A benzopyran enantiomer and its preparation method and application

Abstract

The invention discloses a benzopyran enantiomer and its preparation method and application. The present invention extracts and separates a pair of benzopyran enantiomers from the rhizome of Gentiana macrophylla, and evaluates the hepatoprotective activity of Gentiana macrophylla benzopyran by detecting the inhibitory effect of the benzopyran enantiomer on acetaminophen-induced human hepatocyte (L-O2) damage in vitro. It was found that the benzopyran enantiomer has good hepatoprotective activity. The preparation method of the benzopyran enantiomer provided by the present invention is simple to operate and low in cost, and the obtained benzopyran has high purity, a clear structure, and a variety of potential application values, which lays the foundation for the development of the medicinal value of Gentiana macrophylla benzopyran.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural product chemistry, and particularly relates to a benzopyran enantiomer and a preparation method and application thereof. Background Art

[0002] Gentiana macrophylla Gentiana macrophylla Pall. is a perennial herbaceous plant of the Gentianaceae family, Gentiana genus, Gentiana group, first recorded in "Shennong's Herbal Classic", also known as Zuo Qinjiao, large-leaf Gentiana. Gentiana straminea Maxim.), Gentiana macrophylla ( Gentiana crassicaulis Duthie ex Burk.), Gentiana macrophylla ( Gentiana dahurica Fisch.) is generally referred to as " Gentiana macrophylla ". Plantation is all arranged in Xinjiang, Ningxia, Shaanxi, Shanxi, Hebei and other places. It is mostly grown in riverbanks, hillside grasslands, meadows, under forests and forest edges at an altitude of 400-2400m. It is bitter and pungent in the mouth, and its nature is flat and slightly cold. It returns to the stomach, liver and gallbladder meridians. It has the effects of dispelling rheumatism, stopping pain due to numbness, relieving asthenic fever, relaxing tendons and tendons, promoting dampness and jaundice. It is usually used in diseases such as systemic rheumatic pain, whole body or joint cramps, limb paralysis, hot flashes due to steaming of the bones, damp-heat jaundice. The iridoid glycosides contained in Gentiana macrophylla are its main active ingredients. It also contains natural ingredients such as lignans, flavonoids, alkaloids, polysaccharides, and has pharmacological actions such as anti-inflammatory and analgesic, liver protection, antiviral, antitumor, immunosuppression, and blood pressure reduction. At present, the chemical composition and biological activity reports of the Chinese medicine Gentiana macrophylla are mostly concentrated on the iridoids, and other components such as benzopyran compounds are reported less. Summary of the Invention

[0003] The first object of the present invention is to provide a pair of benzopyran enantiomers, the second object of the present invention is to provide a method for preparing the benzopyran enantiomers, and the third object of the present invention is to provide applications of the benzopyran enantiomers.

[0004] The first object of the present invention is achieved by providing a pair of benzopyran enantiomers having the structures of Formula 1a and Formula 1b:

[0005] .

[0006] The second object of the present invention is achieved by the method for preparing the benzopyran enantiomers, which is specifically achieved by the following steps:

[0007] 1) Wash and dry Gentiana macrophylla, crush, and soak in 70% ethanol for 3-4 times, each time for 24-48 hours. Concentrate the extract under reduced pressure to obtain an extract. Add water to the extract, stir well, and extract with petroleum ether, ethyl acetate, and n-butanol in sequence. Concentrate and recover the n-butanol extract to obtain the n-butanol extract.

[0008] 2) The n-butanol extract was subjected to silica gel column chromatography using a gradient elution method with dichloromethane / methanol in volume ratios of 80:1, 50:1, 30:1, 15:1, and 0:1. The eluate with a volume ratio of 15:1 was collected and concentrated under reduced pressure. The product was then separated by semi-preparative HPLC using an Agilent Eclipse XDB-C18 (9.4 × 250 mm, 5 μm, 3 ml / min) to obtain benzopyran compound 1.

[0009] 3) The obtained compound 1 was subjected to HPLC chiral separation using a CHIRALPAK IH chiral column to obtain compound 1a and compound 1b.

[0010] In step 2), the semi-preparative HPLC separation is performed using isocratic elution with methanol / water in a volume ratio of 30:70.

[0011] In step 3), HPLC chiral separation was performed using isocratic elution with n-hexane / isopropanol in a volume ratio of 50:50, an injection volume of 20 μL, and an injection rate of 1.5 ml / min.

