CN111732621B - Extraction method of new flavonoid compound Hip A - Google Patents

Abstract

The invention discloses an extraction method of a new flavonoid compound Hip A, which comprises the steps of eluting sea buckthorn seed dreg extract through an MCI gel column, and then semi-preparing and separating the sea buckthorn seed dreg extract through a C18 chromatographic column, a glycol-based chromatographic column and a C18 chromatographic column in sequence to obtain the new flavonoid compound Hip A in sea buckthorn.

Description

Extraction method of new flavonoid compound Hip A

Technical Field

The invention relates to the field of extraction methods of new flavonoid compounds, in particular to an extraction method of new flavonoid compound Hip A.

Background

Sea buckthorn (Latin brand: hippophae rhamnoides Linn.) is a deciduous shrub of the genus Hippophae of the family Elaeagnaceae, and has the characteristics of drought tolerance and wind sand resistance, and can survive on salinized lands, so that the sea buckthorn shrub is widely used for water and soil conservation. Sea buckthorn is planted in great quantity in northwest of China and is used for desert greening.

Fructus Hippophae is nutritious, contains vitamins, flavonoids, triterpenes, oil and fatty acids, phenols, volatile oils, microelements, phospholipids, 5-hydroxytryptamine, and various amino acids and proteins required by human body.

Sea buckthorn seeds are commonly used for squeezing oil to prepare sea buckthorn oil, wherein the sea buckthorn oil contains 206 active substances beneficial to human bodies, 46 bioactive substances, and a large amount of vitamin E, vitamin A, flavone and the like. After oil extraction, a large amount of seabuckthorn seed meal is produced and is often discarded as waste or produced into dry powder to be used as feed, and the price is very low. The method has great economic significance if the utilization value of the sea buckthorn seed meal can be developed more.

In addition, the sea buckthorn contains abundant active substances which are not separated and identified, and if the new chemical components and the pharmacological actions of the new chemical components in the sea buckthorn can be subjected to deeper and finer research and active mechanism discussion, more natural plant-derived medicaments which can treat diseases are expected to be developed safely and effectively.

Disclosure of Invention

The invention provides a method for extracting novel flavonoid compounds from sea buckthorn seed meal, which can obtain novel compounds Hip A with various biological activities.

The invention provides an extraction method of flavonoid compounds, wherein the structure of the compounds is shown as a formula XVII (also called Hip A in the invention):

the extraction method comprises the following steps:

(1) The sea buckthorn seed meal extract is eluted by an MCI gel column, and is eluted by 15 to 25 percent of methanol aqueous solution, 35 to 45 percent of methanol aqueous solution, 55 to 65 percent of methanol aqueous solution, 75 to 85 percent of methanol aqueous solution and methanol in sequence, wherein each eluent uses 4 to 6 column volumes, and is preferably eluted by 20 percent of methanol aqueous solution, 40 percent of methanol aqueous solution, 60 percent of methanol aqueous solution, 80 percent of methanol aqueous solution and methanol in sequence;

(2) Semi-preparative separation is carried out on substances obtained by eluting 55-65% methanol aqueous solution to obtain a component SH5:

The conditions for the semi-preparative separation in step (2) include:

chromatographic column: a C18 chromatographic column; preferably 20mm x 250mm,5 μm;

mobile phase: the acidic aqueous solution was mobile phase a and acetonitrile was mobile phase B, and gradient elution was performed using the following procedure: 0min, 85-95% of mobile phase A, 15-5% of mobile phase B;60min, 75-85% of mobile phase A, 25-15% of mobile phase B; the components with retention time of 37-46 min are the components SH5;

(3) Semi-preparative separation is carried out on the component SH5 to obtain a component SH5-8:

the conditions for the semi-preparative separation in step (3) include:

chromatographic column: a glycol-based chromatographic column; preferably 21mm x 250mm,5 μm;

mobile phase: the acidic aqueous solution was mobile phase a and acetonitrile was mobile phase B, and gradient elution was performed using the following procedure: 0min, 5-0% of mobile phase A, 95-100% of mobile phase B;60min, 15-25% of mobile phase A, 85-75% of mobile phase B, and the components with retention time of 55-57 min are components SH5-8;

(4) Semi-preparative separation of component SH5-8 gives compound XVII:

the conditions for the semi-preparative separation in step (4) include:

chromatographic column: a C18 chromatographic column; preferably 21mm x 250mm,5 μm;

mobile phase: the 0.1% formic acid aqueous solution is a mobile phase A, acetonitrile is a mobile phase B, the mobile phase A and the mobile phase B are mixed according to the volume ratio of 80-90% and 20-10% to be used as the mobile phase for isocratic elution, and the component with the retention time of 25-28 min is the compound XVII.

Further, the gradient elution procedure in step (2) is: 0min,90% mobile phase A-10% mobile phase B;60min,80% mobile phase A-20% mobile phase B;

further, the gradient elution procedure in step (3) is: 0min,100% mobile phase B;60min,20% mobile phase A-80% mobile phase B.

Further, the mobile phase in the step (4) is a mixed solution of 85 percent of mobile phase A and 15 percent of mobile phase B in volume ratio, and the component with the retention time of 26-27 min is the compound XVII.

In a specific embodiment of the invention, the acidic aqueous solution in mobile phase a is a 0.1% formic acid aqueous solution.

Further, the semi-preparative separation in steps (2) to (4) further comprises at least one of the following conditions:

detection wavelength: 254nm;

flow rate: 19-21 mL/min.

Further, the sea-buckthorn seed meal extract is an ethanol extract of sea-buckthorn seed meal;

further, the preparation method of the sea buckthorn seed dreg extractum comprises the following steps: extracting sea buckthorn seed meal with a solvent at 50-80 ℃, wherein the solvent is ethanol or ethanol water solution;

further, the solvent is 60-80% ethanol water solution, preferably 70% ethanol water solution;

further, the feed liquid ratio of the sea buckthorn seed meal to the solvent is 1:15-25 g/ml, preferably 1:20g/ml.

Further, the extraction temperature is 60 ℃;

further, the extraction time is 1 to 5 hours, preferably 2 hours.

Further, the seabuckthorn seed meal extract is obtained by forward silica gel column chromatography of the material obtained by solvent extraction of seabuckthorn seed meal, wherein the eluent is a mixed solution of methylene dichloride and methanol in a water volume ratio of 6-8:1-2:0.2-0.7, preferably a mixed solution of methylene dichloride and methanol in a water volume ratio of 7:3:0.5;

further, in the forward silica gel column chromatography, the eluent is used in an amount of 15 to 30 column volumes, preferably 20 column volumes.

In the present invention, the percentages before the aqueous methanol solution and the aqueous ethanol solution refer to the volume fractions, for example, "40% aqueous methanol solution" refers to the volume fraction of 40% aqueous methanol solution, and the rest is the same.

The beneficial effects of the invention are as follows:

(1) The method adopts alcohol extraction, column chromatography and a combined semi-preparative liquid chromatograph to separate and prepare a brand new flavonoid compound Hip A from the seabuckthorn seed meal, thereby more comprehensively developing the active substances of seabuckthorn.

(2) The invention more fully digs the medicinal value of the sea buckthorn, expands the clinical application of the sea buckthorn and provides more reference basis for developing the medicine for treating the potential plant source of the neurodegenerative diseases.

Drawings

FIG. 1 is a chromatogram of analysis of each component of seabuckthorn seed meal;

FIG. 2 is a one-dimensional preparation diagram of a sample of SH5 component of sea buckthorn seed meal;

FIG. 3 is a two-dimensional analysis chart of each component sample in SH 5;

FIG. 4 is a purity analysis of compounds from peaks 1 to 19 in seabuckthorn seed meal;

FIG. 5 is a diagram of the compound Hip A ¹ H NMR spectrum;

FIG. 6 is a graphic illustration of the compound Hip A ¹³ C NMR spectrum;

FIG. 7 is a HSQC diagram of Compound Hip A;

FIG. 8 is an HMBC diagram of compound Hip A;

FIG. 9 is a HHCSY diagram of compound Hip A;

FIG. 10 is a NOESY diagram of Compound Hip A;

FIG. 11 is the effect of Oleic Acid (OA) on HepG2 cell viability;

FIG. 12 is the effect of Oleic Acid (OA) on lipid accumulation in HepG2 cells;

FIG. 13 is the effect of Oleic Acid (OA) on FAS protein expression in HepG2 cells;

FIG. 14 is the effect of compound Hip A on HepG2 cell viability;

FIG. 15 is the effect of compound Hip A on lipid accumulation in HepG2 cells;

FIG. 16 is the effect of compound Hip A on HepG2 cell adipogenic protein;

fig. 17 is a microscopic image of DoX induced H9c2 cardiomyocyte morphology (100×, n=3);

FIG. 18 shows the effect of DoX on H9c2 cardiomyocyte viabilityn＝3)；

FIG. 19 shows the NO content of DoX to H9c2 cardiomyocyte culture supernatantn＝3)；

FIG. 20 shows the LDH content level of DoX on H9c2 cardiomyocyte culture supernatant n＝3)；

FIG. 21 shows the effect of Hip A on H9c2 cardiomyocyte viabilityn＝3)；

FIG. 22 shows the level of H9c2 myocardial cell injury LDH induced by the compound Hip A vs DoXn＝3)；

FIG. 23 shows the level of NO induced by the compound Hip A on DoX to H9c2 cardiomyocyte injuryn＝3)；

FIG. 24 shows the effect of Hip A on DoX-induced H9c2 myocardial apoptosis proteinn＝3)；

FIG. 25 is the effect of 5. Mu.M ATRA and 40nM OA on SH-SY5Y cell synaptic length;

FIG. 26 is the effect of compound Hip A on SH-SY5Y cell viability;

FIG. 27 is the effect of compound Hip A on morphology of AD cell model;

FIG. 28 is the effect of sea buckthorn neoflavonoid on tau protein phosphorylation in SH-SY5Y cells.

Detailed Description

The preparation and application of the flavonoid compound are further described by specific examples and specific experiments, and the verification and description of the flavonoid compound on inhibiting the accumulation of cell lipid, protecting the myocardial cell injury and neuroprotection are performed.

In the embodiment of the invention, the seabuckthorn (Hippophae rhamnoides Linn.) seed meal material is supercritical CO utilized by seabuckthorn seeds produced in Shang-county of Qinghai province and Xining City ₂ Seed meal after extracting seed oil.

