Preparation method and application of N1, N10-bisdihydrodicaffeoyl spermidine

Abstract

The invention belongs to the technical field of biology, and mainly relates to a preparation method and application of N1, N10-bisdihydrodicaffeoyl spermidine. According to the invention, the spermidine component N1, N10-bisdihydrodicaffeoyl spermidine of lycium ruthenicum is obtained through water extraction, macroporous resin separation and preparative liquid chromatography purification. The purity of the N1, N10-bisdihydrodicaffeoyl spermidine obtained by the preparation method disclosed by the invention is high and reaches 98% or above. The prepared N1, N10-bisdihydrodicaffeoyl spermidine can directly activate the activity of G6PD, improve the NADPH (nicotinamide adenine dinucleotide phosphate) level, improve the heart function in a dose-dependent manner, and reduce the formation and fibrosis of scar tissues. Under a high dose (20mg / kg / day), the protection effect of the N1, N10-bisdihydrodicaffeoyl spermidine on myocardial deficiency-reperfusion injury is equivalent to that of metoprolol tartrate (MTA), and the N1, N10-bisdihydrodicaffeoyl spermidine can be used for preparing a product for preventing and treating ischemia-reperfusion injury.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of N1, N10-dihydrodicaffeoylspermidine and application thereof. Background Art

[0002] Ischemic heart disease (IHD) is one of the leading causes of death in humans. Treatments such as angioplasty and coronary artery bypass surgery can restore blood flow to the ischemic myocardium. However, reperfusion can cause additional myocardial damage by increasing reactive oxygen species (ROS) and inducing calcium overload. Currently, there is no effective treatment for ischemia / reperfusion (I / R) injury. Searching for active monomer components from natural products that have protective or preventive effects on myocardial ischemia-reperfusion injury has become a research hotspot.

[0003] Lycium ruthenicum Murr., also known as "Pangma" in Tibetan medicine, is a perennial shrub of the Lycium genus of the Solanaceae family. It has strong vitality, can tolerate salt, cold and drought, and is mainly distributed in high mountain sand forest areas. Lycium ruthenicum is included in classic Tibetan medical works such as "Jingzhu Bencao" and "Sibu Yidian". It is sweet, flat in nature, and can clear the heart and heat. It is a plant that can be used as medicine and food. Lycium ruthenicum is rich in spermidine alkaloid components, and has multiple physiological activities such as anti-inflammatory, antioxidant, hypolipidemic, anti-AD, anti-depression, and anti-aging. It is a resource chemical substance worthy of attention and in-depth research. N1, N10-dihydrodicaffeoyl spermidine is the main spermidine alkaloid component in Lycium ruthenicum. At present, there is no relevant preparation process and research report on the protective effect of myocardial ischemia-reperfusion injury. How to prepare high-purity N1, N10-dihydrodicaffeoyl spermidine and explore its application is a problem that technicians in this field need to solve urgently. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for preparing N1,N10-dihydrodicaffeoylspermidine, which can extract high-purity Lycium barbarum spermidine component N1,N10-dihydrodicaffeoylspermidine.

[0005] Another object of the present invention is to provide an application of N1,N10-dihydrodicaffeoylspermidine prepared by the above-mentioned preparation method in the preparation of a product for preventing and treating ischemia-reperfusion injury. The N1,N10-dihydrodicaffeoylspermidine provided by the present invention can effectively prevent and treat myocardial ischemia-reperfusion injury.

[0006] The present invention provides a method for preparing N1,N10-dihydrodicaffeoylspermidine, comprising the following steps:

[0007] The black wolfberry juice was centrifuged and the supernatant was collected;

[0008] The supernatant is added to a macroporous resin column 1 for sample loading, the macroporous resin column 1 is eluted with water, and the effluent is discarded; the macroporous resin column 1 is eluted with an ethanol solution with a volume fraction of 5% to 15%, the effluent is collected, and the effluent is added to a macroporous resin column 2 for sample loading; the macroporous resin column 2 is eluted with an ethanol solution with a volume fraction of 5% to 15%, the effluent is collected, concentrated, and dried to obtain a spermidine extract of Lycium ruthenicum;

[0009] The spermidine extract of Lycium ruthenicum was purified by preparative liquid chromatography to obtain N1,N10-dihydrodicaffeoylspermidine.