[0012] The third object of the present invention is achieved in that the benzopyran enantiomers are used in the preparation of liver protection drugs.

[0013] The present invention extracts and separates a pair of benzopyran enantiomers from the rhizome of Gentiana macrophylla. These benzopyran enantiomers have good hepatoprotective activity. The method for preparing the benzopyran enantiomers provided by the present invention is simple to operate and low in cost. The obtained benzopyrans are high in purity, have a clear structure, and have multiple potential applications, laying a foundation for developing the medicinal value of benzopyran compounds in Gentiana macrophylla. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the infrared spectrum of compound 1 prepared in Example 1;

[0015] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of compound 1 prepared in Example 1;

[0016] Figure 3 The carbon NMR spectrum of compound 1 prepared in Example 1;

[0017] Figure 4 COSY diagram of compound 1 prepared in Example 1;

[0018] Figure 5 HSQC pattern of compound 1 prepared in Example 1;

[0019] Figure 6 This is the HMBC pattern of compound 1 prepared in Example 1;

[0020] Figure 7 ROESY diagram of compound 1 prepared in Example 1;

[0021] Figure 8 ECD quantum calculation diagrams of compound 1a and compound 1b prepared in Example 1;

[0022] Figure 9 Figures A, B, and C are the results of the cytotoxicity test of benzopyran compounds 1, 1a, and 1b of the present invention on L-O2 cells (the NC group is the normal group; **p<0.01 compared with the normal group);

[0023] Figure 10 Figures A, B, and C show the effects of the benzopyran compounds 1, 1a, and 1b of the present invention on APAP-induced L-O2 cell injury (NC group is the normal group, AP is the model group, and NAC is the positive group; #p<0.05, ##p<0.01, ###p<0.001, and ####p<0.0001 compared with the normal group; *p<0.05, **p<0.01,

[0024] ***p<0.001 and ****p<0.0001 compared with the model group). DETAILED DESCRIPTION

[0025] The present invention is further described below, but is not intended to limit the present invention in any way. Any changes made based on the present invention fall within the scope of protection of the present invention.

[0026] The present invention provides a pair of benzopyran enantiomers having the structures of Formula 1a and Formula 1b:

[0027] .

[0028] The present invention also provides a method for preparing the benzopyran enantiomers, which is specifically achieved by the following steps:

[0029] 1) Wash and dry Gentiana macrophylla, crush, and soak in 70% ethanol for 3-4 times, each time for 24-48 hours. Concentrate the extract under reduced pressure to obtain an extract. Add water to the extract, stir well, and extract with petroleum ether, ethyl acetate, and n-butanol in sequence. Concentrate and recover the n-butanol extract to obtain the n-butanol extract.

[0030] 2) The n-butanol extract was subjected to silica gel column chromatography using a gradient elution method with dichloromethane / methanol in volume ratios of 80:1, 50:1, 30:1, 15:1, and 0:1. The eluate with a volume ratio of 15:1 was collected and concentrated under reduced pressure. The product was then separated by semi-preparative HPLC using an Agilent Eclipse XDB-C18 (9.4 × 250 mm, 5 μm, 3 ml / min) to obtain benzopyran compound 1.

[0031] 3) The obtained compound 1 was subjected to HPLC chiral separation using a CHIRALPAK IH chiral column to obtain compound 1a and compound 1b.

[0032] In step 2), the mobile phase for semi-preparative HPLC separation was methanol / water with a volume ratio of 30:70, the injection volume was 40 μL, and the UV detection wavelength was 254 nm.

[0033] In step 3), HPLC chiral separation was performed using isocratic elution with n-hexane / isopropanol in a volume ratio of 50:50, an injection volume of 20 μL, an injection speed of 1.5 ml / min, and an ultraviolet detection wavelength of 254 nm.

[0034] The present invention also provides the use of the benzopyran enantiomer or the pharmaceutical composition in preparing liver-protecting drugs.

[0035] The present invention further provides a pharmaceutical composition comprising the benzopyran enantiomer or a mixture thereof and a pharmaceutically acceptable carrier.

[0036] Example 1

[0037] 1. Dry and crush 3 kg of Gentiana macrophylla, soak and extract in 70% ethanol (g / mL=1 / 13) three times, each time for 48 h, filter, combine the extracts, and concentrate under reduced pressure until there is no alcohol taste. After drying, 658 g of Gentiana macrophylla extract was obtained.

[0038] 2. Dissolve the extract in water, then extract with petroleum ether, ethyl acetate, and n-butanol in sequence, 3 times each, and concentrate and recover the n-butanol extract phase to obtain 68g of n-butanol phase extract.

