Method for extracting and separating 10 monomeric substances from Chinese knotweed herb

Abstract

The invention discloses a method for extracting and separating 10 kinds of monomeric substances from Polygonum chinense L.. The method comprises the following steps: extracting 10 kinds of monomeric substances from Polygonum chinense L.; comprising the following steps: 1, drying a Chinese knotweed herb, cutting into sections, taking 75% ethanol as an extracting agent, and extracting by a heating reflux method to obtain an extracting solution; 2, concentrating the 75% alcohol extracting solution into dry paste; 3, carrying out column chromatography silica gel elution separation on the Chinese knotweed herb dry paste; 4, eluting and separating the crude component through a C18 column; and 5, separating and purifying the components by adopting semi-preparative high performance liquid chromatography to obtain 10 monomeric substances. The method disclosed by the invention is simple, clear in process, excellent in separation effect and high in preparation speed, and the purity of the purified product is high.

Description

Technical Field

[0001] The invention relates to the extraction, purification, separation and application fields of ethnic medicine materials, in particular to a method for extracting and separating 10 monomer substances from charcoal mother. Background Art

[0002] The source of Polygonum chinense is the dried whole herb of Polygonum chinense L., a plant of the Polygonum genus of the Polygonaceae family. It is sour, sweet and cold in nature, and enters the liver and spleen meridians. It has the effects of clearing away heat and dampness, cooling blood and detoxifying, benefiting the liver and improving eyesight, and relaxing the meridians and activating blood circulation. It is mainly used to treat diarrhea, dysentery, lung heat cough, sore throat, hepatitis, eczema, otitis media, etc. It is mainly distributed in Fujian, Guangxi, Guangdong and other places. In Guangxi, it is included in the "Guangxi Zhuang Autonomous Region Yao Medicinal Materials Quality Standards Volume 1" and the "Guangxi Zhuang Autonomous Region Zhuang Medicine Quality Standards Volume 1". It can be seen that Polygonum chinense has played an important role in the medical field of Guangxi.

[0003] Firewood mainly contains flavonoids and flavonoid glycosides, phenolic acids, steroids and other chemical components, and has pharmacological effects such as anti-hepatitis B virus, liver protection, anti-inflammatory and analgesic, antioxidant, and antibacterial. At present, the methods reported in the literature for extracting, separating, and purifying monomers from firewood include silica gel column chromatography and supercritical fluid extraction. Column chromatography does not require high technical requirements, requires a large amount of reagents, and is time-consuming. Supercritical fluid extraction requires high technology and is fast, but it is mainly used for the extraction and purification of volatile components. Semi-preparative high-performance liquid chromatography has a clear process, excellent separation effect, fast preparation speed, and the purified product has high purity. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, the present invention provides a method for extracting and separating 10 monomer substances from fire charcoal mother. The method is simple, has a clear process, excellent separation effect, fast preparation speed, and the purified product has high purity.

[0005] The technical problem of the present invention is achieved through the following technical solutions:

[0006] S1. Dry the charcoal mother medicinal material, cut it into pieces, soak it in 75% ethanol for 2 hours, and extract it by heating reflux method to obtain an extract;

[0007] S2, concentrating the 75% ethanol extract to a dry paste, and recovering ethanol;

[0008] S3, the dry extract of the charcoal mother extract was separated by column chromatography on silica gel (300-400 mesh), and gradient eluted in different ratios of dichloromethane:methanol to obtain a crude component;

[0009] S4, separating the above crude components by C18 silica gel (50um), and gradient eluting with different ratios of methanol: water in sequence;

[0010] S5. Separate and purify the above components by semi-preparative high performance liquid chromatography, and gradient elute in different ratios of acetonitrile: water to obtain 10 monomer substances.

[0011] In the present invention, the 10 monomer substances are: ellagic acid (309.0 mg), hyperoside (15.0 mg), gallic acid (14.4 mg), rutin (14.2 mg), β-sitosterol (12.0 mg), quercetin (9.6 mg), corilagin (6.1 mg), brevifolia calendula acid (5.3 mg), brevifolia calendula phenol (5.0 mg), and quercetin (4.8 mg). BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Attached Figure 1 It is the structural formula of shortleaf sappanol acid.