[0010] Preferably, the models of the macroporous resin column 1 include: D101, HPD-100, HPD-450, HPD-750, AB-8 or S-8.

[0011] Preferably, the models of the macroporous resin column 2 include: D301R, D301, D296, D315 or D201.

[0012] Preferably, when eluting with water, the volume of water is 1 to 3 BV.

[0013] Preferably, when the macroporous resin column 1 is eluted with an ethanol solution, the volume of the ethanol solution is 1 to 5 BV.

[0014] Preferably, when the macroporous resin column 2 is eluted with an ethanol solution, the volume of the ethanol solution is 1 to 5 BV.

[0015] Preferably, the elution flow rate when eluting the macroporous resin column 1 is 2 to 4 BV / h, and the elution flow rate when eluting the macroporous resin column 2 is 1 to 3 BV / h.

[0016] Preferably, the conditions for the preparative liquid chromatography purification are: the chromatographic column is a C18 chromatographic column, the mobile phase is an acetonitrile solution with a volume fraction of 5% to 20%, and the detection wavelength is 280nm.

[0017] Preferably, the mobile phase is isocratic elution, and the flow rate of the mobile phase is 2 to 20 mL / min.

[0018] The present invention also provides the use of N1,N10-dihydrodicaffeoylspermidine prepared by the preparation method in preparing a product for preventing and treating ischemia-reperfusion injury.

[0019] Beneficial effects of the present invention:

[0020] The invention provides a method for preparing N1, N10-dihydrodicaffeoyl spermidine. Through water extraction, macroporous resin separation and preparative liquid chromatography purification, a high-purity spermidine component N1, N10-dihydrodicaffeoyl spermidine of black wolfberry is obtained, and the purity thereof reaches more than 98%. The prepared high-purity N1, N10-dihydrodicaffeoyl spermidine can directly activate G6PD activity, increase NADPH level, improve cardiac function in a dose-dependent manner, and reduce the formation and fibrosis of scar tissue. Under a high dose (20 mg / kg / day), the protective effect of the prepared N1, N10-dihydrodicaffeoyl spermidine on myocardial ischemia-reperfusion injury is equivalent to that of metoprolol tartrate (MTA), and can be used to prepare products for preventing and treating ischemia-reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the HPLC chart of the spermidine extract of Lycium barbarum obtained in Example 3.

[0022] Figure 2 This is a preparative liquid chromatographic separation diagram of the spermidine extract of Lycium barbarum obtained in Example 3.

[0023] Figure 3 This is a purity detection chart of N1,N10-dihydrodicaffeoylspermidine prepared in Example 3.

[0024] Figure 4 HE staining images of mouse organs after oral administration of different doses of N1,N10-dihydrodicaffeoylspermidine for 7 days. Scale bar: 20 μm.

[0025] Figure 5 Masson staining of mouse organs after oral administration of different doses of N1,N10-dihydrodicaffeoylspermidine for 7 days (scale bar: 20 μm).

[0026] Figure 6 G6PD activity in neonatal rat cardiomyocytes after treatment with N1,N10-dihydrodicaffeoylspermidine (10 μmol / L, 24 h); * indicates P < 0.05 compared with the Con group.

[0027] Figure 7 NADPH activity in neonatal rat cardiomyocytes after treatment with N1,N10-dihydrodicaffeoylspermidine (100 μmol / L, 0.5 h); * indicates P < 0.05 compared with the Con group.

[0028] Figure 8 Echocardiogram of I / R mice after treatment with N1,N10-dihydrodicaffeoylspermidine.

[0029] Fig. 9The left ventricular ejection fraction (EF) and left ventricular fractional shortening (FS) detection graphs of I / R mice after treatment with N1,N10-dihydrodicaffeoylspermidine; the abscissas of the EF bar graph or FS bar graph are Con group, I / R group, I / R+LS-10 group, I / R+LS-20 group, and I / R+MTA group from left to right; * indicates p<0.05 compared with the Con group, and # indicates p<0.05 compared with the I / R group.