EXAMPLE 1 preparation of novel flavonoid Compounds

1. Preparation of the Components

(1) 20.00kg of seabuckthorn (Hippophae rhamnoides Linn.) seed meal is extracted for 2 hours at 60 ℃ each time by adopting 70% ethanol solution with the feed-liquid ratio of 1:20kg/L, the extraction is carried out for 3 times, the extraction solutions of 3 times are combined and then decompressed and concentrated, and 3.50kg of seabuckthorn seed meal alcohol extract is obtained.

(2) Freeze drying the alcohol extract, crushing, sieving with 120 mesh sieve, adding small amount of methanol, mixing with 200-300 mesh normal phase silica gel in 1:1, and volatilizing until no alcohol smell. Adding 7kg of 200-300 mesh normal phase silica gel into a glass chromatographic column, eluting with dichloromethane/methanol/water=7:3:0.5 as mobile phase, eluting with 20 column volumes, and concentrating the eluent under reduced pressure to obtain extract.

(3) Dissolving the extract in 13L of 50% methanol solution, concentrating 2L under reduced pressure to 0.5L, eluting with MCI gel column, eluting with 20%, 40%, 60%, 80% and 100% methanol solution sequentially, eluting with 5 column volumes of each concentration, repeatedly sampling 2L, concentrating under reduced pressure for 3 times, and eluting to obtain 20% methanol eluate with mass of 59.12g,40% methanol eluate with mass of 71.86g,60% methanol eluate with mass of 22.35g,80% methanol eluate with mass of 20.81g, and 100% methanol eluate with mass of 35.54g.

(4) 22.35g of a 60% methanol solution elution site was dissolved in 110mL of 60% methanol solution and analyzed by HPLC under the following chromatographic conditions: chromatographic column Dubhe C18 (4.6X250 mm,5 μm), sample loading 20. Mu.L, mobile phase A:0.1% formic acid in water, mobile phase B: acetonitrile, gradient elution was performed using the following procedure: 0min,90% mobile phase A-10% mobile phase B;60min,80% mobile phase A-20% mobile phase B; the flow rate was 1mL/min and the wavelength was 254nm. The preparation method adopts a Hanbang N7010 semi-preparative liquid chromatograph to prepare a sample, and comprises the following preparation conditions: chromatographic column Dubhe C18 (20X 250mm,5 μm), sample loading 1.5mL, mobile phase A:0.1% formic acid in water, mobile phase B: acetonitrile, gradient elution was performed using the following procedure: 0min,90% mobile phase A-10% mobile phase B;60min,80% mobile phase A-20% mobile phase B; the flow rate is 19mL/min, the wavelength is 254nm, and finally 6 components are obtained, which are respectively named SH 1-SH 6, SH1 component is 0-14 min, SH2 component is 14-23 min, SH3 component is 23-29 min, SH4 component is 29-37 min, SH5 component is 37-46 min, and SH6 component is 46-60 min, as shown in figure 1, wherein the mass of SH5 component is 3.34g.

(5) 3.34g of SH5 component was dissolved in 8mL of dimethyl sulfoxide (DMSO) and analyzed by HPLC using Agilent1260 under the following chromatographic conditions: chromatographic column Kromasil 60-5-Diol (4.6X105 mm,5 μm), sample injection amount 0.1. Mu.L, mobile phase A:0.1% formic acid in water, mobile phase B: acetonitrile, gradient elution was performed using the following procedure: 0min,100% mobile phase B;60min,20% mobile phase A-80% mobile phase B; the flow rate was 1mL/min and the wavelength was 254nm. The preparation method adopts a Hanbang N7010 semi-preparative liquid chromatograph to prepare a sample, and comprises the following preparation conditions: kromasil 60-5-Diol (21X 250mm,5 μm), sample loading 0.4mL, mobile phase A:0.1% formic acid in water, mobile phase B: acetonitrile, gradient elution was performed using the following procedure: 0min,100% mobile phase B;60min,20% mobile phase A-80% mobile phase B, flow rate 21mL/min, wavelength 254nm, and retention time of 9 subfractions (SH 5-1-SH 5-9), SH 5-1-SH 5-9, respectively 0-6 min, 6-8 min, 8-22 min, 22-27 min, 27-37 min, 37-42 min, 42-55 min, 55-57 min, 57-60 min, as shown in figure 2.

2. Preparation of monomeric Compounds

(1) Separation of SH5-3 component

0.107g of SH5-3 component was dissolved in 5mL of methanol, added with DMSO to aid dissolution, and HPLC analysis was performed using Agilent1260 under the following chromatographic conditions: chromatographic column Kromasil 100-5-C18 (4.6X105 mm,5 μm) analytical column, sample size 0.1. Mu.L, mobile phase A:0.1% formic acid in water, mobile phase B: acetonitrile, elution condition 15% B, time 60min, flow rate 1mL/min, wavelength 254nm. The preparation of the sample by using a Hanbang N7010 semi-preparative liquid chromatograph is carried out under the following conditions: chromatographic column Kromasil 100-5-C18 (21X 250mm,5 μm) column was prepared with an amount of sample introduction of 0.2mL, mobile phase A:0.1% formic acid in water, mobile phase B: acetonitrile, elution condition 15% B, flow rate 21mL/min, wavelength 254nm. The 3 peaks under SH5-3 component were obtained as peak 2, peak 3, and peak 4, and the retention time of peak 4 was 22.707min.

(2) Separation of SH5-8 components

0.158g of SH5-8 component was dissolved in 2mL of methanol, and 400. Mu. Of LDMSO was added to aid dissolution, and HPLC analysis was performed using Agilent1260, under the following chromatographic conditions: kromasil 100-5-C18 (4.6X105 mm,5 μm), sample loading 0.5. Mu.L, mobile phase A:0.1% formic acid in water, mobile phase B: acetonitrile, elution condition 15% B, time 60min, flow rate 1mL/min, wavelength 254nm. The preparation is carried out by using a Hanbang N7010 semi-preparative liquid chromatograph under the following preparation conditions: kromasil 100-5-C18 (21X 250mm,5 μm), sample loading 0.3mL, mobile phase A:0.1% formic acid in water, mobile phase B: acetonitrile, elution condition 15% B, time 60min, flow rate 21mL/min, wavelength 254nm. 1 peak at SH5-8 was obtained as peak 17 and retention time was 26.641min.

(3) Separation of SH5-9 component

0.153g of SH5-9 component was dissolved in 2mL of methanol, 400. Mu. Of LDMSO was added to aid dissolution, HPLC analysis was performed using Agilent1260, chromatographic conditions were: kromasil 100-5-C18 (4.6X105 mm,5 μm), sample loading 0.5. Mu.L, mobile phase A:0.1% formic acid in water, mobile phase B: acetonitrile, elution condition 15% B, time 60min, flow rate 1mL/min, wavelength 254nm. The preparation method is carried out by using a Hanbang N7010 semi-preparative liquid chromatograph, and the preparation conditions are as follows: kromasil 100-5-C18 (21X 250mm,5 μm), sample loading 0.2mL, mobile phase A:0.1% formic acid in water, mobile phase B: acetonitrile, elution condition 15% B, time 60min, flow rate 21mL/min, wavelength 254nm, peak 18 and peak 19 under SH5-9, retention time 53.891min and 57.300min, respectively.

The preparative separation patterns of peaks 2, 3, 4, 17, 18, 19 are shown in FIG. 3.

The compounds obtained from peaks 2, 3, 4, 17, 18, 19 were subjected to purity detection, each having a purity of greater than 92%, the purity detection chart being shown in fig. 4.

2. Structure identification of monomeric compounds

The compounds obtained by the experiment are treated by UV, HR-ESI-MS, ESI-MS, ¹ H-NMR、 ¹³ The structure analysis was carried out by the organic spectrum analysis method such as C-NMR, HMBC, HSQC, HHCOSY, TOCSY, and the results were as follows:

Compound IV (peak 4): light yellow powder with a mass of 4mg, a novel compound named (6S, 9R) -9-hydro ox-4, 7-megastingmadien-3-one 9-O-beta-D-apiofuranosyl- (1- & gt 6) -beta-D-glucopyranoside (called Hyd for short), with a chemical formula of C ₂₄ H ₃₈ O ₁₁ By high resolution mass spectrometry (HR-ESI-MS) analysis of the mass-to-core ratio (M/Z) 503.2414[ M+H ]] ⁺ 。 ¹ H NMR(600MHz，CD ₃ OD):δ：5.88(1H，br，s，H-4)，5.75(1H，dd，J＝15.4，6.5，H-8)，5.63(1H，ddd，J＝15.4，9.4，0.7，H-7)，4.98(1H，d，J＝2.5Hz，H-1’)，4.63(1H，m，H-9)，4.31(1H，d，J＝7.8Hz，H-1’)，3.95(1H，d，J＝9.7Hz，H-4’)，3.93(1H，dd，J＝11.3，1.8，H-6’)，3.87(1H，d，J＝2.5Hz，H-2’)，3.74(1H，d，J＝9.7Hz，H-4’)，3.57(1H，dd，J＝11.3，5.9，H-6’)，3.56(1H，d，J＝11.5Hz，H-5’)，3.54(1H，d，J＝11.5Hz，H-5’)，3.33(1H，m，H-5’)，3.32(1H，m，H-3’)，3.27(1H，t，J＝9.2，H-4’)，3.16(1H，dd，J＝9.1，7.8，H-2’)，2.68(1H，d，J＝9.4，H-6)，2.41(1H，d，J＝16.7，H-2)，2.06(1H，d，J＝16.7，H-2)，1.96(3H，d，J＝1.1Hz，H-13)，1.29(3H，d，J＝6.4，H-10)，1.02(3H，s，H-11)，0.96(3H，s，H-12)； ¹³ C NMR(600MHz，CD ₃ OD)：δ：202.1(C-3)，166.1(C-5)，138.3(C-8)，129.1(C-7)，126.1(C-4)，111.0(C-1’)，102.6(C-1’)，80.5(C-3’)，78.0(C-3’，C-2’)，77.2(C-9)，77.0(C-5’)，75.2(C-2’)，75.0(C-4’)，71.6(C-4’)，68.6(C-6’)，65.5(C-5’)，56.8(C-6)，48.6(C-2)，37.2(C-1)，28.1(C-11)，27.3(C-12)，24.0(C-13)，21.2(C-10)。