[0013] Attached Figure 2 It is the structural formula of β-sitosterol.

[0014] Attached Figure 3 It is the corilagin structure.

[0015] Attached Figure 4 It is the structural formula of succinic acid.

[0016] Attached Figure 5 This is the structural formula of ellagic acid.

[0017] Attached Figure 6 This is the structural formula of rutin.

[0018] Attached Figure 7 This is the structural formula of quercetin.

[0019] Attached Figure 8 This is the structural formula of hyperoside.

[0020] Attached Fig. 9 It is the structural formula of gallic acid.

[0021] Attached Fig.10 This is the structural formula of quercetin. DETAILED DESCRIPTION

[0022] 1. Chemical component extraction

[0023] 15 kg of charcoal mother powder was extracted with 75% ethanol (medicinal material / 75% ethanol=1 / 15, W / W) by heating under reflux for 2 times, each time for 2 hours, filtered, the extracts were combined, recovered under reduced pressure until there was no alcohol taste, and dried to obtain 455 g of charcoal mother dry paste.

[0024] 2. Rough classification

[0025] Take 400g of dry charcoal mother paste, mix with 2 times the amount of column chromatography silica gel (extract / silica gel, W / W), and dry. Fill the glass column with 10 times the amount of column chromatography silica gel (300-400 mesh) (W / W) of the extract, and dry sample; use dichloromethane and methanol in a ratio of 100:1-75:1-50:1-25:1-15:1-10:1-5:1-2:1-1:1-0:1 to gradient elute, each gradient is 6000ml, each 2000ml is a crude fraction, collect each eluent, recover the eluent, and obtain 30 crude fractions.

[0026] 3. Separation of crude components

[0027] Take the above 1-30 crude fractions, mix with 2 times the amount of C18 silica gel (extract / silica gel, W / W), and dry. Fill the glass column with 10 times the amount of C18 silica gel (50μm, 120A) (W / W) of the extract, and wet sample loading; use methanol and water in a gradient elution ratio of 1:1-3:2-7:3-4:1-9:1-1:0, each gradient is 500ml, and each 500ml is a fraction. Collect each eluent, recover the eluent, and obtain 180 fractions.

[0028] 4. Preparation of Components

[0029] According to the components obtained under item 3, on the basis of analytical liquid analysis, a preparative C18 column (10 mm×250 mm, 10 μm) and preparative HPLC were used, with acetonitrile and water as mobile phases at a flow rate of 3.0 mL / min, to separate 10 compounds. The preparation process is shown in Table 1. Table 1 Information of compounds prepared by different ratios of acetonitrile and water

[0030] 5. Ingredient identification

[0031] Compound 1 Brassica scutellariae phenolic acid

[0032] Yellow amorphous powder, easily soluble in water, soluble in methanol and ethanol, molecular formula C 13 H8O8. ESI-MS: m / z 293.03[M+H] + . 1 H NMR (500MHz, DMSO-d6) δ7.19 (s, 1H), 4.37 (d, J=6.2Hz, 1H), 2.99 (d, J=18.0Hz, 1H), 2.60-2.52 (m, 2H). 13C NMR (126MHz, DMSO) δ195.13(C-1), 173.25(3-COOH), 161.01(5-C), 148.68(7-C), 147.49(9-C), 143.37(8- C), 142.88(3a-C), 140.88(3b-C), 115.79(9a-C), 112.69(5a-C), 108.37(6-C), 42.48(3-C), 37.45(2a-C).