[0030] Fig.10 HE staining of the effect of N1,N10-dihydrodicaffeoylspermidine on cardiac tissue of I / R mice. Scale bar: 20 μm.

[0031] Fig.11 This is a Sirius red staining image showing the effect of N1,N10-dihydrodicaffeoylspermidine on the heart tissue of I / R mice. Scale bar: 20μm.

[0032] Fig.12 This is a quantitative analysis of the effect of N1,N10-dihydrodicaffeoylspermidine on cardiac tissue fibrosis in I / R mice; the horizontal axes of the bar graph are Con group, I / R group, I / R+LS-10 group, I / R+LS-20 group, and I / R+MTA group from left to right; * indicates p<0.05 compared with the Con group, and # indicates p<0.05 compared with the I / R group. DETAILED DESCRIPTION

[0033] The present invention provides a method for preparing N1,N10-dihydrodicaffeoyl spermidine, which can obtain N1,N10-dihydrodicaffeoyl spermidine with a purity of more than 98%, comprising the following steps:

[0034] The black wolfberry juice was centrifuged and the supernatant was collected;

[0035] The supernatant is added to a macroporous resin column 1 for sample loading, the macroporous resin column 1 is eluted with water, and the effluent is discarded; the macroporous resin column 1 is eluted with an ethanol solution with a volume fraction of 5% to 15%, the effluent is collected, and the effluent is added to a macroporous resin column 2 for sample loading; the macroporous resin column 2 is eluted with an ethanol solution with a volume fraction of 5% to 15%, the effluent is collected, concentrated, and dried to obtain a spermidine extract of Lycium ruthenicum;

[0036] The spermidine extract of Lycium barbarum was purified by preparative liquid chromatography to obtain N1, N10-dihydrodicaffeoyl spermidine. The structure of the N1, N10-dihydrodicaffeoyl spermidine is shown in Formula I:

[0037]

[0038] In the present invention, there is no special limitation on the source of Lycium ruthenicum Murr., and conventional commercial products in the art can be used. The present invention has no special limitation on the preparation method of Lycium ruthenicum juice, and fresh fruit or dried fruit can be selected for preparation. As an embodiment, fresh fruit can be directly squeezed for juice preparation, and the dried fruit preparation preferably includes the following steps: adding water to Lycium ruthenicum dried fruit, wherein the volume (mL) of the water is 10 to 20 times (such as 10 times, 13 times, 15 times, 18 times or 20 times) the mass (g) of the dried fruit, soaking at room temperature for 0.5 to 3h (such as 0.5, 1h, 1.5h, 2h, 3h), and then directly squeezing the juice with a juicer to obtain Lycium ruthenicum juice.

[0039] After obtaining the black wolfberry juice, centrifuge to remove impurities. The present invention has no particular limitation on the centrifugation method, and conventional centrifugation methods in the art can be used. Preferably, centrifugation is performed at 15000 r / min to 20000 r / min, such as 15000 r / min, 18000 r / min or 20000 r / min. After centrifugation, collect the supernatant.