Compound XVII (peak 17): yellow powder with a mass of 50mg, novel compound named Hippophandine A (short for Hip A) with a chemical formula of C ₅₀ H ₆₀ O ₂₉ . By high resolution mass spectrometry (HR-ESI-MS), the mass-to-core ratio (M/Z) was 1123.3146[ M+H ]] ⁺ And C ₅₀ H ₆₁ O ₂₉ Is consistent with calculated value 1124.3220 of (c). ¹ H NMR(600MHz，CD ₃ OD):δ：8.03(2H，d，J＝8.8Hz，H-2′，H-6′)，7.25(1H，d，J＝15.9Hz，H-7″″″)，6.89(2H，d，J＝8.8Hz，H-3′，H-5′)，6.42(2H，s，H-2″″″，H-6″″″)，6.34(1H，d，J＝2.0Hz，H-8)，6.30(1H，d，J＝2.0Hz，H-6)，5.99(1H，d，J＝15.9Hz，H-8″″″)，5.51(1H，br s，H-1″)，5.13(1H，d，J＝7.5Hz，H-1″″)，4.66(1H，d，J＝7.7Hz，H-1″″′)，4.63(1H，d，J＝7.8Hz，H-1″′)，4.47(1H，dd，J＝11.7，7.3Hz，H-6″″′)，4.38(1H，dd，J＝11.7，2.0Hz，H-6″″′)，4.09(1H，br s，H-2″)，4.05(1H，dd，J＝9.4，3.4Hz，H-3″)，3.85(1H，dd，J＝11.9，1.8Hz，H-6″′)，3.74(6H，s，H-OCH ₃ )，3.72(1H，t，J＝9.4Hz，H-4″)，3.68(1H，m，H-6″′)，3.67(1H，m，H-5″″′)，3.63(1H，m，H-2″″)，3.62(1H，m，H-5″)，3.60(1H，m，H-6″″)，3.51(1H，t，J＝9.1Hz，H-3″″)，3.47(1H，t，J＝9.1Hz，H-3″″′)，3.45(1H，m，H-6″″)，3.40(1H，dd，J＝9.1，7.7Hz，H-2″″′)，3.38(1H，t，J＝9.2Hz，H-3″′)，3.35(1H，t，J＝9.2Hz，H-4″′)，3.32(1H，t，J＝9.1Hz，H-4″″)，3.30(1H，m，H-4″″′)，3.29(1H，m，H-5″′)，3.21(1H，dd，J＝9.2，7.8Hz，H-2″′)，3.07(1H，m，H-5″″)，1.31(3H，d，J＝6.1Hz，H-6″)； ¹³ C NMR(600MHz，CD ₃ OD):δ：179.9(C-4)，168.7(C-9″″″)，163.2(C-7)，162.6(C-5)，161.7(C-4′)，158.7(C-2)，157.5(C-9)，149.0(C-3″″″，C-5″″″)，146.7(C-7″″″)，139.1(C-4″″″)，135.3(C-3)，132.7(C-2′，C-6′)，126.2(C-1″″″)，122.5(C-1′)，116.3(C-3′，C-5′)，115.3(C-8″″″)，107.2(C-10)，106.8(C-1″″′)，106.1(C-2″″″，C-6″″″)，105.6(C-1″′)，100.9(C-1″″)，100.2(C-6)，99.7(C-1″)，95.3(C-8)，85.5(C-2″″)，82.8(C-4″)，78.3(C-5″″)，78.2(C-3″′)，78.1(C-5″′)，77.8(C-3″″′)，77.6(C-3″″)，76.4(C-2″″′)，76.1(C-2″′)，75.6(C5″″′)，72.2(C-4″′)，72.1(C-3″)，71.6(C-4″″′)，71.5(C-2″)，71.1(C-4″″)，69.8(C-5″)，64.8(C-6″″′)，62.8(C-6″′)，62.3(C-6″″)，56.5(C-OCH ₃ )，18.3(C-6″)。

Compound XVIII (peak 18): yellow powder with mass of 41mg, new compound named Hippophandine B (short for Hip B) and chemical formula of C ₅₁ H ₆₆ O ₂₉ . By high resolution mass spectrometry (HR-ESI-MS), the mass-to-core ratio (M/Z) was 1141.3614[ M+H ]] ⁺ And C ₅₁ H ₆₇ O ₂₉ Is consistent with calculated value 1142.3690 of (c). ¹ H NMR(600MHz，CD ₃ OD):δ：8.09(2H，d，J＝8.8Hz，H-2′，H-6′)，7.02(1H，d，J＝15.7Hz，H-3″″′)，6.88(2H，d，J＝8.8Hz，H-3′，H-5′)，6.71(1H，d，J＝1.6Hz，H-8)，6.47(1H，d，J＝1.6Hz，H-6)，5.63(1H，br t，J＝7.0Hz，H-5″″′)，5.59(1H，br s，H-1″)，5.50(1H，d，J＝15.7Hz，H-2″″′)，5.15(1H，d，J＝7.4Hz，H-1″′)，4.70(1H，d，J＝7.6Hz，H-1″″)，4.36(1H，d，J＝7.8Hz，H-1″″″)，4.33-4.39(2H，m，H-6″″)，4.05(1H，br s，H-2″)，3.97(1H，m，H-8″″′)，3.85(2H，m，H-3″，H-6″″″)，3.69(1H，m，H-6″″″)，3.67(1H，m，H-2″′)，3.66(1H，m，H-6″′)，3.60(1H，m，H-5″)，3.59(1H，m，H-5″″)，3.52(1H，t，J＝9.1Hz，H-3″′)，3.49(1H，m，H-6″′)，3.48(1H，m，H-4″)，3.44(1H，t，J＝9.0Hz，H-3″″)，3.37(1H，m，H-2″″)，3.36(1H，m，H-3″″″)，3.34(3H，m，H-4″″″，H-4″″，H-4″′)，3.26(1H，m，H-5″″″)，3.21(1H，dd，J＝9.0，7.8Hz，H-2″″″)，3.11(1H，m，H-5″′)，2.72(1H，m，H-9″″′)，2.16-2.25(2H，m，H-6″″′)，1.52-1.59(2H，m，H-7″″′)，1.51(3H，br s，H-12″″′)，1.25(3H，d，J＝6.1Hz，H-6″)，1.12(3H，d，J＝6.1Hz，H-11″″′)； ¹³ C NMR(600MHz，CD ₃ OD):δ：180.1(C-4)，169.0(C-1″″′)，163.4(C-7)，163.0(C-5)，161.8(C-4′)，159.1(C-2)，158.0(C-9)，151.2(C-3″″′)，143.8(C-5″″′)，135.4(C-3)，134.0(C-4″″′)，132.8(C-2′，C-6′)，122.3(C-1′)，116.4(C-3′，C-5′)，115.5(C-2″″′)，107.5(C-10)，106.2(C-1″″)，103.6(C-1″″″)，101.0(C-1″′)，100.6(C-6)，99.7(C-1″)，95.6(C-8)，84.8(C-2″′)，81.3(C-8″″′)，78.3(C-5″′)，78.0(C-3″″″)，77.9(C-5″″″)，77.8(C-3″″)，77.7(C-3″′)，76.2(C-2″″)，75.7(C-5″″)，75.3(C-2″″″)，73.6(C-4″)，72.0(C-3″)，71.8(C-4″″″)，71.7(C-2″，C-4″″)，71.3(C-5″)，70.9(C-4″′)，64.6(C-6″″)，62.9(C-6″″″)，62.4(C-6″′)，45.3(C-9″″′)，31.7(C-7″″′)，25.6(C-6″″′)，18.2(C-6″)，12.8(C-11″″′)，12.1(C-12″″′)。

Compound XIX (peak 19): brown yellow powder with mass of 15mg, new compound named Hippophandine C (short for Hip C) and chemical formula of C ₅₁ H ₆₆ O ₂₉ . By high resolution mass spectrometry (HR-ESI-MS), the mass-to-core ratio (M/Z) was 1141.3614[ M+H ]] ⁺ And C ₅₁ H ₆₇ O ₂₉ Is consistent with calculated value 1142.3690 of (c). ¹ H NMR(600MHz，CD ₃ OD):δ：8.06(2H，d，J＝8.8Hz，H-2′，H-6′)，6.96(1H，br d，J＝11.5Hz，H-3″″′)，6.87(2H，d，J＝8.8Hz，H-3′，H-5′)，6.74(1H，d，J＝1.8Hz，H-8)，6.47(1H，d，J＝1.8Hz，H-6)，6.10(1H，dd，J＝14.8，11.5Hz，H-4″″′)，5.80(1H，dt，J＝14.8，7.1Hz，H-5″″′)，5.59(1H，br s，H-1″)，5.21(1H，d，J＝7.5Hz，H-1″′)，4.73(1H，d，J＝7.7Hz，H-1″″)，4.38(1H，dd，J＝12.0，2.1Hz，H-6″″)，4.36(1H，d，J＝7.8Hz，H-1″″″)，4.34(1H，dd，J＝12.0，5.6Hz，H-6″″)，4.06(1H，m，H-8″″′)，4.05(1H，br s，H-2″)，3.85(1H，m，H-3″)，3.84(1H，m，H-6″″″)，3.70(2H，m，H-6″″″，H-2″′)，3.66(1H，dd，J＝12.1，1.7Hz，H-6″′)，3.60(2H，m，H-5″，H-5″″)，3.54(1H，t，J＝9.1Hz，H-3″′)，3.50(1H，m，H-6″′)，3.48(1H，t，J＝9.3Hz，H-4″)，3.45(1H，t，J＝9.0Hz，H-3″″)，3.38(1H，m，H-2″″)，3.36(3H，m，H-4″″，H-3″″″，H-4″″″)，3.35(1H，m，H-4″′)，3.25(1H，m，H-5″″″)，3.19(1H，dd，J＝9.0，7.8Hz，H-2″″″)，3.12(1H，m，H-5″′)，2.53(1H，dd，J＝15.4，6.8Hz，H-9″″′)，2.46(1H，dd，J＝15.4，4.1Hz，H-9″″′)，，2.36(1H，m，H-6″″′)，1.79(1H，m，H-7″″′)，1.78(1H，m，H-6″″′)，1.62(3H，br s，H-12″″′)，1.25(3H，d，J＝6.1Hz，H-6″)，0.82(3H，d，J＝6.1Hz，H-11″″′)； ¹³ C NMR(600MHz，CD ₃ OD):δ：180.0(C-4)，176.7(C-10″″′)，169.9(C-1″″′)，163.4(C-7)，162.9(C-5)，161.7(C-4′)，159.1(C-2)，158.0(C-9)，143.8(C-5″″′)，140.4(C-3″″′)，135.4(C-3)，132.6(C-2′，C-6′)，127.9(C-4″″′)，125.4(C-2″″′)，122.4(C-1′)，116.4(C-3′，C-5′)，107.5(C-10)，105.8(C-1″″)，103.8(C-1″″″)，100.9(C-1″′)，100.6(C-6)，99.8(C-1″)，95.7(C-8)，84.1(C-2″′)，80.7(C-8″″′)，78.3(C-5″′)，78.1(C-3″″″)，77.8(C-5″″″，C-3″″)，77.7(C-3″′)，76.1(C-2″″)，75.7(C-5″″)，75.3(C-2″″″)，73.6(C-4″)，72.1(C-3″)，71.7(C-2″，C-4″″″)，71.6(C-4″″)，71.3(C-5″)，71.1(C-4″′)，64.9(C-6″″)，62.9(C-6″″″)，62.4(C-6″′)，38.3(C-7″″′)，38.1(C-9″″′)，37.0(C-6″″′)，18.1(C-6″)，12.5(C-12″″′)。