[0033] Compound 2β-sitosterol

[0034] White flocculent crystals, soluble in methanol, ethanol and chloroform, molecular formula C 29 H 50 O. ESI-MS: m / z 415.21 [M+H] + ,437.19[M+Na] + , 453.17[M+K] + . 1 H NMR (500MHz, Chloroform-d) δ5.35 (dt, J=5.5, 2.0Hz, 1H), 3.57-3.47 (m, 1H), 2.29 (ddd, J=13.1, 5.1, 2.2Hz, 1H) , 2.23 (tdd, J=12.9, 5.2, 2.5Hz, 1H), 2.06-1.92 (m, 2H), 1.90-1.78 (m, 3H), 1.73-1.55 (m, 4H), 1.55-1.39 (m, 6H) , 1.35 (dddd, J=14.3, 7.8, 6.0, 3.2Hz, 2H), 1.32-1.23 (m, 4H), 1.23-1.11 (m, 4H), 1.11-0.97 (m, 8H), 0.95 (d, J=5 .8Hz, 1H), 0.92 (dd, J=6.1, 3.7Hz, 4H), 0.86 (d, J=1.6Hz, 1H), 0.85-0.81 (m, 6H), 0.81-0.75 (m, 3H), 0.68 (s, 3H). 13C NMR (126MHz, CDCl3) δ140.90(C-5), 121.87(C-6), 71.95(C-3), 56.91(C-14), 56.20(C-17), 50.28(C-9), 4 5.98(C-24), 42.47(C-4), 42.45(C-13), 39.92(C-12), 37.40(C-1), 36.65(C-10), 36.29(C-20), 34.09(C-2 2), 32.06(C-2), 32.05(C-8), 31.81(C-7), 29.29(C-25), 28.40(C-16), 26.21(C-23), 24.45(C-15), 23.21( C-28), 21.23(C-11), 19.97(C-26), 19.55(C-18), 19.18(C-27), 18.93(C-21), 12.13(C-29), 12.01(C-19).

[0035] Compound 3 Corilagin

[0036] Yellow-white amorphous powder, easily soluble in water, methanol and ethanol, molecular formula C 27 H 22 O 18 . ESI-MS: m / z 657.36 [M+Na] + , 657.36[M+K] + . 1 H NMR (500MHz, Deuterium Oxide) δ7.00 (s, 2H), 6.76 (s, 1H), 6.63 (s, 1H), 6.32 (s, 1H), 4.93-4.83 (m, 2H), 4.67 -4.60 (m, 1H), 4.47 (d, J=3.4Hz, 1H), 4.26 (dd, J=10.9, 8.2Hz, 1H), 4.22-4.17 (m, 1H). 13C NMR (126MHz, Deuterium Oxide) δ169.43(C-7′), 167.85(C-7″), 166.21(C-7″′), 144.77(C-5′), 144.61(C-5″), 144.51(C-″′ and C-5″′), 143.80(C-3′), 143.68(C-3″), 139.03(C-4″′), 136.52(C-4″), 135.38(C-4′), 125.06(C-2 ′), 123.50(C-2″), 119.34(C-1″′), 115.27(C-1″), 114.41(C-1′), 110.46(C-2″′ and C-6″′), 110.05(C -6″), 107.25(C-6′), 94.64(C-1), 73.97(C-5), 68.69(C-3), 65.87(C-2), 63.58(C-6), 60.05(C-4).

[0036] Compound 4: Brassica brevifolia

[0037] Yellow amorphous powder, easily soluble in water, methanol and ethanol, molecular formula C 12 H8O6. ESI-MS: m / z 249.03[M+H] + . 1 H NMR (400MHz, DMSO-d6) δ7.28 (s, 1H), 3.39-3.04 (m, 2H), 2.50 (dq, J=3.9, 1.9Hz, 5H). 13 C NMR (101MHz, DMSO) δ195.52(C-3), 160.54(C-5), 149.27(C-3a), 144.96(C-9), 144.23(C-7), 141 .43(C-8), 140.27(C-9a), 115.42(C-3b), 113.06(C-5a), 107.93(C-6), 32.98(C-2), 23.81(C-1).