[0039] 3. The n-butanol phase extract was mixed with 1 times the sample amount of column chromatography silica gel, and 10 times the sample amount of column chromatography silica gel (W / W) was dry-loaded onto the column, and eluted with a gradient of dichloromethane / methanol in a volume ratio of 80:1→50:1→30:1→15:1→0:1. The eluent of dichloromethane / methanol (15:1) was collected by thin layer chromatography.

[0040] 4. The above fractions were passed through a semi-preparative high performance liquid chromatography (methanol / water = 30 / 70, t R= 7.5min, to obtain benzopyran compound 1. According to the literature method, it was subjected to HPLC chiral separation on a CHIRALPAK IH chiral column (n-hexane: isopropanol = 50:50, injection volume = 20 μL) to obtain enantiomers (compound 1a, 1.8 mg, t R =5min, 1.5ml / min; Compound 1b, 1.6mg, t R =6min, 1.5ml / min).

[0041] The following is a correlation spectrum analysis of Compound 1, Compound 1a and Compound 1b obtained in Example 1.

[0042] Compound 1, white amorphous powder. Planar structure: [α] QUOTE : -4.5 (c 0.200, MeOH). (+)-HR-ESI-MS m / z 267.0879 [M–H] - (C 13 H 16 O6H-, calculated value 267.0874), suggesting that its molecular formula is C 13 H 16 O6, unsaturation is 6.

[0043] Infrared spectroscopy ( Figure 1 ) suggests the presence of hydroxyl groups (3371 cm -1 ), carbonyl acid (1622 cm -1 ) and methyl (1383 cm -1 ), and the two methyl groups are on the same carbon. 13 The C-NMR spectrum shows a total of 13 carbon signals, including 5 quaternary carbons, 1 carboxyl group, 4 methine groups, 2 methyl groups, and 1 methoxy group. 1 H-NMR, 13 C-NMR and HSQC spectra ( Figure 2 , 3, 5) suggest that there are two isolated aromatic protons 7.81 (s, 1H, H-5) and 6.37 (s, 1H, H-8) in the low field, suggesting the presence of 3,4-phenyl ring -OCH3 and -COOH substitution; there are geminal dimethyl groups at δH 1.44 (s, 3H, H-9) and δH 1.19 (s, 3H, H-10) in the high field region; there are resonances at 4.43 (d, J=8.3Hz, 1H, H-4) and 3.49 (d, J=8.2Hz, 1H, H-3). 1H-1H COSY spectrum ( Figure 4), δH 3.49 (d, J=8.2 Hz, 1H ,H-3) is correlated with 4.43 (d, J=8.3 Hz, 1H ,H-4); δH 1.19 (s, 3H,H-10) is correlated with δH 1.44 (s, 3H, H-9); δH 4.43 (d, J=8.3 Hz, 1H ,H-4) is correlated with 7.81 (s, 1H, H-5). Based on the above spectral information, it is speculated that the compound is a 2,2-dimethyl-2H-benzopyran compound.

[0044] HMBC spectrum shows ( Figure 6 ), δH 7.81 (s, 1H, H-5), 6.37 (s, 1H, H-8)) are related to δC 176.26(C-11), δH 3.49 (d, J=8.2 Hz, 1H, H-3), 7.81 (s, 1H, H-5) are related to δC69.79(C-4), and the 1H-1H ROESY spectrum ( Figure 7 ), δH 3.79 (s, 3H, -OCH3) correlates with 6.37 (s, 1H, H-8), indicating that the C-6 substituent is -COOH and the C-7 substituent is -OCH3. The planar structure of compound 1 is deduced as shown in Figure 1.

[0045]

[0046] Absolute configuration: The optical rotation of compound 1 is [α] QUOTE :-4.5 (c 0.200, MeOH), it is speculated that the compound may be a racemate, that is, there are two possible pairs of enantiomers, "3S, 4S", "3R, 4R" and "3R, 4S", "3S, 4R". In order to determine the configuration, the ECD spectra of compound 1a and compound 1b were calculated and fitted ( Figure 8 ). The results show that the ECD spectrum of compound 1a is consistent with the "3R,4R" configuration, and compound 1b is consistent with the "3S,4S" configuration. Therefore, the two absolute configurations of compound 1 are determined to be 3R,4R-(1a) and 3S,4S-(1b). The experimentally measured specific rotations are: [α] QUOTE :32.53(c0.080, MeOH, 1a), [α] QUOTE : -60.57 (c 0.060, MeOH, 1b).

