CN103169727B - General-flavonoid compound in chionanthus as well as preparation method and application thereof - Google Patents

Abstract

The total flavonoids of tassel flower and its preparation method and application relate to the extraction, separation and application technology of active ingredients of traditional Chinese medicine, including the following steps: extraction with ethanol solution: separation by macroporous adsorption resin column chromatography: separation by silica gel column chromatography: separation by gel column chromatography: Semi-preparative high performance liquid chromatography purification: high performance liquid chromatography detection: finally, the purified sample was detected by high performance liquid chromatography, and the specific composition and content of total flavonoids in tassel flower were accurately determined, and its antioxidant capacity was systematically studied. Effect. The method used in the invention has high extraction purity, is easy to industrialize, and the total flavonoids obtained by extraction have excellent antioxidant activity.

Description

Total flavonoids of tassel flower and its preparation method and application

technical field

The invention relates to extraction, separation and application technology of effective components of traditional Chinese medicines, in particular to total flavonoids of tassel flower and its preparation method and application.

Background technique

Tassel is a plant of the genus Tassel in the family Oleaceae, and it is a national second-class protected plant. Tassels are mainly distributed in the middle and lower reaches of the Yellow River and its south [Yang Yumei, Hu Jiang. Propagation, cultivation and garden application of tassel tree[J]. Shandong Forestry Science and Technology, 2005(1):56]. Tassel is a medicinal and edible plant. It is recorded in "Chinese Economic Flora" that the buds and leaves of Oleaceae plant tassel tree have the effect of relieving heat and thirst, and are mainly used to treat heatstroke. The folks have a history of picking young leaves and flowers of tassels in spring to make tea for drinking. The tea made from tassel flowers is called "glutinous rice flower tea", which is fragrant and refreshing, and has a unique flavor [Hu Shicai. Branches and leaves brewing method [J]. Forestry Science and Technology Development, 1991, 3:16; Fang Shaokun, Du Qingzhi, Zheng Dong. Breeding and application of tassel tree[J]. Land Greening, 2005, 9:45-46]. Tassel "glutinous rice scented tea" has a history of more than one thousand years according to historical records. Lu Yu said in "The Classic of Tea": "The top one produces rotten rocks, the middle one produces oak soil, and the bottom one produces loess. The wild ones go up, and the garden ones come next." . "Glutinous rice scented tea" grows naturally and contains rich substances beneficial to human health. The content of vitamins, amino acids, catechin polyphenols, caffeine, and tea tannins are all high. Drinking this tea can eliminate accumulated food, clear internal fire, sterilize and treat dysentery, Smooth Qi and collaterals, relieve heat and diuresis, remove greasy blood pressure, eliminate fatigue, lose weight and skin care. Tea dregs can cure stomach problems and diarrhea in children, and have medicinal value. Tassel flower, as a precious folk tea raw material, is receiving more and more attention because of its special flavor and function. Existing studies have clarified that tassel flowers contain more flavonoids, and it has been proved that these substances have better antioxidant effects. However, only the content of the total flavonoids has been extracted and measured, and the extraction purity is not high. It is not clear how to determine the specific substances and composition ratios in it, and the anti-aging and antioxidant effects of the total flavonoids are also uncertain. It is not clear; and in the preparation method of total flavonoids, more toxic solvents with higher cost are used, which is not only unfavorable for industrial production but also harmful to human health.

Contents of the invention

The technical problem to be solved by the present invention is to provide the total flavonoids of tassel flower with definite components and its preparation method and application, so that the preparation method is economical and simple, easy for industrial production, and the extracted total flavonoid product of tassel flower is high in purity, specifically The composition ratio of the substances is clear, and the antioxidant effect of the total flavonoids is clear.

In order to achieve the above object, the technical scheme adopted in the present invention is: a kind of preparation method of total flavonoids of tassel flower, comprises the following steps:

(1) Ethanol solution extraction:

Dry and pulverize the tassel flower medicinal material, add ethanol with a mass concentration of 60-80% to soak, heat and reflux for extraction for 3-5 hours, filter to obtain the filtrate and residue, and then heat and reflux the residue with a mass concentration of 60-80% ethanol for 2 Once, each time for 3-5 hours, the filtrate was combined, and the filtrate was evaporated and concentrated under reduced pressure to obtain an extract;

(2) Macroporous adsorption resin column chromatographic separation:

Dissolve the obtained extract in ethanol with a mass concentration of 10-30%, filter, fully absorb the filtrate with a macroporous adsorption resin, pack it into a column, and use volume concentrations of 10%, 20%, 30%, 40%, 50% %, 60%, 80% and 95% ethanol-water system eluent for gradient elution, collect ethanol volume concentration of 50-60% ethanol-water eluted fractions, concentrate under reduced pressure to an extract, and extract The paste is dried under vacuum and reduced pressure at 50-60°C to obtain the crude total flavonoids of tassel flower;

(3) Silica gel column chromatographic separation:

The crude flavonoids from Tassel Flowers were separated by silica gel column chromatography, followed by gradient elution with CHCl ₃ : CH ₃ OH volume ratios of 1: 9, 1: 7, 1: 5, and 1: 3 as mobile phases. Rutin was used as a control, and the content of the total flavonoids of Tassel Flower was measured by ultraviolet spectroscopy, and the fractions containing more than 60% of the total flavonoids of Tassel Flower were collected;

(4) Gel column chromatography separation:

