume, and the flow rate is 3ml / min. It is detected by spraying with anisaldehyde - concentrated sulfuric acid, once every 100ml. The same fractions are combined to obtain 5 components M14 - 2 - 1 - M14 - 2 - 5; 8) Separate the fraction M14-2-4 obtained in step 7) by semi-preparative HPLC using a 5C 18 18 -MS-II chromatographic column with the specification model: 250×10 mm, particle size 5 μm, pore size 12 nm. The mobile phase is methanol: water = 42:58, the flow rate is 3 ml / min, and collect the fraction with retention time t R R = 52 - 54 min, concentrate and dry to obtain compound 2 ephedra root phenolic acid C; 9) The fraction M11 obtained in step 2) is concentrated under reduced pressure, and then separated by MCI gel CHP 20P column chromatography. It is eluted successively with 50%, 70%, 80%, 90%, and 100% methanol. The amount of each elution part is 10 times the column volume, and the flow rate is 3ml / min. It is detected by spraying with anisaldehyde - concentrated sulfuric acid, once every 100ml. The same fractions are combined to obtain 9 components M11 - 1 - M11 - 8; 10) The fraction M11-8 obtained in step 9) was separated by semi-preparative HPLC using a 5C-MS-Ⅱ chromatographic column with the specifications of 250×10 mm, particle size of 5 μm, and pore size of 12 nm. The mobile phase was methanol:water = 60:40, the flow rate was 3 ml / min, and the fraction with a retention time t 18 = 7 - 9 min was collected, concentrated and dried to obtain compound 3 ephedra root phenolic acid D. R ​ 3. The preparation method of the arylnaphthalene phenolic acid compound extracted from Ephedra root according to claim 2, characterized in that, It includes the following steps: 1) Take 40 kg of the dried roots and rhizomes of Ephedra sinica Stapf, and extract them twice by water decoction method with 15 times the amount of water for 2 hours each time. After water decoction extraction, the Ephedra sinica Stapf roots are further extracted twice with 95% ethanol using a 10 L heating reflux device. Each time, 4 L of ethanol is added per 1 kg of Ephedra sinica Stapf roots, and heated under reflux for 2 hours. The extract is concentrated under reduced pressure to obtain 10.0 kg of total extract, which is dispersed and dissolved in water, and successively extracted with petroleum ether 5 times, 7 L each time; dichloromethane 10 times, 10 L each time; ethyl acetate 12 times, 10 L each time; and n-butanol 6 times, 6 L each time. After recovering the solvents, petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, and n-butanol fraction are obtained. 2) Separate the dichloromethane fraction obtained in step 1) by silica gel column chromatography. Among them, 2000 g of blank silica gel and 270 g of sample-mixing silica gel are used, and gradient elution is carried out 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 15 corresponding fractions M1 - M15. 3) Separate the fraction M12 obtained in step 2) by silica gel column chromatography, and carry out gradient elution with petroleum ether - ethyl acetate in a volume ratio of 30:1, 20:1, 10:1, 5:1 and dichloromethane - methanol in a volume ratio of 30:1, 20:1, 10:1 to obtain 6 corresponding fractions M12-1 - M12-6. 4) Concentrate the fraction M12-5 obtained in step 3) under reduced pressure, and carry out column chromatography on MCI gel CHP 20P column, eluting successively with 70%, 80%, 90%, 100% methanol. The amount of each elution part is 10 times the column volume, the flow rate is 3 ml / min, and it is detected by spraying with anisaldehyde - concentrated sulfuric acid. Detection is carried out once every 100 ml, and the same fractions are combined to obtain 8 components M12-5-1 - M12-5-8. 5) Separate the fraction M12-5-4 obtained in step 4) by semi-preparative HPLC using a 5C 18 18 -MS-II chromatographic column with the specifications: 250×10 mm, particle size 5 μm, pore size 12 nm. The mobile phase is methanol:water = 47:53, the flow rate is 3 ml / min, and collect the fraction with a retention time t R R = 28.4 min, concentrate and dry to obtain compound 1 ephedra root phenolic acid B; 6) Separate the fraction M14 obtained in step 2) by silica gel column chromatography, and carry out gradient elution with petroleum ether - ethyl acetate in a volume ratio of 50:1, 30:1, 15:1, 5:1, 3:1 to obtain 5 corresponding fractions M14-1 - M14-5. 7) Concentrate the fraction M14-2 obtained in step 6) under reduced pressure, and carry out column chromatography on MCI gel CHP 20P column, eluting successively with 70%, 80%, 90%, 100% methanol. The amount of each elution part is 10 times the column volume, the flow rate is 3 ml / min, and it is detected by spraying with anisaldehyde - concentrated sulfuric acid. Detection is carried out once every 100 ml, and the same fractions are combined to obtain 5 components M14-2-1 - M14-2-5. 8) The fraction M14-2-4 obtained in step 7) was separated by semi-preparative HPLC using a 5C 18 -MS-Ⅱ chromatographic column with the specification model of 250×10 mm, particle size 5 μm, and pore size 12 nm. The mobile phase was methanol:water = 42:58, and the flow rate was 3 ml / min. The fraction with a retention time t R = 53.3 min was collected, concentrated and dried to obtain compound 2 ephedra root phenolic acid C; 9) Concentrate the fraction M11 obtained in step 2) under reduced pressure, and carry out column chromatography on MCI gel CHP 20P column, eluting successively with 50%, 70%, 80%, 90%, 100% methanol. The amount of each elution part is 10 times the column volume, the flow rate is 3 ml / min, and it is detected by spraying with anisaldehyde - concentrated sulfuric acid. Detection is carried out once every 100 ml, and the same fractions are combined to obtain 9 components M11-1 - M11-8. 10) The fraction M11-8 obtained in step 9) was separated by semi-preparative HPLC using a 5C-MS-Ⅱ chromatographic column with the following specifications: 250×10 mm, particle size 5 μm, pore size 12 nm. The mobile phase was methanol:water = 60:40, and the flow rate was 3 ml / min. The fraction with a retention time t 18 = 8.6 min was collected, concentrated and dried to obtain compound 3 ephedra root phenolic acid D. R ​ 4. Use of the arylnaphthalene phenolic acid compound extracted from Ephedra root as claimed in claim 1 in the preparation of antidepressant drugs.

5. Use of the arylnaphthalene phenolic acid compound extracted by the extraction method as claimed in claim 2 or 3 in the preparation of antidepressant drugs.

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