The following test examples prove the beneficial effects of the novel compound Hip A:

Test example 1 Compound Hip A inhibits lipid accumulation in HepG2 cells

1. Experimental method

1.1HepG2 cell culture technique

(1) In vitro culture of HepG2 cells

HepG2 cells were cultured in DMEM high-sugar medium containing 10% FBS and 1% Streptomyces lividans at 37℃and 5% CO ₂ Culturing in a saturated humidity incubator.

(2) Passage of HepG2 cells

HepG2 cells were incubated at 37℃with 5% CO ₂ Culturing in an incubator with saturated humidity, and when the cell fusion degree reaches more than 80%, carrying out 1:5 ratio passage on the cells.

(3) Counting of HepG2 cells

The usual tool for cell counting is a blood cell counting plate, each counting plate of the blood cell counting plate is formed by 2H-shaped grooves to form a counting pool with the height of 0.10mm, 9 large squares are arranged in the counting plate, and the cell counting area is 4 large squares at four corners. And (3) adjusting the cell count density according to the experimental requirement, recording the total number of cells of 4 large squares, and finally calculating the cell number according to a formula 1.1:

cell number = total number of cells in 4 cells 4 x 10 ⁴ X dilution factor

After the cell number is determined, the cell density is then adjusted according to the experimental requirements.

(4) Cryopreservation of HepG2 cells

Selecting cells in logarithmic growth phase, rapidly digesting the cells, adding the cell cryopreservation solution prepared in advance, subpackaging into sterile cell cryopreservation tubes at proper cell density, and simultaneously making time marks on tube walls so as to be capable of inquiring at any time, placing at 4 ℃ for 10min, -20 ℃ for 1h, -80 ℃ for overnight, and storing in a liquid nitrogen tank.

(5) Resuscitating HepG2 cells

And rapidly taking out the frozen cell strain from the liquid nitrogen tank, rapidly placing the frozen cell strain in a water bath at 37 ℃ for melting, rapidly culturing the cell transfer culture solution after the cells are completely fused, and replacing the fresh culture solution after the cells are attached to the wall (generally for 6 h).

1.2 Effect of varying concentrations of Oleic Acid (OA) on HepG2 cell viability

The influence of oleic acid on the cell viability of HepG2 cells is determined by adopting an SRB method, and the specific steps are as follows: (1) HepG2 cells at 5×10 ⁴ When the cells were grown to about 70-80% in a high-sugar DMEM medium containing 10% FBS, the OA concentrations were set to 0, 0.1, 0.25, 0.5, 0.75, 1 and 2mM, respectively, and the cells were treated for 24 hours and 48 hours, respectively. (2) After the treatment of the drug, the stock solution was discarded, 100. Mu.L of fresh DMEM was added to each well, and then 25. Mu.L of 50% TCA (light-shielding, available in a gun) was added thereto, and the mixture was left at room temperature for 5min at 4℃for 1h; (3) The stock solution was discarded, each well was rinsed 5 times with flowing ddH2O, and forced out after each rinsing pass. Inverted on an ultra-clean workbench for 15min for drying (high wind); (4) 70 μl of 0.4% SRB was added to each well and shaken for 30min (protected from light). (5) The SRB dye liquor is discarded, 1% glacial acetic acid is added into each hole to be washed for 4 times, and the solution is thrown out forcefully after each washing. Inverted on an ultra-clean workbench for 15min for drying (high wind); (6) mu.L of 100mM Tribase was added to each well and the mixture was incubated with shaking at 37℃for 20min. (7) detecting the OD value at 540nm wavelength by using an enzyme-labeled instrument. Cell viability was calculated according to the formula.

Cell viability (%) = (drug group OD/blank group OD) ×100%

1.3 Effect of different time OA treatments on HepG2 cell oil Red O staining

Measurement of lipid droplets in cells after 0.5mM OA treatment HepG2 cells for 0h, 24h and 48h the lipid droplets in cells were measured after 0.5mM OA and test compound were co-incubated for 48h, which was specifically performed by placing a sterile cover glass in a 24-well plate, adding 1mL DMEM, and placing in an incubator for 30 min. The medium was aspirated and the cells were incubated at 5X 10 ⁴ Inoculating 1mL of cell suspension to a slide, adding 1mL of cell suspension to each hole, pressing the slide by a needle head, removing bubbles below the slide, culturing in an incubator, performing OA induction treatment when the cell density reaches about 90% -95%, fixing after the treatment is finished, adding an oil red O working solution prepared in advance to dye the surface of the cells, and standing in a dark place for 60min. The cells were washed with 70% ethanol, excess dye was removed, washed 3-4 times with double distilled water, observed under a microscope and photographed.

1.4 Effect of OA treatments at various times on triglyceride content in HepG2 cells

Measurement of Triglycerides in cells after 0.5mM OA treatment of HepG2 cells for 0h, 24h and 48h and measurement of Triglycerides in cells after 0.5mM OA co-incubation with test Compound for 48h, which is specifically operated by subjecting HepG2 cells to 5X 10 ⁴ The density of each hole is inoculated into a 24-hole plate, OA induction treatment is carried out after the cell density reaches about 70% -80%, and the TG content is measured after the treatment time is over, wherein the specific method is as follows: (1) cell treatment: after the induction treatment is finished, the cell culture solution is sucked away, and after the cell culture solution is washed twice by cold PBS, the cell lysate PIPA is added for 5min to lyse (the operation is carried out on ice); (2) cell collection: after cell lysis, scraping and transferring cells from the bottom of the pore plate to a 1.5ml centrifuge tube by using a cell scraper, and directly measuring the prepared homogenate without centrifugation; (3) Adding 2 mu L of sample to be detected into each well of a 96-well plate, adding 2 mu L of distilled water into a blank well, adding 2 mu L of standard substance into a standard well, adding 160 mu L of reagent R1 into each well, uniformly mixing, incubating at 37 ℃ for 5min, and measuring the A1 value by using an enzyme-labeled instrument under the condition of 546nm wavelength; immediately thereafter, 40. Mu.L of reagent R2 was added to each sample, mixed well, and incubated at 37℃for 5min, after which the second spot absorbance A2 was read. Absorbance change of each tube _△ a=a2-A1. And finally, measuring the protein concentration in the sample to be tested by means of a BCA method, and correcting.

TG content= (OD _{Sample of} -OD _{Blank space} )/(OD _{Proof mass} -OD _{Blank space} ) X calibrator concentration (mM)/protein concentration of sample to be measured (gprot/L)

1.5 Effect of various time OA treatments on Fatty Acid Synthase (FAS) protein expression in HepG2 cells

HepG2 cells were grown at 5X 10 ⁴ Inoculating each hole into a 6-hole plate, performing OA induced differentiation when cells are completely grown to about 80% -90%, extracting cytoplasmic proteins after the induction time point is over, and performing protein electrophoresis, wherein the specific method is as follows:

(1) Extraction of total cell proteins:

after 0.5mM OA treatment of HepG2 cells for 0h, 24h and 48h and 0.5mM OA co-incubation with test compounds for 48h, the cells were collected and total proteins were extracted, and the total proteins were finally stored at-80 ℃.

(2) Protein content determination by BCA method

Taking out the frozen protein sample, diluting the protein standard with PBS buffer solution to 0.5mg/mL according to the requirements of the BCA protein quantification kit instruction, and mixing the reagent A in a ratio of 50:1: and (3) uniformly mixing the reagent B to prepare a working solution. Standards and PBS were added to 96-well plates as shown in table 3.3, with 20 μl per well, typically 3 multiplex wells per concentration, to draw standard curves. 1 μl of protein sample was added to a 96-well plate and 20 μl of PBS was added. Adding 200 mu LBCA working solution into each hole, fully and uniformly mixing, oscillating and incubating the samples at 37 ℃ for 30min by a microplate incubation oscillator, measuring the luminosity value at 562nm by an enzyme-labeling instrument, and calculating the protein concentration of the samples according to a standard curve. After the protein concentration was measured, 30. Mu.g of the protein sample was calculated, SDS-PAGE loading buffer was added, and the mixture was thoroughly mixed, and boiled in boiling water for 10 minutes to completely denature the protein. And before protein loading, the sample is centrifugally and uniformly mixed for use.

TABLE 1 BCA method protein quantification Table

(1) SDS-PAGE electrophoresis

The specific operation method of protein electrophoresis is the same as the conventional method in the prior art, and according to the molecular weight of target protein, proper concentration of separating gel is selected according to table 3, according to the gel preparation description table 2, a proper solution system is prepared, finally 5% of concentrated gel on the upper layer is prepared according to table 4, after gel preparation, electrophoresis is performed as soon as possible, and electrophoresis conditions are: s1, 80V for 30min; and S2, 120V, about 2h, and ending electrophoresis when bromophenol blue in the buffer solution runs to the lowest end of the separation gel.

TABLE 2 optimal separation ranges for SDS-PAGE separating gels of different concentrations

TABLE 3 preparation of the volumes of the components required for different volumes of 10% SDS-PAGE separating gel

TABLE 4 preparation of the various Components required for different volumes of 5% SDS-PAGE concentrate gel

(2) Transfer film

And (3) immediately performing transfer membrane after the protein electrophoresis is finished, wherein the specific experimental operation method is the same as the conventional method in the prior art.