The sample separated by silica gel column chromatography was separated by gel column chromatography, and was eluted isocratically with CHCl ₃ : CH ₃ OH volume ratio of 2: 1. The eluate was collected, concentrated under reduced pressure, and dried;

(5) Semi-preparative HPLC purification:

The sample separated by gel column chromatography was purified by semi-preparative high-performance liquid chromatography, the eluate was collected, concentrated under reduced pressure, and dried to obtain the total flavonoids of tassel flower. The chromatographic conditions were column ODS-A, 20×250mm, flow The phase is CH ₃ OH: H ₂ O, the volume ratio is 7:3;

(6) High performance liquid chromatography detection:

Finally, the purified sample is detected by high performance liquid chromatography, and the chromatographic conditions are:

Chromatograph: Agilent1100 high performance liquid chromatograph,

Chromatographic column: Agilent Zorbax SB C ₁₈ column, 4.6×250 mm, 5 μm,

Mobile phase: methanol-acetonitrile-0.4% glacial acetic acid, the volume ratio is 30: 10: 60,

Flow rate: 1.0 mL/min,

Column temperature: 25°C,

Detection wavelength: 350 nm.

The ethanol soaking time in the step (1) is 8-12 hours.

In the step (1), when heating and refluxing for the first time, the amount of ethanol added is 10-12 times the mass of the medicinal material of Tassel Flowers; 8-10 times.

The macroporous adsorption resin in the step (2) is D101 or AB-8 type macroporous adsorption resin.

Total flavonoids from tassel flower, consisting of quercetin-3-methoxy-7-O-α-L-arabino(1-6)-β-D-glucoside, eriodictin-7-O-β -D-glucoside, myricetin-3-O-β-D-glucoside, luteolin-7-O-β-D-glucoside and kaempferol-3-O-β-D-glucoside 5 kinds The composition of flavonoids is 5-8%, 8-12%, 3-6%, 30-50% and 20-30% by mass respectively.

The use of the total flavonoids of Tassel Flowers, the total flavonoids of Tassel Flowers has antioxidant activity, and has the purposes of scavenging superoxide anion free radicals, DPPH free radicals, hydroxyl free radicals and resisting liposome peroxidation.

The beneficial effects brought by the present invention are:

1. The present invention uses ethanol extraction, macroporous adsorption resin column chromatography, silica gel column chromatography, gel column chromatography and semi-preparative high-performance liquid chromatography to extract, separate and purify the total flavonoids in Tassel Flowers, and the extraction purity is high , the content of impurities is less, and the content of total flavonoids after separation and purification is greater than 90%;

2. The purified sample was detected by high performance liquid chromatography, and the specific material components and composition ratios of the total flavonoids in Tassel Flowers were accurately determined. The obtained total flavonoids had clear components and controllable quality. Active ingredients and mass percentages are (1): quercetin-3-methoxy-7-O-α-L-arabino(1-6)-β-D-glucoside (5-8 %), (2): North American eriodictine-7-O-β-D-glucoside (8-12%), (3): Myricetin-3-O-β-D-glucoside (3-6%), (4): Luteolin-7-O-β-D-glucoside (30-50%) and (5): Kaempferol-3-O-β-D-glucoside (20-30%);

3. The macroporous adsorption resin used can be recycled repeatedly. The extraction with organic solvent ethanol has the characteristics of large extraction amount and simple process. It is eluted with water and ethanol as solvent, which is economical and simple, easy to industrialize, and has little harmful effect on human body;

4. The total flavonoids extracted from Tassel Flowers have excellent antioxidant activity, and have significant ability to scavenge superoxide anion free radicals, DPPH free radicals, hydroxyl free radicals and anti-liposome peroxidation.

Description of drawings

Fig. 1 is the chemical structural formula of quercetin-3-methoxy-7-O-α-L-arabino (1-6)-β-D-glucoside;

Fig. 2 is the chemical structural formula of North American eriodictine-7-O-β-D-glucoside;

Fig. 3 is the chemical structural formula of myricetin-3-O-β-D-glucoside;

Fig. 4 is the chemical structural formula of luteolin-7-O-β-D-glucoside;

Fig. 5 is the chemical structural formula of kaempferol-3-O-β-D-glucoside;

Fig. 6 is the HPLC spectrogram of total flavonoids reference substance;

Fig. 7 is the HPLC spectrogram of total flavonoids of tassel flower obtained in Example 1 of the present invention;

Fig. 8 is the HPLC spectrogram of total flavonoids of tassel flower obtained in Example 2 of the present invention;

Fig. 9 is the HPLC spectrogram of the total flavonoids of Tassel Flower obtained in Example 3 of the present invention.

1, 2, 3, 4 and 5 in Figure 6-9 represent quercetin-3-methoxy-7-O-α-L-arabino(1-6)-β-D-glucoside, North American Erionidin-7-O-β-D-glucoside, Myricetin-3-O-β-D-glucoside, Luteolin-7-O-β-D-glucoside and Kaempferol-3-O - beta-D-glucoside.

Detailed ways

Composition and Structure Determination of Flavonoids from Tassel Flower

(1) Extraction, separation and purification of flavonoids in tassel flower

Take 10 kg of naturally dried tassel flowers, crush them, pass through a 40-mesh sieve, soak and extract with 95% ethanol for 3 times, each time for 6 days, and collect the extract obtained each time. The extract was concentrated under reduced pressure at a temperature lower than 60°C to obtain 3200 grams of the total extract. Suspend 3000 g of the obtained extract in 20 L of distilled water, and repeatedly extract with petroleum ether, ethyl acetate, and n-butanol in sequence. The amount of each extract is 50 L, and the extracts are respectively concentrated under reduced pressure. Obtained 2.3 grams of petroleum ether extracts, 107 grams of ethyl acetate extracts, and 1500 grams of n-butanol extracts.