[0047] Table 1 One-dimensional NMR data of compound 1 prepared in Example 1

[0048]

[0049]

[0050] In vitro hepatoprotective activity assay of compound 1, compound 1a and compound 1b prepared in Example 1

[0051] 1. L-O2 cytotoxicity assay

[0052] Take L-O2 cells with stable growth status and add 100 μL (about 5×10 4 Cells were plated in 96-well plates and incubated for 24 hours at 37°C in a 5% CO2 incubator. A control group (1640 complete medium containing 10% fetal bovine serum, 1% penicillin and streptomycin) and benzopyran compound-treated groups (5, 10, 20, 40, and 80 μmol / L) were set up. After 24 hours of incubation, 10 μL of CCK-8 working solution was added to each well and incubated for 1.5 hours. The absorbance (OD) value at a wavelength of 450 nm was measured using a microplate reader to determine the cytotoxic activity of each compound against L-O2.

[0053] Results: As Figure 9 As shown, the benzopyran compounds 1, 1a and 1b prepared by the present invention have no cytotoxic effect, and their optimal administration concentration ranges are 20, 40 and 80 μmol / L.

[0054] 2. Activity detection of APAP-induced L-O2 cell damage

[0055] L-O2 cells in the logarithmic growth phase were seeded into 96-well plates and cultured for 24 hours. Compounds 1, 1a, and 1b, along with a positive agent (NAC, 0.25 mmol / L), were administered at varying concentrations (20, 40, and 80 μmol / L). After 24 hours of culture, APAP (10 mmol / L) was added to each well and incubated for 24 hours. Cell supernatants were collected, and 10 μL of CCK-8 reagent was added to each well. After incubation for 1.5 hours, the absorbance (OD) of each group was measured, and cell viability was calculated.

[0056] Results: As Figure 10 As shown (NC group is the normal group, AP is the model group, NAC positive group; #p<0.05, ##p<0.01, ###p<0.001 and ####p<0.0001 compared with the normal group; *p<0.05, **p<0.01,

[0057] ***p<0.001 and ****p<0.0001 compared with the model group). Compounds 1, 1a, and 1b prepared in the present invention all have certain anti-APAP-induced L-O2 cell injury activities, and their activities are superior to those of the positive control drug N-acetylcysteine.

[0058] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A pair of benzopyran enantiomers, characterized in that The enantiomers have the structures of Formula 1a and Formula 1b: 。 2. The method for preparing the benzopyran enantiomers according to claim 1, wherein: To do this, follow these steps: 1) Wash and dry Gentiana macrophylla, crush, and soak in 70% ethanol for 3-4 times, each time for 24-48 hours. Concentrate the extract under reduced pressure to obtain an extract. Add water to the extract, stir well, and extract with petroleum ether, ethyl acetate, and n-butanol in sequence. Concentrate and recover the n-butanol extract to obtain the n-butanol extract. 2) The n-butanol extract was subjected to silica gel column chromatography using a gradient elution method with dichloromethane / methanol in volume ratios of 80:1, 50:1, 30:1, 15:1, and 0:

1. The eluate with a volume ratio of 15:1 was collected and concentrated under reduced pressure. The benzopyran compound 1 was then separated by semi-preparative high performance liquid chromatography on an Agilent Eclipse XDB-C18 column (9.4 × 250 mm, 5 μm) at a flow rate of 3 ml / min. 3) The obtained benzopyran compound 1 was subjected to HPLC chiral separation using a CHIRALPAK IH chiral column to obtain compound 1a and compound 1b.

3. The method for preparing benzopyran enantiomers according to claim 2, wherein: In step 2), the mobile phase for semi-preparative HPLC separation was methanol / water with a volume ratio of 30:70, the injection volume was 40 μL, and the UV detection wavelength was 254 nm.

4. The method for preparing benzopyran enantiomers according to claim 2, wherein: In step 3), HPLC chiral separation was performed using isocratic elution with n-hexane / isopropanol in a volume ratio of 50:50, an injection volume of 20 μL, an injection speed of 1.5 ml / min, and an ultraviolet detection wavelength of 254 nm.

5. Use of the benzopyran enantiomer according to claim 1 in the preparation of hepatoprotective drugs.

6. A pharmaceutical composition comprising the benzopyran enantiomer or a mixture thereof according to claim 1 and a pharmaceutically acceptable carrier.

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