[0040] The supernatant is separated by a macroporous resin to enrich the spermidine component of Lycium ruthenicum. Specifically, the supernatant is added to a macroporous resin column 1 for loading, and the models of the macroporous resin column 1 preferably include: D101, HPD-100, HPD-450, HPD-750, AB-8 or S-8; the present invention does not specifically limit the loading method, and the conventional loading method in the art can be used. After loading, the macroporous resin column 1 is eluted with water, and the volume of water is preferably 1 to 3 BV, more preferably 1.5 to 2.5 BV, and further preferably 2 BV. After elution, the effluent is discarded. Then, the macroporous resin column 1 is eluted with an ethanol solution having a volume fraction of 5% to 15%, and the concentration (volume fraction) of the ethanol solution is preferably 8% to 12%, more preferably 10%; after elution, the effluent is collected, and the effluent is added to the macroporous resin column 2 for loading, and the model of the macroporous resin column 2 preferably includes: D301R, D301, D296, D315 or D201. The present invention does not specifically limit the loading method, and the conventional loading method in the art can be used. After loading, the macroporous resin column 2 is eluted with an ethanol solution having a volume fraction of 5% to 15%, and the concentration (volume fraction) of the ethanol solution is preferably 8% to 12%, more preferably 10%. After elution, the effluent is collected, concentrated, and dried to obtain the black wolfberry spermidine extract. The present invention does not specifically limit the concentration and drying methods, and the conventional concentration and drying methods in the art can be used. As an embodiment, vacuum concentration can be selected for concentration, and freeze drying can be selected for drying. In the present invention, when eluting the macroporous resin column 1 with an ethanol solution, the volume of the ethanol solution is preferably 1 to 5 BV, more preferably 2 to 4 BV, and further preferably 3 BV; when eluting the macroporous resin column 2 with an ethanol solution, the volume of the ethanol solution is preferably 1 to 5 BV, more preferably 2 to 4 BV, and further preferably 3 BV. The elution flow rate when eluting the macroporous resin column 1 is preferably 2 to 4 BV / h, more preferably 2.5 to 3.5 BV / h, and further preferably 3 BV / h; the elution flow rate when eluting the macroporous resin column 2 is preferably 1 to 3 BV / h, more preferably 1.5 to 2.5 BV / h, and further preferably 2 BV / h. The present invention does not specifically limit whether the macroporous resin column 1 and the macroporous resin column 2 are connected and the connection method. As an embodiment, the macroporous resin column 1 and the macroporous resin column 2 can be selected to be connected in series.

[0041] The obtained black wolfberry spermidine extract is prepared into a sample solution and then purified by preparative liquid chromatography to obtain high-purity N1,N10-dihydrodicaffeoyl spermidine. The present invention has no special limitation on the method of sample preparation, and a conventional preparation method in the art can be used. As an implementation method, deionized water can be added for dissolution and preparation. The conditions for the preparative liquid chromatography purification are as follows: the chromatographic column is preferably a C18 chromatographic column, such as Bonnasil-BS AQ-C18 (250×10mmi.d., 5μm), Supersil AQ-C18 (250×10mm id, 5μm) or Nucifera C18A (250×10mm id, 5μm); the mobile phase is preferably an acetonitrile solution with a volume fraction of 5% to 20%, more preferably an acetonitrile solution with a volume fraction of 8% to 15%, and further preferably a 10% acetonitrile solution; the mobile phase is preferably eluted by isocratic elution; the flow rate of the mobile phase is preferably 2 to 20 mL / min, more preferably 10 to 18 mL / min, and further preferably 15 mL / min; the detection wavelength is preferably 280 nm.

[0042] The present invention also provides the use of N1,N10-dihydrodicaffeoylspermidine prepared by the preparation method in preparing a product for preventing and treating ischemia-reperfusion injury.

[0043] The N1, N10-dihydrodicaffeoylspermidine prepared by the invention can directly activate G6PD activity, increase NADPH level, improve cardiac function in a dose-dependent manner, reduce scar tissue formation and fibrosis, and has a protective effect on myocardial ischemia-reperfusion injury comparable to that of metoprolol tartrate (MTA), and can be used to prepare products for preventing and treating ischemia-reperfusion injury.

[0044] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0046] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0047] Example 1

[0048] Preparation of N1, N10-dihydrodicaffeoylspermidine from black wolfberry:

[0049] Step 1, weighing 20 kg of dried wolfberry, adding 200 L of deionized water, soaking at room temperature for 0.5 h, and directly squeezing the juice with a juicer to obtain black wolfberry juice;

[0050] Step 2, centrifuging the black wolfberry juice obtained in step 1 to remove impurities using a centrifuge at a speed of 15000 r / min, and collecting the supernatant;

[0051] Step 3, add the supernatant obtained in step 2 to macroporous resin column 1, where macroporous resin column 1 is D101, and add the effluent to macroporous resin column 1 again, and repeat 3 times. After standing for 24 hours, first elute the macroporous resin column 1 with 1BV of pure water, the elution flow rate is 2BV / h, and discard the effluent; then elute the macroporous resin column 1 with 1BV of 5% ethanol solution, the flow rate is 2BV / h; collect the effluent, and directly pass the effluent through the macroporous resin column 2 for sample loading, the flow rate is 1BV / h, and the macroporous resin 2 is D201; then elute the macroporous resin column 2 with 1BV of 5% ethanol solution, the elution flow rate is 1BV / h, collect the effluent, and obtain the eluate of the spermidine part of Lycium barbarum;