The obtained n-butanol extract was separated by silica gel column chromatography (1200 mm×70 mm) to obtain 52 fractions. The 13th fraction, namely fraction 13, was eluted with chloroform-methanol (volume ratio 2:1), and 15 fractions were obtained, of which fraction 8 was recrystallized to obtain compound 3 (100.3 mg), and the remaining fraction after recrystallization Compound 2 (84.5 mg) was obtained from the mother liquor by PTLC; Fractions 19-22 were combined and then separated by a chromatographic column (1200 mm×40 mm) and separated with chloroform-methanol (9:1, 7:1, 5:1, 3: 1, 2:1, 1:1, volume ratio) for gradient elution, and 21 fractions were obtained. Fractions 8-11 were combined and subjected to semi-preparative liquid chromatography (20 mm×250 mm, 5 μm, methanol : water=40:60, volume ratio) to obtain compound 1 (12.1 mg).

The obtained ethyl acetate extract was applied to a silica gel column (1200 mm×70 mm), and gradient elution was carried out with different concentrations of petroleum ether-ethyl acetate (4:1-0:1, volume ratio). 500 mL received 1 portion, detected by TLC, combined to obtain 43 fractions. The 36th fraction, namely fraction 36, was separated by a chromatographic column (1200 mm×35 mm), and gradient eluted with chloroform-methanol (9:1-5:1, volume ratio), and finally combined with Sephadex LH-20 chromatographic (1200 mm×40 mm) were purified to obtain compound 4 (85.3 mg) and compound 5 (451 mg).

(2) Structural identification of the extracted flavonoids

Compound 1 is a yellow amorphous powder, mp 181~183°C, magnesium hydrochloride powder reaction is positive, molish reaction is positive, and glucose and arabinose are detected by paper chromatography after hydrochloric acid hydrolysis. ESI-MS m/z : 593 [MH] ^- (molecular formula is C ₂₇ H ₃₀ O ₁₅ ). ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 12.72 (5-OH), 7.61 (1H, d, J = 2.0 Hz, 2'-H), 7.49 (1H, dd, J = 8.4 Hz, 2.0 Hz , 6'-H), 6.95 (1H, d, J = 8.4 Hz, 5'-H), 6.76 (1H, d, J = 2.0 Hz, 8-H), 6.46 (1H, d, J = 2.0 Hz , 6-H), 5.11 (1H, d, J = 7.6 Hz, 1"-H), 4.74 (1H, d, J = 2.0 Hz, 1"'-H), 3.81 (3H, s, 3-OCH ₃ ). ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ: 156.2 (C-2), 138.1 (C-3), 178.3 (C-4), 161.2 (C-5), 99.4 (C-6), 163.1 (C-7), 94.6(C-8), 156.2(C-9), 106.1(C-10), 121.0(C-1'), 115.9(C-2'), 145.4(C-3') , 1491(C-4'), 116.1(C-5'), 120.9(C-6'), 100.0(C-1''), 73.3(C-2"), 76.4(C-3"), 70.0 (C-4"), 75.7(C-5"), 67.1(C-6"), 108.7(C-1'''), 82.23(C-2"'), 77.4(C-3"' ), 84.2 (C-4"'), 61.5 (C-5"'). The above data and literature [Choi Y H, Lim Y H, Yeo H, et al. A flavonoid diglycoside from Lepisorus ussuriensis . Phytochemistry, 1996, 43( 5): 1111-1114] reported basically the same, so identify compound 1 as quercetin-3-methoxyl-7-O-α-L-arabino (1-6)-β-D-glucoside, structural formula As shown in Figure 1.

Compound 2 was obtained as yellow crystals (obtained by crystallization from methanol). mp 156~158°C, magnesium hydrochloride powder reaction was positive, molish reaction was positive, and glucose was detected by paper chromatography after hydrochloric acid hydrolysis. ESI-MS m/z : 449 [MH] ^- (molecular formula is C ₂₁ H ₂₂ O ₁₁ ). ¹ H-NMR (DMSO-d ₆ , 400 MHz) δ: 6.91 (1H, s, H-5'), 6.72 (2H, s, H-2', 6'), 6.19 (1H, s, H- 6), 6.1 (1H, s, H-8), 5.33 (1H, dd, H-2), 2.76 (1H, dd, J =17.1 Hz, H-3), 4.98 (1H, d, glc-1 '). ¹³ C-NMR (100 MHz, DMSO-d ₆ ) δ: 80.7 (C-2), 44.1 (C-3), 198.5 (C-4), 164.9 (C-5), 97.9 (C-6), 167.0(C-7), 96.9(C-8), 164.5(C-9), 103.75(C-10), 131.5(C-1'), 116.2(C-2'), 146.9(C-3' ), 114.7(C-4'), 119.3(C-5'), 104.9(C-6'), 101.2(C-1"), 74.6(C-2"), 77.8(C-3"), 71.1 (C-4"), 78.2 (C-5"), 62.3 (C-6"). The above data and literature [Zhao Lei, Wu Dinghui, Yu Xiaohui, et al. Dihydroflavonoids in Rhododendron Xiuya[J]. Chinese Journal of Traditional Chinese Medicine, 2010, 35 (6): 722-724] is relatively consistent, so compound 2 is identified as eriodictydin-7-O-β-D-glucoside, and the structural formula is shown in Figure 2.