(3) Closure

After the transfer, the PVDF membrane was gently removed from the transfer plate with tweezers, rinsed in TBST buffer for 5min, and immediately blocked with a TBST room temperature shaker containing 5% nonfat milk powder for 1 h.

(4) Incubation with primary antibody

After the end of the blocking, the PVDF membrane was placed in the diluted primary antibody solution and incubated overnight at 4℃on a horizontal shaking table with slow shaking.

(5) Second antibody incubation

After the incubation of the primary antibody was completed and the membrane washing was completed, the PVDF membrane was placed in a solution containing HRP-labeled murine secondary antibody or rabbit secondary antibody and incubated for 1h at room temperature in a shaker.

(6) Development process

After the incubation of all antibodies is finished, the ECL method is adopted for detection, developing solution is prepared according to the proportion of 1:1, the mixed ECL is added on a PVDF film for luminescence, and a gel imaging system is used for photographing.

2. Experimental results

2.1 Effects of OA on HepG2 cell viability

The effect of Oleic Acid (OA) on the cell viability of HepG2 cells was determined using SRB, with OA concentrations of 0, 0.1, 0.25, 0.5, 0.75, 1 and 2mM, and cells treated for 24h and 48h, respectively, and the results are shown in figure 11. The optimal treatment concentration for OA was finally determined to be 0.5mM.

2.2 Effect of OA treatment at various times on lipid accumulation in HepG2 cells

After 0.5mM OA treatment of HepG2 cells for 0h, 24h and 48h, the lipid drop content and the triglyceride content in the cells were measured, and as a result, see FIG. 12, it was found that the lipid drop and triglyceride content in the cells were significantly increased with the increase of the treatment time, and the final treatment time of OA was determined to be 48h.

2.3 Effect of OA on FAS protein expression in cells at various times

The expression level of Fatty Acid Synthase (FAS) protein in the cells was detected by Western blot after 0.5mM OA treatment of HepG2 cells for 0h, 24h and 48h, and the result (fig. 13) shows that the lipid accumulation level was highest after 0.5mM OA treatment of cells for 48h, which is consistent with the results of oil red O staining and TG content measurement, and thus the OA treatment time was 48h in the subsequent treatment.

2.4 Effect of Compound Hip A on HepG2 cell viability

The effect of the compound Hip A on the activity of HepG2 cells was examined by the SRB method. As a result of setting 6 treatment concentrations (0, 0.1, 1, 10, 50 and 100. Mu.M) for each compound, as shown in FIG. 14, the viability of the cells was high at the Hip A concentration of the compound of 0 to 50. Mu.M, and significantly decreased at the Hip A concentration of the compound of 100. Mu.M or more, so that 10. Mu.M was selected as the optimum acting concentration of the compound in the subsequent test.

2.5 Effect of Compound Hip A on lipid accumulation in HepG2 cells

The effect of the compound Hip a on lipid accumulation in HepG2 cells was examined by oil red O staining and measurement of triglyceride content, and the results are shown in fig. 15. Compared with the normal control group, there was a large accumulation of lipid droplets and a significant increase in triglyceride content in the OA group after oleic acid treatment, whereas both lipid droplets and triglyceride content were reduced to different extents after compound Hip a treatment.

2.6 Effect of Compound Hip A on HepG2 cell adipogenic proteins

SCD-1 and FAS are important enzymes in de novo fatty acid synthesis. SREBP1 is an important transcription factor in the regulation of adipogenesis genes, involved in fatty acid chain synthesis by activating FAS expression. The effect of Hip A on HepG2 cell lipoproteins as shown in FIG. 16 shows that the protein expression levels of FAS, SREBP1 and SCD-1 were significantly increased after oleic acid treatment, and the protein expression levels of FAS, SREBP1 and SCD-1 were decreased to different extents after Hip A treatment.

Test example 2 protective Effect of Compound Hip A on doxorubicin-induced myocardial cell injury

1 experiment materials, reagents and instruments

1.1 H9c2 rat cardiomyocytes

H9c2 (2-1) rat cardiomyocytes were purchased from Shanghai cell bank, national academy of sciences. H9c2 cardiomyocytes were initially subcloned from b.kimes and b.brandt by selective subculture using ventricular cells derived from BD1X rat embryonic heart tissue as the cell culture source. It exhibits specific cardiomyocyte lines with skeletal muscle and myocardial function at the same time.

1.2 materials and reagents

(1) DoX mother liquor preparation: 10mgDoX is dissolved in 3.680mL PBS to prepare mother solution with a final concentration of 5mmol/L, and the mother solution is preserved at-20 ℃ and can be diluted to 100 mu mol/L for split charging, thus avoiding repeated freezing and thawing. Note that DoX is preferably a glass vessel during the formulation process, if EP tubing concentrations are not less than 1mmol/L.

(2) Preparation of mother liquor of 5mg/mL

MTT is sensitive to bacteria and is easy to denature in light, and is required to be sterile and light-proof during preparation. Fully dissolving 0.5g MTT powder in 100mLPBS until the final concentration of MTT solution is 5mg/mL, filtering and sterilizing with 0.22 mu M microporous filter membrane, packaging into 1.5mLEP tube, and storing at-20deg.C for a long time in dark place to avoid repeated freeze thawing. Note that: when the MTT solution turns a grey green color, it is not usable.

(3) Preparation of reagent for Western blot

1) BCA working solution preparation: the required volume is prepared according to the ratio of the reagent A to the reagent B of 50:1.

2) Protein standard solution preparation: the concentration of the protein standard solution is 50mg/mL, the protein standard solution is diluted by PBS to a final concentration of 0.5mg/mL,200 mu L of the protein standard solution is split charging, and the protein standard solution is preserved at-20 ℃ and is taken at present.

3) 10% (W/V) Ammonium Persulfate (AP) solution: and (3) dissolving 0.5g of ammonium persulfate in 5.0mL of ultrapure water, subpackaging after dissolving, and preserving at-20 ℃ in a dark place for taking at present.

4) Preparing 5 XSDS-PAGE electrophoresis liquid: after Tris 15.1g,Glycine 94.0g is weighed and added with ultrapure water for ultrasonic dissolution, 5.0g of SDS is added at last to prevent foaming, and the volume is fixed to 1000mL, so that the solution can be stored at normal temperature. When the solution is used, 100mL of 5 Xelectrophoretic solution is measured, ultrapure water is added to fix the volume to 500mL, and the solution is diluted to 1 Xelectrophoretic solution.

5) Preparing 10 x membrane transferring liquid: tris 30.3g,Glycine 144.0g is weighed, and after being dissolved by ultrapure water, the volume is fixed to 1000mL. Preparing a 1X membrane transferring liquid: 50mL of 10 Xtransfer solution (1 part) was taken, 100mL of methanol (2 parts) and ultrapure water (7 parts) was added thereto to make up to 500mL, thereby obtaining 1 Xtransfer solution. Can be repeatedly used for 3-5 times and stored in a refrigerator at the temperature of 4 ℃.

6) 10×tbs solution preparation: adding ultrapure water to dissolve 12.12g of Tris and 40.03g of NaCl which are weighed, adjusting the pH to 7.6, adding ultrapure water to 500mL, and preserving in a refrigerator at 4 ℃ for later use.

7) 1 XTBST buffer preparation: the pipette sucks 500 mu L of Tween 20, adds the Tween 20 into 50mL of 10 XTBS, mixes the Tween 20 with the 50mL of 10 XTBS, adds ultrapure water to fix the volume to 500mL, mixes the Tween 20 with the 500mL of ultrapure water, and stores the Tween 20 in a refrigerator at 4 ℃ for standby. Note that: tween 20 is sticky and needs to cut off the gun head of the pipetting gun, so that the pipetting gun is convenient to suck, and the whole gun head is pumped into buffer solution.

8) Preparing a sealing liquid: weighing 0.5g of skimmed milk powder (or BSA), adding 10mL of TBST solution, mixing, and preferably mixing at a desired amount according to the proportion, and storing in refrigerator at 4deg.C. Note that only the supernatant is used in the using process, and the lower-layer particles are not used together, so that incomplete sealing is prevented, and black spots can appear on the membrane.

9) Preparing an anti-solution: antibodies such as beta-actin, COX-2, caspase-3, etc. were diluted 1:1000 with antibody dilutions. The refrigerator is stored at 4 ℃ when in use and at-20 ℃ when not in use. To extend the life of the antibody, an appropriate amount of sodium azide (NaN ₃ )。

10 Preparation of secondary antibody solution: according to goat anti-rabbit or goat anti-mouse antibody: antibody dilutions = 1:2000. The antibody is stored in a refrigerator at 4 ℃ when being used, and is stored in a refrigerator at-20 ℃ when not being used.

1.3 In vitro culture of H9c2 cardiomyocytes

DMEM high sugar culture with 10% FBS and 1% diabody was used at 37℃on 5% CO ₂ H9c2 myocardial cells are cultured in an incubator with saturated humidity, liquid is changed every other day, and subculture is carried out when the cell growth density reaches 70% -80%.

1.4 H9c2 cardiomyocyte cryopreservation

H9c2 myocardial cells with the growth density reaching 70-80% and in the logarithmic growth phase are selected, and the digestion operation of the cells is carried out in the step of passage. Finally, the collected cell suspension is placed into a sterile centrifuge tube, the temperature is 25 ℃ at 1000rpm, the centrifugation is carried out for 6min, the supernatant is discarded, the trypsin effect is thoroughly removed, and the influence of cell impurity particles is removed. After resuspension of the cells with DMEM high sugar medium, the cells were counted and adjusted to the corresponding concentration. Preparing proper cell freezing solution according to the proportion of DMEM high-sugar culture medium to FBS, DMSO=5:4:1, adding the resuspended cells, lightly blowing the mixture to be uniform, and sub-packaging the mixture into sterile cell freezing tubes with proper volume, wherein each tube is 1.0-1.2 mL. After the sealing film of the freezing tube is sealed, the information of cell name, algebra, freezing date, freezing person and the like is marked, and the freezing tube is placed in an isopropanol gradient cooling box and stored for 24 hours at the temperature of minus 80 ℃ and then transferred into a liquid nitrogen tank for storage.