[0038] Compound 5: ellagic acid

[0039] Yellow-green amorphous powder, easily soluble in water, methanol and ethanol, molecular formula C 14 H6O8. ESI-MS: m / z 303.14[M+H] + . 1 H NMR (400MHz, DMSO-d6) δ7.45 (s, 1H). 13C NMR (101MHz, DMSO-d6) δ159.13 (C-5, C-10), 148.10 (C-3, C-8), 139.71 (C-2, C-7), 13 6.34 (C-1, C-6), 112.32 (C-5a, C-10a), 110.13 (C-3a, C-8a), 107.47 (C-10b, C-10c).

[0040] Compound 6 Rutin

[0041] Bright yellow powder, easily soluble in methanol, ethanol and DMSO, molecular formula C 27 H 30 O 16 . ESI-MS: m / z 303.05[M+H] + . 1 H NMR(500MHz Methanol-d4) δ7.70-7.53 (m, 2H), 6.83 (d, J=8.4Hz, 1H), 6.35 (d, J=2.1Hz, 1H), 6 .16 (d, J=2.1Hz, 1H), 5.07 (d, J=7.6Hz, 1H), 4.48 (d, J=1.6Hz, 1H), 3.77 (dd, J=11 .0, 1.5Hz, 1H), 3.60 (dd, J=3.5, 1.7Hz, 1H), 3.51 (dd, J=9.5, 3.4Hz, 1H), 3.46-3. 33 (m, 4H), 3.32-3.29 (m, 1H), 3.23 (dt, J=9.7, 4.4Hz, 2H), 1.08 (d, J=6.2Hz, 3H). 13 CNMR (126MHz, MeOD) δ179.38(C-4), 165.99(C-7), 162.95(C-5), 159.31(C-2), 158.47(C-9), 149.79(C-4 ′), 145.81(C-3′), 135.62(C-3), 123.55(C-1′), 123.09(C-6′), 117.68(C-2′), 116.03(C-5′), 105.60(C- 10), 104.72(C-1″), 102.40(C-1″′), 99.92(C-6), 94.85(C-8), 78.15(C-3″), 77.18(C-5″), 75.71(C-2″), 73.91(C-4″′), 72.21(C-3″′), 72.08(C-2″′), 71.36(C-4″), 69.69(C-5″′), 68.53(C-6″), 17.88(C-6″′).

[0042] Compound 7 Quercetin

[0043] Yellow powder, easily soluble in methanol, ethanol and DMSO, molecular formula C 21 H 20 O 11 . ESI-MS: m / z 303.05[M+H] + . 1 H NMR (500MHz, Methanol-d4) δ7.38-7.26 (m, 2H), 6.91 (d, J = 8.3Hz, 1H), 6.36 (d, J = 2.1Hz, 1H), 6.19 (d, J = 2.1Hz, 1H), 5.35 (d, J = 1.7Hz , 1H), 4.23 (dd, J=3.4, 1.7Hz, 1H), 3.76 (dd, J=9.4, 3.4Hz, 1H), 3.42 (dq, J=9.5, 6.1Hz, 1H), 3.36-3.33 (m, 2H), 0.94 (d, J=6.1Hz, 3H). 13 C NMR (126MHz, Methanol-d4) δ179.61(C-4), 165.87(C-7), 163.17(C-5), 159.28(C-9 ), 158.49(C-2), 149.77(C-4′), 146.38(C-3′), 136.21(C-3), 122.94(C-6′), 122.86 (C-1′), 116.91(C-5′), 116.34(C-2′), 105.86(C-10), 103.51(C-1″), 99.80(C-6), 94.70(C-8), 73.23(C-4″), 72.08(C-3″), 72.01(C-5″), 71.89(C-2″), 17.64(C-6″).