Compound 3 is a yellow powder (obtained by crystallization from methanol), mp 193~195°C. The magnesium hydrochloride powder reaction was positive, the molish reaction was positive, and glucose was detected by paper chromatography after hydrochloric acid hydrolysis. ESI-MS m/z : 479 [MH] ^- (molecular formula is C ₂₁ H ₂₀ O ₁₃ ). ¹ H-NMR (DMSO-d ₆ , 400 MHz) δ: 12.63 (1H, s, 5-OH), 8.68 (1H, s, 3'-OH), 7.81 (1H, s, 4'-OH), 7.23 (1H, s, H-2', 6'), 6.37 (1H, d, J =1.9 Hz, H-8), 6.21 (1H, d, J =1.9 Hz, H-6), 5.47 (1H , d, J =7.6 Hz, H-1''). ¹³ C-NMR (DMSO-d ₆ , 100 MHz) δ: 156.4 (C-2), 133.4 (C-3), 177.4 (C-4), 161.3 (C-5), 98.9 (C-6), 164.6 (C-7), 93.5 (C-8), 156.2 (C-9), 103.7 (C-10), 120.1 (C-1'), 108.6 (C-2'), 145.5 (C-3' ), 136.7 (C-4'), 145.5 (C-5'), 108.6 (C-6'), 100.9 (C-1''), 73.9 (C-2"), 76.6 (C-3") , 69.8 (C-4"), 77.6 (C-5"), 61.1 (C-6"). The above data are basically consistent with those reported in the literature [Zhao Liqin, Zhang Xiaoping, Zhang Chaofeng. Isolation and identification of chemical components of Polygonum vine[J] , Food Science, 2012, 33(7):1-5; Li Chunmei, Wang Tao, Zhang Yi, Gao Xiumei, etc. Isolation and identification of chemical constituents of the traditional Chinese medicine hollyhock flower (Ⅰ) [J]. Journal of Shenyang Pharmaceutical University, 2010, 27(9):711-714], identified compound 3 as myricetin-3-O-β-D-glucoside, the structural formula is shown in Figure 3.

Compound 4 is a yellow powder (obtained by crystallization from methanol), mp 259~261°C. The magnesium hydrochloride powder reaction was positive, the molish reaction was positive, and glucose was detected by paper chromatography after hydrochloric acid hydrolysis. ESI-MS m/z : 447 [MH] ^- (molecular formula is C ₂₁ H ₂₀ O ₁₁ ). ¹ H-NMR (Pyridine-d ₅ , 400 MHz) δ: 12.11 (1H, s, 5-OH), 8.71 (1H, s, 3'-OH), 7.89 (1H, s, 4'-OH), 7.49(1H, dd, J =8.7, 1.7 Hz, H-6'), 7.27(1H, d, J =1.7 Hz, H-2'), 6.98(1H, d, J =8.7 Hz, H-5 '), 6.92 (1H, s, H-3), 6.83 (1H, d, J = 1.9 Hz, H-8), 5.82 (1H, d, J = 1.9 Hz, H-6). ¹³ C-NMR (Pyridine-d ₅ , 100 MHz) δ: 164.0 (C-2), 101.8 (C-3), 182.8 (C-4), 157.9 (C-5), 100.6 (C-6), 165.3(C-7), 95.3(C-8), 162.6(C-9), 104.1(C-10), 122.7(C-1'), 113.30(C-2'), 147.8(C-3' ), 150.1(C-4'), 116.9 (C-5'), 119.7 (C-6'), 95.3(C-1"), 79.2(C-2"), 78.5(C-3"), 74.8(C-4"), 71.1(C-5"), 62.3(C-6"). The above data are basically consistent with those reported in the literature [Long Fei, Deng Liang, Chen Yang. Study on the chemical constituents of privet flower [J]. West China Pharmaceutical Journal, 2011, 26(2):97-100], identified compound 4 as luteolin-7-O-β-D-glucoside, the structural formula is shown in Figure 4.

Compound 5 is a yellow powder (obtained by crystallization from methanol). mp 229~231℃. The magnesium hydrochloride powder reaction was positive, the molish reaction was positive, and glucose was detected by paper chromatography after hydrochloric acid hydrolysis. ESI-MS m/z : 447 [MH] ^- (molecular formula is C ₂₁ H ₂₀ O ₁₁ ). ¹ H-NMR (DMSO-d ₆ , 400 MHz) δ: 6.2 (1H, d, J = 2.0 Hz), 6.43 (1H, d, J = 2.0 Hz), 8.07 (1H, dd, J = 2.0 Hz, 7.2 Hz), 6.86 (1H, dd, J =2.0 Hz, 7.0 Hz), 6.86 (1H, dd, J =2.0 Hz, 7.0 Hz), 8.07 (1H, dd, J =2.0 Hz, 7.2 Hz), 5.35 (1H, d, J = 7.6 Hz), 12.61 (1H, s), 10.87 (1H, s). ¹³ C-NMR (DMSO-d ₆ , 100 MHz) δ: 156.3 (C-2), 133.1 (C-3), 177.4 (C-4), 161.1 (C-5), 98.71 (C-6), 164.3(C-7), 93.65(C-8), 156.1(C-9), 103.8(C-10), 120.8(C-1'), 130.8(C-2'), 115.0(C-3' ), 159.9 (C-4'), 115.0(C-5'), 130.9(C-6'), 100.8(C-1''), 74.17(C-2"), 76.36(C-3") , 69.83 (C-4"), 77.46 (C-5"), 60.77 (C-6"). The above data are basically consistent with those reported in the literature [Long Fei, Deng Liang, Chen Yang. Research on the chemical composition of privet flower [J]．West China Journal of Pharmaceutical Sciences, 2011, 26(2):97-100], identified compound 5 as kaempferol-3-O-β-D-glucoside, the structural formula is shown in Figure 5.