[0052] Step 4, vacuum concentrating the spermidine fraction eluate obtained in step 3, and freeze-drying to obtain 5 g of spermidine extract of Lycium ruthenicum;

[0053] Step 5, dissolving the black wolfberry spermidine extract obtained in step 4 in 25 mL of deionized water to prepare a solution with a concentration of 0.2 g / mL, and purifying it by preparative liquid chromatography, using a chromatographic column of Bonnasil-BSAQ-C18 (250×10 mmi.d., 5 μm), a mobile phase of 5% acetonitrile solution by volume, isocratic elution, a flow rate of 2 mL / min, and a detection wavelength of 280 nm to obtain 300 mg N1, N10-dihydrodicaffeoyl spermidine, which has a purity of 98.8% as determined by HPLC.

[0054] Example 2

[0055] Preparation of N1, N10-dihydrodicaffeoylspermidine from Lycium ruthenicum

[0056] Step 1, weighing 20 kg of dried wolfberry, adding 400 L of deionized water, soaking at room temperature for 1 hour, and directly squeezing the juice with a juicer to obtain black wolfberry juice;

[0057] Step 2, centrifuging the black wolfberry juice obtained in step 1 to remove impurities using a centrifuge at a speed of 20000 r / min, and collecting the supernatant;

[0058] Step 3, add the supernatant obtained in step 2 to macroporous resin column 1, macroporous resin column 1 is HPD-100, add the effluent to macroporous resin column 1 again, and repeat 3 times. After standing for 24 hours, first elute the macroporous resin column 1 with 3BV of pure water, the elution flow rate is 4BV / h, and discard the effluent; then elute the macroporous resin column 1 with 5BV of 15% ethanol by volume, the flow rate is 4BV / h, collect the effluent, and directly pass the effluent through the macroporous resin column 2 for sample loading, the flow rate is 3BV / h, and the macroporous resin column 2 is D301; then elute the macroporous resin column 2 with 5BV of 15% ethanol by volume, the elution flow rate is 3BV / h, collect the effluent, and obtain the eluate of the spermidine part of Lycium barbarum;

[0059] Step 4, vacuum concentrating the spermidine fraction eluate obtained in step 3, and freeze-drying to obtain 9 g of spermidine extract of Lycium ruthenicum;

[0060] Step 5, dissolving the black wolfberry spermidine extract obtained in step 4 in 30 mL of deionized water to prepare a solution with a concentration of 0.3 g / mL, purifying by preparative liquid chromatography, using a chromatographic column of Supersil AQ-C18 (250×10 mmi.d., 5 μm), a mobile phase of acetonitrile solution with a volume fraction of 20%, isocratic elution, a flow rate of 20 mL / min, and a detection wavelength of 280 nm to obtain 350 mg N1, N10-dihydrodicaffeoyl spermidine, which has a purity of 98.2% as determined by HPLC.

[0061] Example 3

[0062] Preparation of N1, N10-dihydrodicaffeoylspermidine from Lycium ruthenicum

[0063] Step 1, weighing 20 kg of dried wolfberry, adding 300 L of deionized water, soaking at room temperature for 3 hours, and directly squeezing the juice with a juicer to obtain black wolfberry juice;

[0064] Step 2, centrifuging the black wolfberry juice obtained in step 1 to remove impurities using a centrifuge at a speed of 20000 r / min, and collecting the supernatant;

[0065] Step 3, add the supernatant obtained in step 2 to macroporous resin column 1, macroporous resin column 1 is AB-8, add the effluent to macroporous resin column 1 again, and repeat 3 times. After standing for 24 hours, first elute the macroporous resin column 1 with 2BV of pure water, the elution flow rate is 3BV / h, and discard the effluent; then elute the macroporous resin column 1 with 3BV of 10% ethanol by volume, the flow rate is 3BV / h, collect the effluent, and directly pass the effluent through macroporous resin column 2 for sample loading, the flow rate is 2BV / h, macroporous resin column 2 is D301R; then elute the macroporous resin column 2 with 3BV of 10% ethanol by volume, the elution flow rate is 2BV / h. Collect the effluent to obtain the eluate of the spermidine part of Lycium barbarum;