1.5 H9c2 cardiomyocyte resuscitation

Preparation: before operation, the ultraviolet lamp is turned on, the ultra-clean bench is irradiated for about 30min, and after the bench is wiped by spraying with the new Jieling, the DMEM high-sugar culture medium is incubated. The water bath kettle is opened in advance to adjust the temperature to 37 ℃ in advance, then H9c2 myocardial cells taken out of the liquid nitrogen tank are rapidly put into a water bath with the temperature of 37 ℃, and shake back and forth during the period to accelerate dissolution, so that rapid dissolution within 1min is achieved. The cells in the cryopreservation tube were aspirated into DMEM high sugar medium containing 10% fbs, taking care to ensure uniform cell distribution. Labeling information such as cell name, algebra and date on culture dish, placing at 7deg.C, 5% CO ₂ Culturing in saturated humidity incubator for about 6 hr, and observing cells under microscopeAfter the culture medium is completely adhered, the culture medium is sucked and removed, PBS is used for washing for 2 to 3 times, residual DMSO is removed, and a new culture medium is replaced. When the cell density reaches 70% -80%, the passage operation can be performed.

1.6 H9c2 cardiomyocyte passage

Culturing H9c2 myocardial cells, and carrying out subculture when the myocardial cells are fused to about 70-80%. Before operation, the ultraviolet lamp is turned on, the ultra-clean workbench is irradiated for about 30min, and the workbench surface is sprayed and wiped by the novel Jiertangling. The old medium in the dish was discarded by the pipette and washed 2 times with PBS phosphate buffer incubated at 37 ℃. A100 mm dish was added with 750. Mu.L of trypsin digestion solution containing 0.25% EDTA and the mixture was placed in an incubator for digestion for 1 to 2 minutes. Observing under a microscope, slightly shaking the wall of the culture dish when most cell gaps become large and cells almost become light-transmitting circles, and slightly falling off small amount of cells in a fine sand shape to finish digestion. The pancreatin is removed by vacuum pumping, a proper amount of culture medium is added into a liquid-transfering gun to stop digestion, and the mixture is gently blown until all the cells fall off, so that uniform cell suspension is prepared. Inoculating to a new culture dish according to the ratio of 1:2-1:4, supplementing corresponding serum and culture medium, and placing into an incubator for continuous culture.

1.7 MTT method for detecting cell viability

H9c2 myocardial cells with the growth density reaching 70-80% and in the logarithmic growth phase are selected, and the digestion operation of the cells is carried out in the step of passage. A homogeneous single cell suspension was prepared with DMEM high-sugar medium containing 10% FBS and inoculated into 96-well cell culture plates at a density of 7500 cells/well, with a volume of 200. Mu.L per well. At 37℃with 5% CO ₂ Culturing under saturated humidity for complete cell adhesion, adding DoX with different concentrations, inducing for 24 hr, adding 20 μl of 5mg/mL MTT solution per well, incubating in incubator for 3 hr, and discarding culture medium to stop culturing. 100 μl DMSO was added to each well, and the mixture was shaken for 10min to dissolve the formazan sufficiently, and absorbance was measured at 490nm using a multifunctional microplate reader.

Cell viability (%) = (drug treated/blank OD) ×100%

1.8 Griess method for detecting NO content of cell supernatant

(1) Preparing a standard substance: 1mol/L NaNO ₂ Standard solution, cell culture solution (DMEM

+10% fbs) was diluted in a gradient to a series of 0,1,2,5, 10, 20, 40, 60, 100 μmol/L standard solutions for standard curve plotting.

(2) Samples were added to 96-well plates at 50 μl/well. Then, 50. Mu.L/well was added to each well, and Griess Reagent I was added at room temperature. Then, 50. Mu.L/well of each well was filled with room temperature Griess Reagent II. Finally, absorbance was measured at 540 nm.

1.9 detection of Lactate Dehydrogenase (LDH) from cell supernatants by microplate method

The specific operation method is shown in the specification.

1.10 Westernblot experimental method

(1) Extraction of cellular proteins

a. Preparation: an ice-crushing bath was prepared, an EP tube was labeled, pre-chilled PBS was used, and lysates (RIPA lysis buffer: pmsf=100:1) were prepared. The vacuum pump sucks the culture medium as much as possible, and the residue of the culture medium can influence the measurement of protein absorbance, thereby influencing the accuracy of protein concentration measurement.

b. Cleaning: the round was slowly turned and cold PBS was added to prevent wash-off and suck away cells. After rinsing for 2 times, PBS is sucked away as much as possible, otherwise the concentration of protein is diluted, so that the concentration is too low and the loading amount is too large.

c. Cell lysis: and adding a proper amount of lysate into each hole, and uniformly spreading the lysate on the bottom of the dish as much as possible. Placing in a refrigerator at 4deg.C or on ice for 5min for cracking.

d. Protein extraction: the cell scraper was rinsed with PBS and the cells were scraped with a scraper on ice. The cell scraper scrapes cells along the same direction as much as possible. Cell extracts in the dish were collected and placed in the EP tube while sucking the liquid on the spatula. When the cell scraper scrapes different groups, the cell scraper should be cleaned and wiped dry.

e. And (3) centrifuging: the protein lysate is cracked on ice for 30min, and the bullet is mixed for 1 time every 5min. 12000g, centrifuging at 4 ℃ for 15min, collecting supernatant, discarding precipitate, and preserving at-80 ℃ until protein is quantified.

(2) BCA method protein content determination

a. Preparation: the extracted protein is placed on an ice bath box to prevent degradation and denaturation.

b. Standard protein preparation: 50mg/mL protein standard was diluted to a concentration of 0.5mg/mL with PBS buffer. 0.5mg/mL protein standard solution 0, 2, 4, 6, 8, 12, 16 and 20. Mu.L were added to 96-well microplates, each set was set in 3 replicates, and the wells were topped up to 20. Mu.L with PBS buffer.

c. Preparing protein liquid to be quantified: the possibility of errors is reduced by adding 76. Mu.L of LPBS buffer to 4. Mu.L of protein test solution, and repeating 3 times every 20. Mu.L, with the remainder. 76 mu L of PBS was added to each EP tube, the proteins were thawed in batches, centrifuged by small vortex, and mixed well. mu.L of the protein solution to be metered was added to 80. Mu.L. mu.L of protein solution was added to each well of the 96-well plate.

d. 200 mu L of working solution is added into each hole, the AB working solution is prepared into solution A, solution B=50:1, and when the working solution is added into a 96-well plate, the gun head is slightly higher than the whole interface so as to avoid cross dyeing.

e. After incubation at 37℃for 30min with a microplate shaker, absorbance was measured at 562nm with an microplate reader.

f. The protein content in the sample was calculated from the standard curve. In Western blot experiments, equal volume and equal amount are adopted for Loading, namely PBS is used for diluting protein samples to consistent concentration, meanwhile, the volume of 5 Xprotein Loading buffer solution (Loading buffer) required by the Loading volume is calculated, and after the concentration is adjusted to be consistent, the volume of 30 mug protein solution is calculated to be the Loading volume.

g. Protein denaturation: after the EP tube sealing film is sealed and marked, the protein is fully denatured in a water bath at 100 ℃ for 10 min. Before sample addition, the sample is centrifugally and evenly mixed for reuse, and the protein is generally stored for 1 month at the temperature of minus 80 ℃ after denaturation and quantification.

(3) SDS-PAGE electrophoresis

a. And (3) glue preparation: selecting corresponding concentration of the separation gel according to the molecular weight of the target protein; cleaning the glass plate, flushing with ultrapure water, and placing the surface contacted with the glue on clean dust-free paper in a downward inclined manner for airing; before the glue is prepared, each solution is placed at room temperature, so that heat generated in the gel process is prevented, and gas dissolved in the storage liquid at low temperature is separated out to generate bubbles. After fixing the rubber plate, the leakage is tested by deionized water, and after testing, the water is wiped clean.

Preparing a separating gel: after the device is determined to be watertight, proper concentration of the separating gel is selected according to the molecular weight of the target protein and gel preparation specification, each component is added according to the required volume, TEMED is added finally, and gel is filled after uniform mixing. After the separating gel is fully and uniformly mixed, the gel is slowly added into the glass at the left side, which is propped against the top end, until the protruding end of the plate is level and slightly higher. After 2/3 of the separating glue is filled, the glue should be sealed immediately, and after the isopropanol is sealed, the glue should be cut and recorded without moving. Generally, a laboratory also often adopts a water seal mode to seal glue, but the flatness of the liquid level is slightly poor. Pouring out the sealing glue solution after gelation, for example, using isopropanol sealing glue to wash the sealing glue with ultrapure water.

Preparing concentrated glue: preparing 5% of concentrated glue according to a glue preparation instruction, filling the concentrated glue along the glass plate, avoiding generating bubbles, enabling the comb to be close to the glass at 45 degrees, quickly inserting the comb into the concentrated glue, slowly inserting the comb when the comb is not solidified, pulling out the comb once the bubbles are generated, and removing the bubbles and reinserting the comb. The gel is solidified for about 1h, and the solidification is favorable for the formation of a gel structure for a long time, because the arrangement of internal molecules is not completed when the gel is observed by naked eyes. Wrapping with ultra-clean paper, adding appropriate amount of ultrapure water for wetting, and sealing and preserving at 4deg.C. Before use, the mixture is put into an electrophoresis liquid to be activated for 10 to 15 minutes and then used. When in use, the glass plate is arranged in an electrophoresis tank, and the electrophoresis buffer solution is added according to the amount. The comb was carefully removed and prepared for loading. Note that observing whether the electrophoresis tank leaks, the leakage can lead to a decrease in conductivity.

b. Loading sample

After the sample is placed in a refrigerator, proteins can be settled and the solution is heterogeneous. Before loading, melting and centrifuging, and repeatedly mixing or blowing with a gun, and paying attention to replacing the gun head. The gun head or the microsyringe slowly adds the sample into the hole, wherein the 1 st hole needs to be added with 2-5 mu L of Marker. After loading, electrophoresis needs to be carried out as soon as possible, and the electrophoresis conditions are as follows: step1, 80V,30min; step2, 120V,180min, stopping electrophoresis when the Loading buffer goes to the lowest end. Note that: the gun head sucks the protein and inserts into the gel sample hole without extending too much so as to prevent the gun head from stretching the glass plate.

c. Transfer film

And cutting PVDF film in advance according to the target protein and the number of samples, and placing the PVDF film in methanol solution for 1-3 min for activation. The glove is dried and the PVDF film is not soaked with water. During the use of the membrane, the membrane is grasped by forceps so as to prevent the PVDF membrane from being polluted by protein on hands. The PVDF film is marked on the upper right when cut to distinguish between the front and back. The sandwich structure II used in the film transfer process is soaked in the film transfer liquid precooled at 4 ℃ until the film is completely soaked. The membrane is wetted in advance in the membrane transferring liquid. Washing the gel plate containing the electrophoresis liquid with tap water, and cutting gel of the target protein gel according to the molecular weight of the Marker. The gel was placed flat on the side of the blackboard and the notch in the upper left corner of the PVDF film was aligned with the glue face, following the principles of-black glue whiteboard. The transfer liquid repeatedly wets the offset plate and the membrane. Until the two are mutually attached without bubbles. And (3) covering the filter paper and silk floss on one side of the membrane, and gently expelling and pressing bubbles by using a test tube until no bubbles are generated between the filter paper and the silk floss. The force is not too great so as to avoid dislocation between the film adhesives. And placing the clamping plate into a membrane electrode groove, and changing black into black and changing red into red. In order to prevent the temperature from being too high during film transfer, an ice bag or the like needs to be placed on one side of the film transfer groove to achieve the purpose of cooling, and the whole film transfer process needs to be carried out in an ice bath. And determining the membrane transfer time and the current required by membrane transfer according to the molecular weight of the target protein.

d. Blocking and hybridization

Closing: at this point, note that the front side of the membrane was distinguished from the back side, the side in contact with the gel was the front side, which was incubated with antibody binding upwards. The membranes were removed, TBST rinsed slightly, immersed in 5% skim milk powder and blocked for 1h. The skimmed milk powder blocks non-specific sites on the PVDF membrane.