Example 1

Take 1 kg of dried and crushed Funiu Mountain Tassel Flowers in Western Henan, add 12 kg of mass concentration 60% ethanol to soak for 8 hours, heat and reflux for extraction for 4 hours, filter to obtain the filtrate and residue, and add 10 kg of 80% ethanol to the residue for reflux extraction 2 times, each time for 4 hours, the combined filtrates were evaporated and concentrated under reduced pressure, and ethanol was recovered to obtain 126 grams of extract. Dissolve the ethanol extract with 10% ethanol, filter, fully absorb the filtrate with 1000 grams of D101 type macroporous adsorption resin, pack it into a column, and use ethanol-water (10%, 20%, 30%, 40%, 50%, 60%, 80% and 95%) gradient elution, collect 50-60% ethanol eluted fractions, concentrate under reduced pressure to an extract, and dry the extract under vacuum at 50°C to obtain dry total flavonoid powder 10 gram. The sample separated by macroporous adsorption resin column chromatography was loaded and separated by silica gel column chromatography, and the mobile phase gradient elution was carried out with a volume ratio of CHCl ₃ : CH ₃ OH of 1: 9, 1: 7, 1: 5, and 1: 3 , adopt rutin as contrast, measure the content of tassel flower total flavonoids with ultraviolet spectrum, collect the distillate section that contains tassel flower total flavonoids greater than 60%; The sample after silica gel column chromatography is separated by gel column chromatography, Use isocratic elution with CHCl ₃ : CH ₃ OH volume ratio of 2: 1, collect the eluent, concentrate under reduced pressure, and dry; use semi-preparative high-performance liquid chromatography to purify the sample separated by gel column chromatography, collect and elute liquid, concentrated under reduced pressure, and dried to obtain the total flavonoids of Tassel Flowers. The chromatographic conditions were chromatographic column ODS-A, 20×250 mm, the mobile phase was CH ₃ OH:H ₂ O, and the volume ratio was 7:3; finally, the purified The final sample was detected by high-performance liquid chromatography, and the chromatographic conditions were as follows: chromatographic instrument: Agilent1100 high-performance liquid chromatograph, chromatographic column: Agilent Zorbax SB C ₁₈ chromatographic column (4.6×250 mm, 5 μm), mobile phase: methanol-acetonitrile -0.4% glacial acetic acid, volume ratio 30: 10: 60, flow rate: 1.0 mL/min, column temperature: 25°C, detection wavelength: 350 nm, quercetin-3-methoxy -7-O-α-L-Arabic (1-6)-β-D-glucoside, Eriodin-7-O-β-D-glucoside, Myricetin-3-O-β-D- The mass percentages of glucoside, luteolin-7-O-β-D-glucoside and kaempferol-3-O-β-D-glucoside flavonoids were 6.2%, 10.1%, 5.2%, respectively, 46.5%, 25.2% (Fig. 1, Fig. 2, Fig. 3, Fig. 4 and Fig. 5 are the chemical structural formulas of these five flavonoid compounds respectively), the total flavonoid content is greater than 93.2%, as shown in Fig. 7, Fig. 6 is the quercetin Cortexin-3-methoxy-7-O-α-L-arabino(1-6)-β-D-glucoside, North American eriodin-7-O-β-D-glucoside, myricetin- 3-O-β-D-glucoside, luteolin-7-O-β-D-glucoside and kaempferol-3-O-β-D-glucoside were formulated into a mixture according to a certain concentration as Reference substance, the high-performance liquid chromatogram of the reference substance obtained by testing.