[0066] Step 4, vacuum concentrating the spermidine fraction eluate obtained in step 3, and freeze-drying to obtain 10 g of spermidine extract of Lycium barbarum; HPLC analysis of the spermidine extract of Lycium barbarum is shown in the HPLC analysis chart. Figure 1 As shown by Figure 1 It can be seen that there are many chemical components in the spermidine extract of Lycium barbarum with similar polarity, and there is a certain difficulty in separation and preparation;

[0067] Step 5, the black wolfberry spermidine extract obtained in step 4 is dissolved in 100 mL of deionized water to prepare a solution with a concentration of 0.1 g / mL, and purified by preparative liquid chromatography. The preparative liquid chromatography separation diagram of the black wolfberry spermidine extract is as follows: Figure 2 As shown, the chromatographic column is Nucifera C18A (250×10mm id, 5μm), the mobile phase is 10% acetonitrile solution by volume, the elution is isocratic, the flow rate is 15mL / min, the detection wavelength is 280nm, and 500mg N1,N10-bis(dihydrodicaffeoyl)spermidine is obtained. The purity of N1,N10-bis(dihydrodicaffeoyl)spermidine is detected by HPLC, and the purity detection diagram is shown in FIG. Figure 3 As shown, the purity was 99.3% as determined by HPLC.

[0068] Test example

[0069] The N1,N10-dihydrodicaffeoylspermidine in the black wolfberry is referred to as LS in the following test examples. The N1,N10-dihydrodicaffeoylspermidine used in the following test examples is prepared in Example 1.

[0070] Test Example 1

[0071] Safety experiment of N1, N10-dihydrodicaffeoylspermidine in black wolfberry

[0072] C57BL / 6J mice (6-8 weeks old) were randomly divided into 3 groups, 8 mice in each group, and intragastrically administered with LS 0mg / kg / d, 10mg / kg / d, and 50mg / kg / d for 7 days, respectively. They were then killed and their organs were removed for HE staining and Masson staining.

[0073] HE staining of organs ( Figure 4 ) and Masson staining ( Figure 5 ) The results showed that there was no significant difference in the heart, liver, lung, kidney and brain of mice after LS intervention compared with the control group, indicating that LS has no obvious toxic side effects.

[0074] Test Example 2

[0075] G6PD promotes cardiomyocyte growth by producing NADPH, and elevated NADPH levels can prevent ischemia-reperfusion injury. The effects of N1,N10-dihydrodicaffeoylspermidine in Lycium barbarum on G6PD and NADPH were determined to evaluate the protective effect of LS against ischemia-reperfusion injury.

[0076] (1) Isolation of primary cardiomyocytes from neonatal rats

[0077] The hearts of 1- to 3-day-old SD rats were removed and immersed in D-Hanks solution (0.137 M NaCl, 5.4 mM KCl, 10% glucose, 0.44 mM KH 2 PO 4 ,0.4mMNa 2 HPO 4 , pH = 7.2), cut the heart into 1 mm 3 The tissue blocks were digested with 0.1% trypsin at 37°C for 10 min each time. The digestion can be repeated until the myocardial tissue is completely digested. The digestion was terminated with DMEM containing 20% ​​FBS, and the separated single cells were collected by centrifugation. The single cells were resuspended in DMEM containing 20% ​​FBS and incubated at 37°C, 95% humidity, and 5% CO. 2 The cells were cultured by differential adhesion method in an incubator at 4 °C for 1.5 h, the supernatant was taken, resuspended and further cultured to obtain primary cardiomyocytes of neonatal rats.