[0044] Compound 8 Hyperoside

[0045] Yellow powder, easily soluble in methanol, ethanol and DMSO, molecular formula C 21 H 20 O 12 . ESI-MS: m / z 303.05[M+H] + . 1H NMR (500MHz, Methanol-d4) δ7.85 (d, J=2.2Hz, 1H), 7.59 (dd, J=8.5, 2.2Hz, 1H), 6.86 (d, J=8.5Hz, 1H), 6.41 (d, J=2.0Hz, 1H), 6.21 (d, J=2.1Hz, 1H), 5.18 (d, J=7.8Hz, 1H), 3.87-3.79 (m, 2H), 3.68-3.52 (m, 4H), 3.48 (td, J=6.2, 1.2Hz, 1H), 3.35 (s, 1H). 13 C NMR (126MHz, MeOD) δ179.55(C-4), 166.09(C-7), 163.04(C-5), 158.77(C-9), 158 .45(C-2), 149.96(C-4′), 145.83(C-3′), 135.76(C-3), 122.91(C-6′), 122.86(C- 1′), 117.76(C-5′), 116.07(C-2′), 105.62(C-10), 105.36(C-1″), 99.88(C-6), 94 .69(C-8), 77.19(C-5″), 75.09(C-3″), 73.17(C-2″), 70.02(C-4″), 61.93(C-6″).

[0046] Compound 9 Gallic acid

[0047] White powder, easily soluble in methanol, ethanol and DMSO, molecular formula C7H6O5. ESI-MS: m / z 171.03[M+H] + . 1 HNMR (500MHz, Methanol-d4) δ7.06 (s, 1H). 13 C NMR (126MHz, Methanol-d4) δ 170.39 (C-7), 146.37 (C-3, 5), 139.57 (C-4), 121.91 (C-1), 110.28 (C-2, 6).

[0048] Compound 10 Quercetin

[0049] Yellow powder, easily soluble in methanol, ethanol and DMSO, molecular formula C 15 H 10 O7. ESI-MS: m / z 303.05 [M+H] + . 1H NMR(500MHz,DMSO-d6)δ12.49(s,1H),10.77(s,1H),9.58(s,1H),9.32(d,J=28.3Hz,2H),7.68(d,J=2.2Hz,1H),7.54(dd,J=8.5,2.2Hz,1H),6.88(d,J=8.5Hz,1H),6.40(d,J=2.1Hz,1H),6.18(d,J=2.0Hz,1H)。 13 C NMR(126MHz,DMSO)δ175.84(C-4),163.88(C-7),160.72(C-9),156.13(C-2),147.70(C-5),146.80(C-4′),145.06(C-3′),135.74(C-3),121.95(C-1′),119.97(C-6′),115.60(C-5′),115.07(C-2′),103.01(C-10),98.18(C-6),93.35(C-8)。

Claims

1. A method for extracting and separating 10 monomer substances from fire charcoal mother, characterized in that: The following steps are involved: S1. Dry the charcoal mother medicinal material, cut it into sections, soak it in 75% ethanol for 2 hours, and extract it by heating reflux method to obtain an extract; S2, concentrating the 75% ethanol extract to a dry paste, and recovering ethanol; S3, the dry extract of the charcoal mother extract was separated by column chromatography on silica gel (300-400 mesh), and gradient eluted in different ratios of dichloromethane:methanol to obtain a crude component; S4, separating the above crude components by C18 silica gel (50um), and gradient eluting with different ratios of methanol: water in sequence; S5. Separate and purify the above components by semi-preparative high performance liquid chromatography, and gradient elute in different ratios of acetonitrile: water to obtain 10 monomer substances.

2. The method according to claim 1, characterized in that The volume ratio of the medicinal material to 75% ethanol is 1:10-15.

3. The method according to claim 1, characterized in that The extraction times are 2 to 3 times and the extraction time is 90 to 150 minutes.

4. The method according to claim 1, characterized in that: The ratio of dichloromethane:methanol is 100:1-75:1-50:1-25:1-15:1-10:1-5:1-2:1-1:1-0:

1.

5. The method according to claim 1, characterized in that: The ratio of methanol: water is 1:1-3:2-7:3-4:1-9:1-1:

0.

6. The method according to claim 1, characterized in that The detection wavelengths of semi-preparative HPLC are 220nm, 254nm, 280nm, and 300nm.

7. The method according to claim 1, characterized in that The ratio of acetonitrile to water is 5% to 30% to 95% to 70%.

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