Example 2

Take 2 kg of dried and crushed Funiu Mountain Tassel Flowers in Western Henan, add 22 kg of mass concentration 70% ethanol to soak for 11 hours, heat and reflux for extraction for 5 hours, filter to obtain the filtrate and residue, and add 18 kg of 70% ethanol to the residue for reflux extraction 2 times, each time for 5 hours, the combined filtrates were evaporated and concentrated under reduced pressure, and ethanol was recovered to obtain 284 grams of extract. The ethanol extract is dissolved with 20% ethanol, filtered, and the filtrate is fully adsorbed with 2000 grams of AB-8 type macroporous adsorption resin, packed into a column, and washed with ethanol-water (10%, 20%, 30%, 40%, 50% %, 60%, 80% and 95%) gradient elution, collect 50-60% ethanol eluted fractions, concentrate under reduced pressure to an extract, and dry the extract under vacuum at 60°C to obtain dry total flavonoids Powder 18.6 g. The sample separated by macroporous adsorption resin column chromatography was loaded and separated by silica gel column chromatography, and the mobile phase gradient elution was carried out with a volume ratio of CHCl ₃ : CH ₃ OH of 1: 9, 1: 7, 1: 5, and 1: 3 , adopt rutin as contrast, measure the content of tassel flower total flavonoids with ultraviolet spectrum, collect the distillate section that contains tassel flower total flavonoids greater than 60%; The sample after silica gel column chromatography is separated by gel column chromatography, Use isocratic elution with CHCl ₃ : CH ₃ OH volume ratio of 2: 1, collect the eluent, concentrate under reduced pressure, and dry; use semi-preparative high-performance liquid chromatography to purify the sample separated by gel column chromatography, collect and elute liquid, concentrated under reduced pressure, and dried to obtain the total flavonoids of Tassel Flowers. The chromatographic conditions were chromatographic column ODS-A, 20×250 mm, the mobile phase was CH ₃ OH:H ₂ O, and the volume ratio was 7:3; finally, the purified The final sample was detected by high-performance liquid chromatography, and the chromatographic conditions were as follows: chromatographic instrument: Agilent1100 high-performance liquid chromatograph, chromatographic column: Agilent Zorbax SB C ₁₈ chromatographic column (4.6×250 mm, 5 μm), mobile phase: methanol-acetonitrile -0.4% glacial acetic acid, volume ratio 30: 10: 60, flow rate: 1.0 mL/min, column temperature: 25°C, detection wavelength: 350 nm, quercetin-3-methoxy -7-O-α-L-Arabic (1-6)-β-D-glucoside, Eriodin-7-O-β-D-glucoside, Myricetin-3-O-β-D- The mass percentages of glucoside, luteolin-7-O-β-D-glucoside and kaempferol-3-O-β-D-glucoside flavonoids were 8.0%, 9.1%, 5.0%, respectively, 44.1%, 27.2%, the total flavonoid content is greater than 93.4%, as shown in Figure 8.

Example 3

Take 4 kg of dried and crushed Funiu Mountain tassel flowers in Western Henan, add 40 kg of mass concentration 80% ethanol to soak for 12 hours, heat and reflux for extraction for 3 hours, filter to obtain the filtrate and residue, and add 32 kg of 60% ethanol to the residue for reflux extraction 2 Once, each time for 3 hours, the combined filtrates were evaporated and concentrated under reduced pressure, and ethanol was recovered to obtain 605 grams of extract. The ethanol extract is dissolved with 30% ethanol, filtered, and the filtrate is fully adsorbed with 5000 grams of D101 type macroporous adsorption resin, packed into a column, and washed with ethanol-water (10%, 20%, 30%, 40%, 50%, 60%, 80% and 95%) gradient elution, collected 50-60% ethanol eluted fractions, concentrated under reduced pressure to an extract, and dried the extract under vacuum at 55°C to obtain dry total flavonoid powder 37.6 gram. The sample separated by macroporous adsorption resin column chromatography was loaded and separated by silica gel column chromatography, and the mobile phase gradient elution was carried out with a volume ratio of CHCl ₃ : CH ₃ OH of 1: 9, 1: 7, 1: 5, and 1: 3 , adopt rutin as contrast, measure the content of tassel flower total flavonoids with ultraviolet spectrum, collect the distillate section that contains tassel flower total flavonoids greater than 60%; The sample after silica gel column chromatography is separated by gel column chromatography, Use isocratic elution with CHCl ₃ : CH ₃ OH volume ratio of 2: 1, collect the eluent, concentrate under reduced pressure, and dry; use semi-preparative high-performance liquid chromatography to purify the sample separated by gel column chromatography, collect and elute liquid, concentrated under reduced pressure, and dried to obtain the total flavonoids of Tassel Flower. The chromatographic conditions were chromatographic column ODS-A, 20×250mm, the mobile phase was CH ₃ OH:H ₂ O, and the volume ratio was 7:3; finally, the purified The samples were detected by high-performance liquid chromatography, and the chromatographic conditions were as follows: chromatographic instrument: Agilent1100 high-performance liquid chromatograph, chromatographic column: Agilent Zorbax SB C ₁₈ chromatographic column (4.6×250 mm, 5 μm), mobile phase: methanol-acetonitrile- 0.4% glacial acetic acid, volume ratio 30: 10: 60, flow rate: 1.0 mL/min, column temperature: 25°C, detection wavelength: 350 nm, and quercetin-3-methoxy- 7-O-α-L-Arabic (1-6)-β-D-glucoside, Eriodin-7-O-β-D-glucoside, Myricetin-3-O-β-D-glucoside The mass percentages of glucoside, luteolin-7-O-β-D-glucoside and kaempferol-3-O-β-D-glucoside flavonoids were 6.9%, 11.3%, 4.9%, 45.1% %, 22.2%, the total flavonoid content is greater than 90.4%, as shown in Figure 9.

pharmacological research

experiment material:

Tassel flower total flavonoids products (quercetin-3-methoxy-7-O-α-L-arabino(1-6)-β-D-glucoside, eriodictin-7-O-β-D The mass percent of -glucoside, myricetin-3-O-β-D-glucoside, luteolin-7-O-β-D-glucoside and kaempferol-3-O-β-D-glucoside The contents are 8.0%, 9.1%, 5.0%, 44.1%, and 27.2%, respectively, and the total flavonoid content is greater than 93.4%. See Example 2 for the preparation method.