[0078] (2) Determination of G6PD activity in primary cardiomyocytes of neonatal rats

[0079] Neonatal rat primary cardiomyocytes were divided into two groups, namely the Con group (blank control group) and the LS group. LS was added to the culture medium of neonatal rat primary cardiomyocytes in the LS group to make the concentration of LS 10μM, and the culture was carried out. LS was not added to the Con group and cultured under the same conditions. After the two groups were cultured for 24 hours, the G6PD activity of neonatal rat primary cardiomyocytes was measured using a high-sensitivity G6PD activity assay kit (Biyuntian, S0189). That is, 1×10 6200 μL of extract solution was added to each neonatal rat primary cardiomyocyte, ground, centrifuged at 12,000 g for 10 min at 4°C, and the supernatant was collected. 50 μL of supernatant was mixed with 50 μL of reaction buffer, incubated at 37°C in the dark for 30 min, and the absorbance was measured at 450 nm.

[0080] (3) Lentiviral vector assembly and transfection

[0081] The lentiviral vector construct (pLVX-cyto-iNap1, pLVX-cyto-iNapc; 16 μg each) was co-transfected into HEK293T cells with the auxiliary vectors 12 μg psPAX2 and 4 μg pMD2.G using 7 μL of transfection reagent. At 48 h and 72 h after transfection, the virus solution was collected and filtered to obtain the cyto-iNap1 and cyto-iNapc lentiviral supernatants.

[0082] (4) Determination of NADPH levels in neonatal rat cardiomyocytes

[0083] The isolated primary cardiomyocytes of neonatal rats were inoculated into 96-well black glass bottom plates and cultured for 12 hours. The cyto-iNap1 and cyto-iNapc lentiviral supernatants collected in (3) and 8 μg / ml polybrene were added and placed in a 37°C, 95% humidity and 5% CO 2 The infected rat primary cardiomyocytes were divided into two groups, namely the Con group (blank control group) and the LS group. In the LS group, LS was prepared into 100 μM and directly added to the neonatal rat primary cardiomyocytes at 37°C and 5% CO. 2 The cells were incubated under the same conditions as those in the Con group, and the cells were directly incubated without LS. After 0.5 h of incubation, dual-excitation fluorescence images were obtained using a high-content analysis system, with excitation wavelengths of 405 nm and 488 nm, and an emission range of 500-560 nm. Images of ≥100 neonatal rat cardiomyocytes were randomly collected, and the fluorescence ratio of 405 nm / 488 nm was analyzed using ImageJ software. The fluorescence ratio of 405 nm / 488 nm is the intracellular NADPH level.

[0084] G6PD activity and NADPH levels in neonatal rat cardiomyocytes after LS treatment Figure 6 and Figure 7 The results showed that compared with the Con group, LS acted on neonatal rat primary cardiomyocytes for 24 hours and significantly increased G6PD activity ( Figure 6 ), and significantly increased the NADPH level in neonatal rat primary cells ( Figure 7 ). The results showed that LS could directly activate G6PD and increase NADPH levels.

[0085] Test Example 3

[0086] N1,N10-dihydrodicaffeoylspermidine in black wolfberry can improve I / R injury.

[0087] (1) Ischemia-reperfusion animal model and drug administration

[0088] C57BL / 6J mice (6-8 weeks old) were fed with regular food and water and kept in a room at 22℃ with 12-hour light and 12-hour dark cycles. They were randomly divided into sham operation group (Con), ischemia reperfusion model group (I / R), 20mg / kg / d metoprolol tartrate group (I / R+MTA), 10mg / kg / d LS group (I / R+LS-10), and 20mg / kg / d LS group (I / R+LS-20), with 8 mice in each group. Mice were anesthetized with isoflurane and connected to a ventilator via endotracheal intubation. The left anterior descending artery (LAD) was ligated with 7-0 suture in the model group and each drug-treated group for 30 minutes. The color of the distal myocardium of the vessel turned pale, the ligature on the LAD was removed, and the ischemic myocardium returned to redness, which was considered a successful reperfusion. The sham operation group only threaded the suture without ligation. After reperfusion, except for the sham operation group (Con) and the ischemia-reperfusion model group (I / R), the rest were intragastrically administered with drugs (MTA or LS) once a day for 4 weeks.

[0089] (2) Echocardiography of mice

[0090] Four weeks after administration, the mice underwent echocardiography using R-VINNO6LAB with Vermon probe (20-23MHz). A heating pad was used to maintain the body temperature of the mice (36.5-37.5°C), and M-mode images were obtained using the parasternal short-axis view. The left ventricular ejection fraction (EF) and left ventricular shortening fraction (FS) were measured, and the average value was obtained by repeating three times.