Washing the film: the TBST solution is washed for 5-10 min each time, and the total time is 3 times.

Incubating primary antibodies: the primary antibody is diluted in a ratio of generally 1:1000 to 1:10000. The PVDF membrane was then primed with primary antibody facing upward, allowed to pass through the membrane as far as possible, and allowed to stand overnight at 4 ℃. And recovering the primary antibody solution after the incubation is completed.

Washing the film: the TBST solution is washed for 5-10 min each time, and the total time is 3 times. Incubating a secondary antibody: and selecting a proper secondary antibody according to the primary antibody, diluting the secondary antibody according to the proportion of 1:1000, and carrying out light shaking incubation for 1-2 h at room temperature. And after the incubation is finished, recovering the secondary antibody and repeatedly using.

Washing the film: the TBST solution is washed for 5-10 min each time, and the total time is 3 times.

e. Development process

The PVDF film was placed on a developing blackboard, placed horizontally, and the bubbles were removed. Equal volumes of solution A and solution B were mixed, the mixed ECL reagent was gently added dropwise to PVDF membrane and photographed with a gel imaging system. Shooting by a developing instrument, prolonging shooting time and the like. Analysis and scanning are performed. After shooting, the film is recovered and soaked in TBST solution, and can be kept for repeated use. If necessary, the protein bands can be observed with ponceau staining (2% acetic acid, 0.5% ponceau in water) and then rinsed off with deionized water and TBST.

2. Experimental results

2.1 Morphological observation of H9c2 cardiomyocytes

Cells were divided into 6 groups when observed in morphological experiments: normal control, 1. Mu. Mol/L, 2. Mu. Mol/L, 3. Mu. Mol/L, 4. Mu. Mol/L and 5. Mu. Mol/L DoX treated groups. After DoX stimulation of cardiomyocytes for 24h, cardiomyocytes were observed under an inverted microscope. As a result, as shown in FIG. 17, it was found that the cardiomyocytes of the normal control group H9c2 were spindle-shaped, triangle-shaped or polygonal, were full, and had protrusions of different lengths. Compared with the normal control group, the DoX treatment group with different concentrations has the advantages that the cell bodies of the myocardial cells shrink, the refractive index is reduced, the volume of the cells is obviously reduced, and the cell density is obviously reduced.

2.2 MTT detection of H9c2 cardiomyocyte viability

After the myocardial cell treatment effect of each concentration DoX treatment group and the normal control group is 2 hours, MTT cell survival rate experiments are carried out, and the results are shown in figure 18, wherein 1 mu mol/L, 2 mu mol/L, 3 mu mol/L, 4 mu mol/L and 5 mu mol/L DoX treatment groups have obvious killing and inhibiting effects on cell viability compared with the normal control group, and the statistical difference P between the groups is less than 0.01. By calculating half-lethality of H9c2 cell viability, doX concentration was IC ₅₀ ＝2.46μM。

2.3 NO content in H9c2 cardiomyocyte culture supernatant

The sulfanilic acid in Griess reagent is usually dissolved in an acidic liquid solution, which upon contact with nitrite forms diazonium compounds. Ethylene diamine in the Griess reagent turns the diazo compound into a pink product with maximum absorption at 540nm, and the reaction yields a product concentration that has a linear relationship with the NO concentration. As a result, as shown in FIG. 19, NO significant difference was found in the NO content levels of the 1. Mu. Mol/L, 2. Mu. Mol/L, 3. Mu. Mol/L, 4. Mu. Mol/L and 5. Mu. Mol/LDoX treated groups as compared with the normal control group.

2.4 LDH content in H9c2 cardiomyocyte supernatant

Disruption of cell membrane structure by apoptosis or necrosis results in release of lactate dehydrogenase in the cytoplasm into the medium, under the action of lactate dehydrogenase, NAD ⁺ Is oxidized and reduced to NADH, NADH and INT are catalyzed by lipoamide dehydrogenase to generate NAD ⁺ And strong color formazan, which produces an absorption peak at 490nm wavelength, whereby lactate dehydrogenase activity can be quantified by occlusion. The results are shown in FIG. 20, where the 3. Mu. Mol/L, 4. Mu. Mol/L and 5. Mu. Mol/LDoX treated groups had a tendency to increase the level of LDH content in the cell culture broth without significant differences, and there was no statistical difference between the groups, as compared with the normal control group.

2.5 Effect of Compound Hip A on H9c2 cardiomyocyte viability

The effect of compound Hip a on H9c2 cardiomyocyte viability was examined by MTT method. The compound Hip A was set to 6 sets of concentration gradients (0, 0.1, 1, 10, 50 and 100. Mu. Mol/L) as can be seen from FIG. 21, the compound Hip A had no toxic effect on H9c2 cardiomyocytes at concentrations of 0 to 10. Mu. Mol/L, and therefore the selection of compound Hip A was 10. Mu. Mol/L when compound Hip A was selected in combination with DoX.

2.6 Compound Hip A vs DoX induces LDH levels in H9c2 cardiomyocytes

Lactate Dehydrogenase (LDH) is an important index for detecting apoptosis or necrosis, and as shown in FIG. 22, the Hip A compound has a remarkable protective effect (P < 0.05) on DoX induced H9c2 myocardial injury.

2.7 Compound Hip A vs DoX induces NO levels in H9c2 cardiomyocytes

The level of NO in the cell culture supernatant is an important indicator for detecting whether myocardial cells are damaged or not. As shown in fig. 23, compound Hip a was able to reduce NO levels in injured cardiomyocytes, indicating that Hip a has a significant protective effect (P < 0.05) against DoX-induced H9c2 cardiomyocyte injury.

2.8 Effect of Compound Hip A on DoX Induction of H9c2 myocardial apoptosis proteins

Bcl-2 and Bax regulate apoptosis activators by controlling the permeability of the mitochondrial membrane, and the occurrence of apoptosis is determined by the ratio of Bcl-2 to Bax. As shown in FIG. 24, the neoflavonoid Hyd, hip A, hip B and Hip C all showed protective effect on DoX induced H9C2 myocardial apoptosis.

Test example 3 neuroprotection of Compound Hip A

1. Experimental materials, reagents and instruments

1.1 preparation of the Main reagents

ATRA solution formulation: 15.0mg of all-trans retinoic acid powder is weighed, placed in a sterilized 1.5mL centrifuge tube, added with 1mL of DMSO, vibrated and dissolved to obtain 50mM ATRA solution, subpackaged with 200 mu L centrifuge tube, preserved in a refrigerator at-20 ℃ for standby, and diluted to the required concentration by DMEM culture medium before use.

Preparing an OA solution: each of the okadaic acid contents was 50. Mu.g, which was dissolved in 593. Mu.L of DMSO to give a 100. Mu.M mother liquor, which was packed in 200. Mu.L centrifuge tubes and stored in a refrigerator at-20℃for further use.

Preparing Giemsa solution:

(1) Preparation of giemsa stock solution: giemsa powder 0.8g, glycerol 50mL, methanol 50mL. Giemsa powder is dissolved in a small amount of glycerin, fully ground in a mortar, added with methanol, uniformly mixed and shaken, kept at a constant temperature of 56 ℃ for 2 hours, placed in a brown bottle and reserved in a refrigerator at 4 ℃.

(2) And (3) preparing a working solution: and (3) solution A: 11.93g of disodium hydrogen phosphate (Na2HPO4.12H2O) was weighed out by a balance and dissolved in 500mL of ultrapure water; and (2) liquid B: 4.535g of potassium dihydrogen phosphate (KH 2PO 4) was dissolved in 500mL of ultrapure water by a balance; when in use, the solution A is prepared by: and mixing the solution B according to a ratio of 1:1 to obtain the working solution with the pH of 7.0.

(3) Giemsa stock: the working solution=1:4, and the Giemsa staining solution is obtained after uniform mixing and is prepared for use.

Preparing MTT solution: 25.0mg of MTT powder is weighed, 5mL of PBS is added to be fully dissolved, the MTT solution with the concentration of 5mg/mL is obtained, and the MTT solution is packaged and stored in a refrigerator at the temperature of minus 20 ℃ in a dark place.

1.2 Experimental methods

(1) Resuscitating, passaging and culturing cells

Before operation, an ultraviolet lamp is turned on to irradiate the ultra-clean workbench for 15min, and alcohol is used for wiping the workbench surface; rapidly placing the cells taken out of the liquid nitrogen tank into a water bath at 37 ℃ for rapid dissolution within 1min, and taking out back and forth to see whether dissolution is performed; transferring the cells in the cryopreservation tube to DMEM medium containing 10% fbs; cell names, times and algebra were marked on petri dishes and incubated in a 5% CO2 incubator at 37 ℃. After the cells are attached, the culture solution is replaced with new culture solution for continuous culture, the cell morphology is observed, and when the cell growth and fusion reach about 80%, the passage operation can be carried out. Discarding old culture solution in the culture dish, and washing for 1 time by PBS phosphate buffer solution; adding trypsin digestion liquid containing 0.25% EDTA, digesting for 30 s-1 min in an incubator, observing under a microscope, adding DMEM culture medium to stop digestion when most cells are almost round and the cell gap is enlarged, and repeatedly and gently blowing off the adherent cells for several times by using a pipetting gun until the cells are completely shed to obtain cell suspension; diluting the uniformly mixed cells according to the required proportion, inoculating the uniformly mixed cells into a new culture medium, and placing the culture medium into an incubator for continuous culture.