Sodium nitrite, aluminum nitrate, sodium hydroxide, 1,1-diphenyl-2-picrylphenylhydrazine (DPPH), reduced nicotinamide adenine dinucleotide (NADH), phenazine methyl sulfate ( PMS), Nitrotetrazolium Chloride (NBT), Absolute Ethanol, Hydrogen Peroxide, Ascorbic Acid (VC), Salicylic Acid, Tris(Tris), Trichloroacetic Acid (TCA), Thiobarbe Tauric acid (TBA), hydrochloric acid (HCl) are analytically pure, soybean lecithin (>90%).

test methods:

(1) Determination method for scavenging superoxide anion radicals (O ^- ₂ ·)

Dilute the total flavonoids of Tassel Flowers into different concentration gradients with Tris-HCl buffer solution at pH 8.0 and concentration 50 mol/L, take 1.5 mL of different gradient flavonoid solutions, and then add 0.5 mL NBT (300 μmol/L, pH8.0 Tris-HCl buffer), 0.5 mL NADH (468 μmol/L, pH8.0 Tris-HCl buffer), 0.5 mL PMS (60 μmol/L, pH8.0 Tris -HCl buffer solution), after mixing, put it in a water bath at 25°C for 5 minutes, take it out and measure the absorbance at a wavelength of 560 nm, and replace the flavone solution with buffer solution in the blank control group. The formula for calculating superoxide anion radical scavenging rate is as follows:

E(O ^- ₂ )(%)=(1－A ₁ /A ₀ )×100

Where: E(O ^- ₂ ·) is the scavenging rate (%) of flavonoids to O ^- ₂ ·; A ₁ is the absorbance of flavonoids; A ₀ is the absorbance of blank.

(2) Determination method for scavenging DPPH free radicals

Dilute the total flavonoids of Tassel Flower into different concentration gradients, take 2 mL of the flavonoid solutions of the above concentrations in a test tube, then add 2 mL of DPPH solution with a concentration of 0.04 mg/mL, mix well, and react for 20 minutes, 3500 r/ Min centrifuge for 10 minutes, take the supernatant and measure its absorbance at a wavelength of 517 nm as A _i ; take 2 mL of the flavonoid solution of the above concentration in a test tube, add 2 mL of absolute ethanol, react for 20 minutes, 3500 r /min centrifugation for 10 minutes, the supernatant was taken at a wavelength of 517 nm to measure its absorbance as A _j ; taking 2 mL of 0.04 mg/mL DPPH and 2 mL of absolute ethanol as a reference, its absorbance was recorded as A ₀ . The formula for calculating the DPPH free radical scavenging rate is as follows:

E(DPPH)(%)=[1－(A _i -A _j )/A ₀ ]×100

In the formula: E(DPPH) is the scavenging rate (%) of flavonoids to DPPH free radicals; A ₀ is the absorbance of 2 mL DPPH solution+2 mL absolute ethanol; _Ai is the absorbance of 2 mL DPPH solution+2 mL flavone solution ; A _j is the absorbance of 2 mL absolute ethanol + 2 mL flavonoids.

(3) Determination method for scavenging hydroxyl radical ( OH)

Prepare the total flavonoids of Tassel Flowers into different concentration gradients with double-distilled water, take 2 mL of the above-mentioned flavonoid solutions, add 2 mL of 6 mmol/L FeSO ₄ and 2 mL of 6 mmol/L H ₂ O ₂ in sequence, and mix well After standing still for 10 minutes, add 2 mL of 6 mmol/L salicylic acid, mix well, let stand for 30 minutes, measure its absorbance at a wavelength of 510 nm and record it as A _i , when double distilled water is used instead of salicylic acid The absorbance is recorded as A _j . For the blank control group, double distilled water was used instead of the flavone solution, and the absorbance was recorded as A ₀ . The formula for calculating the hydroxyl radical scavenging rate is as follows:

E(·OH)(%)= [1－(A _i -A _j )/A ₀ ]×100

In the formula: E(·OH) is the scavenging rate of hydroxyl radicals (%); A _i is the absorbance of the total flavonoids of Tassel Flower; A ₀ is the blank absorbance; A _j is the total flavonoids of Tassel Flower without salicylic acid The absorbance of the product.

(4) Determination of anti-liposome peroxidation activity

Add 1.00 mL of tassel flower total flavonoid product solution of different mass concentrations, 1.0 mL of lecithin solution, 1.0 mL of 0.4 mmol/L FeSO ₄ into a 10 mL colorimetric tube, mix well, place in a water bath at 37°C for 60 minutes, then add Mix 2.0 mL of trichloroacetic acid (TCA)-thiobarbituric acid (TBA)-hydrochloric acid (HC1), rapidly cool in a water bath at 100°C for 15 minutes, centrifuge at 3000 r/min for 10 minutes, and take the supernatant at a wavelength of 532 The absorbance was measured at nm, with distilled water as a reference and VC as a control, and the inhibition rate was calculated.

Inhibition rate/%＝[1-(A _X -A _X0 )/A ₀ ]×100

In the formula: A ₀ is the absorbance of the blank control solution; A _X is the absorbance of the flavone solution added; A _X0 is the absorbance of the flavone solution without lecithin.

result:

(1) Scavenge superoxide anion radicals (O ₂ ^- ·)

Total flavonoid concentration (mg/mL) 0.01 0.02 0.04 0.08 0.16 VC clearance 25% 29% 37% 51% 83% total ketone clearance 17% 19% 26% 39% 53%

Within the selected mass concentration range, the total flavonoids of Tassel Flowers has the ability to scavenge superoxide anion free radicals. The scavenging effect on superoxide anion free radicals was enhanced with the increase of the mass concentration of total flavonoids of tassel flower, showing a dose-effect relationship. Although the O ^- ₂ · scavenging effect of the total flavonoids of Tassel Flowers is not as strong as that of VC, it has already shown a strong O ^- ₂ · scavenging effect.