[0091] Echocardiography in mice Figure 8 and Fig. 9 As shown, compared with the sham operation group (Con), I / R injury led to a decrease in left ventricular ejection fraction (EF) and left ventricular fractional shortening (FS), and a decrease in left ventricular systolic function. After MTA and LS intervention, EF and FS increased, and the high-dose LS group had an effect comparable to that of MTA. The results showed that LS could improve cardiac function in a dose-dependent manner.

[0092] (3) After echocardiography, the mice were killed and the hearts were removed for subsequent analysis. The hearts were cut into 1 mm thick slices and stained with HE and Sirius red. The slices were analyzed using ImageJ software.

[0093] Mouse heart histopathological results Fig.10 , Fig.11 and Fig.12As shown in the results, myocardial cells in I / R mice were disordered, interstitial components increased, and scar tissue and fibrosis formed. Excessive proliferation of fibrous connective tissue will destroy the normal structure of the heart. Scar tissue cannot contract like normal myocardial cells, thereby weakening the heart's ability to pump blood. These structural changes will further affect the function of the heart. LS intervention can reduce scar tissue formation ( Fig.10 ) and fibrosis ( Fig.11 and Fig.12 ), the effect of high-dose LS group was comparable to that of MTA.

[0094] The above results show that LS can improve cardiac function in a dose-dependent manner, reduce the formation of scar tissue and fibrosis, and at a high dose (20 mg / kg / day), its protective effect on myocardial ischemia-reperfusion injury is equivalent to that of metoprolol tartrate (MTA).

[0095] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing N1, N10-bis(dihydrodicaffeoyl)spermidine, characterized in that: The following steps are involved: The black wolfberry juice was centrifuged and the supernatant was collected; The supernatant is added to the macroporous resin column 1 for sample loading, the macroporous resin column 1 is eluted with water, and the effluent is discarded; the macroporous resin column 1 is eluted with an ethanol solution with a volume fraction of 5% to 15%, the effluent is collected, and the effluent is added to the macroporous resin column 2 for sample loading; The macroporous resin column 2 is eluted with an ethanol solution having a volume fraction of 5% to 15%, and the effluent is collected, concentrated, and dried to obtain a spermidine extract of Lycium ruthenicum; The spermidine extract of Lycium ruthenicum was purified by preparative liquid chromatography to obtain N1,N10-dihydrodicaffeoylspermidine.

2. The preparation method according to claim 1, characterized in that: The models of the macroporous resin column 1 include: D101, HPD-100, HPD-450, HPD-750, AB-8 or S-8.

3. The preparation method according to claim 1, characterized in that: The models of the macroporous resin column 2 include: D301R, D301, D296, D315 or D201.

4. The preparation method according to claim 1, characterized in that: When eluting with water, the volume of water is 1 to 3 BV.

5. The preparation method according to claim 1, characterized in that: When the macroporous resin column 1 is eluted with an ethanol solution, the volume of the ethanol solution is 1 to 5 BV.

6. The preparation method according to claim 1, characterized in that: When the macroporous resin column 2 is eluted with an ethanol solution, the volume of the ethanol solution is 1 to 5 BV.

7. The preparation method according to claim 1, characterized in that: The elution flow rate when eluting the macroporous resin column 1 is 2 to 4 BV / h, and the elution flow rate when eluting the macroporous resin column 2 is 1 to 3 BV / h.

8. The preparation method according to claim 1, characterized in that: The conditions for the preparative liquid chromatography purification are as follows: the chromatographic column is a C18 chromatographic column, the mobile phase is an acetonitrile solution with a volume fraction of 5% to 20%, and the detection wavelength is 280nm.

9. The preparation method according to claim 8, characterized in that: The mobile phase is isocratic during elution, and the flow rate of the mobile phase is 2-20 mL / min.

10. Use of N1,N10-dihydrodicaffeoylspermidine prepared by the preparation method according to any one of claims 1 to 9 in the preparation of products for preventing and treating ischemia-reperfusion injury.

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