(2) Cell cryopreservation

Selecting cells in logarithmic growth phase, carrying out passage according to the steps, transferring the collected cells into a sterile centrifuge tube, and centrifuging at the normal temperature of 860rpm for 6min; the supernatant was discarded and DMEM medium was used: FBS: adding the cell cryopreservation solution prepared by DMSO according to a ratio of 5:4:1, lightly blowing and uniformly mixing, and sub-packaging into sterile cell cryopreservation tubes, wherein each tube is 1.2mL; placing in a refrigerator at 4 ℃ for 10min, placing in a refrigerator at-20 ℃ for 1h, and after overnight in a refrigerator at-80 ℃, transferring the cells into a liquid nitrogen tank for preservation.

2. Experimental results

2.1 AD in vitro cell model establishment

As shown in FIG. 25, SH-SY5Y cells in the logarithmic growth phase were taken at 3X 10 ⁴ Each mL was seeded in 6-well plates, and 3mL of cell suspension was added to each well. Culturing in DMEM medium containing 10% FBS at 37deg.C in 5% CO2 incubator for 24 hr, and changing to ATRA with final concentration of 5 μm and DMEM medium containing 2% FBSCulturing. The morphology was observed every day, the liquid was changed every other day, and the cells were induced for 4 days to become differentiated and mature nerve cells. The crude medium was aspirated and the final concentration of OA, 2% FBS in DMEM medium was added for 6h. The cell synapse length was then measured to the cell body length by Giemsa staining and phase contrast microscopy, the ratio calculated using Image probe 6.0, and the model was evaluated for success (Wang YY, song, XY, liu, DD, et al, IMM-H004 reduced okadaic acid-induced neurotoxicity byinhibiting Tau pathology in vitro and in vivo.neuro-toxicity 2019,75:221-232.; medeiros LM, bastani MD, rico, EP, et al, chorinergic Differentiation of Human Neuroblastoma SH-SY5Y Cell Line and Its Potential Use as an In Vitro Model for Alzheimer's Disease Studies.mol Neurobiol2019,56:7355-7367.; zhangZ, simple, JW, okadaic Acid Induces Tau Phosphorylation in SH-SY5Y Cells in an Estrogen-presvant able Mantid 2010,1345:176-181.

2.2 Effect of Hip A on SH-SY5Y cell viability

MTT cell viability principle: succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water insoluble blue-violet formazan and deposit in cells, whereas dead cells do not. The formazan deposited in the cells was dissolved in DMSO, and the number of living cells was determined by measuring its absorbance at 490nm using a microplate reader. The greater OD value indicates greater cellular activity, i.e., less drug toxicity. When the cell viability is higher than 80%, the drug is considered to have no toxic effect on the cells, thereby determining the optimal concentration range for the drug effect. As can be seen from fig. 26, compound Hip a significantly decreased cell viability at 100 μm. Therefore, 10. Mu.M was chosen as the optimal concentration for this experiment and subsequent studies were performed.

2.3 Effect of Compound Hip A on morphology of AD cell model

Giemsa's dye solution is a mixture of azurin and eosin, and can dye nuclei into mauve or bluish violet, and cytoplasm into pink, and shows clear cell images under a microscope. The experiment utilizes the method to study the mutation and the transformation of SH-SY5Y cells, and is divided into an ATRA group (normal group), an OA group (damaged group) and a drug treatment group. After 4d induction, ATRA groups continued to be cultured with normal medium containing 2% serum; adding OA culture with final concentration of 40nM into normal culture solution; drug treatment groups were incubated with OA at a final concentration of 40nM and Compound Hip A at a final concentration of 10. Mu.M simultaneously in culture. After 6h Giemsa staining was performed and the change in cell synaptic length was observed, and the experimental results are shown in FIG. 27.

As can be seen from FIG. 27, cells were rounded, shrunken, and the synaptic ratio was significantly shortened after OA treatment. Hip A can significantly improve synaptic atrophy caused by OA, and has repair effect on damaged nerve to maintain normal cell morphology.

2.4 Western Blotting detection of tau protein phosphorylation-associated protein expression

The brain of an AD patient has obviously raised tau protein phosphorylation level, and has huge prospect for researching and developing medicines for preventing and treating AD aiming at tau protein targets, so that the experiment researches the influence of flavonoid compounds on the expression of p-tau Ser 262, p-tau Ser396 and tau 5 through a Western blotting method. Wherein the ratio of p-tau Ser 262, p-tau Ser396 to tau 5 represents the degree of phosphorylation of tau protein at serine 262, 396 sites, respectively, the higher the ratio, the higher the degree of phosphorylation. As can be seen from fig. 28, the compound Hip a of the present invention can significantly reduce the phosphorylation level of tau protein at Ser 262 and Ser396 sites, and is superior to other compounds, which indicates that the compound Hip a has better effect of improving tau protein hyperphosphorylation, and suggests that the compound Hip a has the potential of treating AD.

In conclusion, the novel flavonoid compound extracted by the invention can effectively inhibit lipid accumulation in cells, has the effects of repairing myocardial injury, protecting myocardial cells and protecting nerves, and provides more reference basis for developing potential plant-derived drugs for preventing and/or treating related diseases.

The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related technical fields are included in the scope of the present invention.

Claims (17)

1. The extraction method of the flavonoid compound is characterized in that the structure of the compound is shown as a formula XVII:

；

the extraction method comprises the following steps:

(1) Eluting the sea buckthorn seed meal extract by an MCI gel column, and eluting by 15-25% of methanol aqueous solution, 35-45% of methanol aqueous solution, 55-65% of methanol aqueous solution, 75-85% of methanol aqueous solution and methanol in sequence, wherein each eluent uses 4-6 column volumes; the sea-buckthorn seed meal extract is an ethanol extract of sea-buckthorn seed meal;

(2) Semi-preparative separation is carried out on substances obtained by eluting 55-65% methanol aqueous solution to obtain a component SH5:

the conditions for the semi-preparative separation in step (2) include:

chromatographic column: c18 column, 20mm x 250mm,5 μm;

mobile phase: the acidic aqueous solution was mobile phase a and acetonitrile was mobile phase B, and gradient elution was performed using the following procedure: 0min, 85-95% of mobile phase A, 15-5% of mobile phase B;60min, 75-85% of mobile phase A, 25-15% of mobile phase B; the components with retention time of 37-46 min are the components SH5;

(3) Semi-preparative separation is carried out on the component SH5 to obtain a component SH5-8:

the conditions for the semi-preparative separation in step (3) include:

chromatographic column: a glycol-based chromatographic column; specification is 21mm x 250mm,5 μm;

mobile phase: the acidic aqueous solution was mobile phase a and acetonitrile was mobile phase B, and gradient elution was performed using the following procedure: 0min, 5-0% of mobile phase A, 95-100% of mobile phase B;60min, 15-25% of mobile phase A, 85-75% of mobile phase B; the components with retention time of 55-57 min are components SH5-8;

(4) Semi-preparative separation of component SH5-8 gives compound XVII:

the conditions for the semi-preparative separation in step (4) include:

chromatographic column: a C18 chromatographic column; specification is 21mm x 250mm,5 μm;

mobile phase: the acid aqueous solution is a mobile phase A, acetonitrile is a mobile phase B, the mobile phase A is mixed with the mobile phase B in a volume ratio of 80-90 percent and 20-10 percent as the mobile phase for isocratic elution, and the component with the retention time of 25-28 min is the compound XVII;

the acidic aqueous solution is selected from formic acid aqueous solution, acetic acid aqueous solution and phosphoric acid aqueous solution; the concentration of the acid is 0.05 to 0.5 percent.

2. The method according to claim 1, wherein in the step (1), the elution is performed with 20% aqueous methanol, 40% aqueous methanol, 60% aqueous methanol, 80% aqueous methanol, and methanol in this order.

3. The extraction method according to claim 1, wherein the gradient elution procedure in step (2) is: 0min,90% mobile phase a,10% mobile phase B;60min,80% mobile phase A,20% mobile phase B.

4. The extraction method according to claim 1, wherein the gradient elution procedure in step (3) is: 0min,100% mobile phase B;60min,20% mobile phase A,80% mobile phase B.

5. The extraction method according to claim 1, wherein the mobile phase in the step (4) is a mixed solution of 85% by volume of mobile phase A and 15% by volume of mobile phase B, and the component with the retention time of 26-27 min is a compound XVII.

6. The extraction method according to any one of claims 1 to 5, wherein the semi-preparative separation in steps (2) to (4) further comprises at least one of the following conditions:

detection wavelength: 254nm;

flow rate: 19-21 mL/min.

7. The extraction method according to claim 1, wherein the preparation method of the sea buckthorn seed dreg extract comprises the following steps: extracting sea buckthorn seed meal with a solvent at 50-80 ℃, wherein the solvent is ethanol or ethanol water solution.

8. The method according to claim 7, wherein the solvent is 60 to 80% ethanol aqueous solution.

9. The method according to claim 8, wherein the solvent is 70% ethanol aqueous solution.

10. The extraction method according to claim 7, wherein the feed liquid ratio of the seabuckthorn seed meal to the solvent is 1:15-25 g/ml.

11. The extraction method according to claim 7, wherein the feed liquid ratio of the seabuckthorn seed meal to the solvent is 1:20g/ml.

12. The extraction method according to claim 7, wherein the extraction temperature is 60 ℃.

13. The extraction method according to claim 7, wherein the extraction time is 1 to 5 hours.

14. The extraction method according to claim 7, wherein the extraction time is 2h.

15. The extraction method according to claim 7, wherein the extract of seabuckthorn seed meal is obtained by subjecting the material obtained by solvent extraction of seabuckthorn seed meal to forward silica gel column chromatography, and the eluent is a mixed solution of dichloromethane and methanol in a volume ratio of 7:3:0.5.

16. The method according to claim 15, wherein the amount of eluent used in the forward silica gel column chromatography is 15 to 30 column volumes.

17. The method of claim 16, wherein the eluent is used in an amount of 20 column volumes.