(2) Scavenging DPPH free radicals

Total flavonoid concentration (μg/mL) 5 10 15 20 25 VC clearance 46% 73% 87% 90% 91% total ketone clearance 41% 57% 71% 80% 83%

The DPPH free radical is a very stable nitrogen-centered free radical, and its scavenging effect shows that the tested drug has the ability to reduce the effective concentration of hydroxyl free radicals, alkyl free radicals or peroxyl free radicals and interrupt lipid peroxidation. chain reaction effect. Although the scavenging effect of DPPH free radicals by the total flavonoids of Tassel Flowers was not as strong as that of VC, the total flavonoids of Tassel Flowers still showed a strong ability to scavenge DPPH free radicals.

(3) Scavenging hydroxyl radicals (·OH)

Total flavonoid concentration (mg/mL) 0.05 0.1 0.2 0.4 0.8 VC clearance 45% 49% 66% 74% 92% total ketone clearance twenty one% 34% 47% 56% 71%

Hydroxyl free radicals are the strongest oxidants, which can react with almost all cell components in living organisms, and are very harmful to the body. Compared with VC, the total flavonoids of tassel flower had the ability to remove ·OH, but its effect was weaker.

(4) Anti-liposome peroxidation activity

Total flavonoid concentration (μg/mL) 50 100 150 200 250 VC clearance none none none none none total ketone clearance 39% 47% 51% 60% 63%

Lipid peroxidation damage is related to the occurrence of many diseases, such as tumors, aging, cardiovascular and cerebrovascular diseases, autoimmune diseases and so on. The total flavonoids of Tassel Flowers can inhibit the peroxidation of lecithin liposomes induced by Fe ²⁺ , and the inhibition rate increases with the increase of flavonoid mass concentration. Within the range of experimental mass concentration, VC has no anti-liposome peroxidation ability, which may be because VC is a water-soluble polar substance, which is mainly concentrated in the water phase in the emulsified lipid system, thereby affecting its inhibitory effect.

Claims (1)

1. a preparation method of tassel flower total flavonoids, is characterized in that: comprise the following steps: (1) Ethanol solution extraction: Dry and pulverize the tassel flower medicinal material, add ethanol with a mass concentration of 60-80% to soak for 8-12 hours, heat and reflux for extraction for 3-5 hours, filter to obtain the filtrate and residue, and then extract the residue with a mass concentration of 60-80% ethanol Heated and refluxed for 2 extractions, 3-5 hours each time, combined the filtrates, evaporated and concentrated the filtrates under reduced pressure to obtain extracts, when heated and refluxed for the first time, the amount of ethanol added was 10-12 times the mass of Tassel Flower medicinal material, and for the second time When heating and refluxing for the first time and the third time, the amount of ethanol added is 8-10 times of the quality of the tassel flower medicinal material; (2) Macroporous adsorption resin column chromatographic separation: Dissolve the obtained extract in ethanol with a mass concentration of 10-30%, filter, fully absorb the filtrate with D101 or AB-8 macroporous adsorption resin, pack it into a column, and use volume concentrations of 10%, 20%, 30% %, 40%, 50%, 60%, 80% and 95% ethanol-water system eluent for gradient elution, collect ethanol volume concentration of 50-60% ethanol-water eluted fractions, concentrate under reduced pressure to For the paste, the extract is dried under vacuum and reduced pressure at 50-60°C to obtain the crude product of total flavonoids of Tassel Flower; (3) Silica gel column chromatographic separation: The crude flavonoids from Tassel Flowers were separated by silica gel column chromatography, followed by gradient elution with CHCl ₃ :CH ₃ OH volume ratios of 1: 9, 1: 7, 1: 5, and 1: 3 as the mobile phase. Rutin was used as a control, and the content of the total flavonoids of Tassel Flower was measured by ultraviolet spectroscopy, and the fractions containing more than 60% of the total flavonoids of Tassel Flower were collected; (4) Gel column chromatography separation: The sample separated by silica gel column chromatography was separated by gel column chromatography, and was eluted isocratically with CHCl ₃ :CH ₃ OH volume ratio of 2: 1, and the eluate was collected, concentrated under reduced pressure, and dried; (5) Semi-preparative HPLC purification: The sample separated by gel column chromatography was purified by semi-preparative high-performance liquid chromatography, the eluate was collected, concentrated under reduced pressure, and dried to obtain the total flavonoids of tassel flower. The chromatographic conditions were column ODS-A, 20×250mm, flow The phase is CH ₃ OH:H ₂ O, the volume ratio is 7:3; (6) High performance liquid chromatography detection: Finally, the purified sample is detected by high performance liquid chromatography, and the chromatographic conditions are: Chromatograph: Agilent1100 high performance liquid chromatograph, Chromatographic column: Agilent Zorbax SB C ₁₈ column, 4.6×250 mm, 5 μm, Mobile phase: methanol-acetonitrile-0.4% glacial acetic acid, the volume ratio is 30: 10: 60, Flow rate: 1.0 mL/min, Column temperature: 25°C, Detection wavelength: 350 nm; The tassel flower total flavonoids prepared by the above method are composed of quercetin-3-methoxy-7-O-α-L-arabino(1-6)-β-D-glucoside, eriodictin- 7-O-β-D-glucoside, Myricetin-3-O-β-D-glucoside, Luteolin-7-O-β-D-glucoside and Kaempferol-3-O-β-D - Glucoside composition, the mass percentages are respectively 5-8%, 8-12%, 3-6%, 30-50% and 20-30%, and the above-mentioned total mass percentages are 100%.