Preparation method of specnuezhenide and nuezhenide G13 based on sweet osmanthus seeds

Abstract

The invention provides a preparation method of specnuezhenide and nuezhenide G13 based on sweet osmanthus seeds, and belongs to the technical field of biological medicines. The preparation method disclosed by the invention comprises the following steps: extracting by adopting a percolation extraction mode to obtain an osmanthus fragrans seed extract; petroleum ether, dichloromethane and n-butyl alcohol are adopted for sequentially extracting the osmanthus fragrans seed extract, and an n-butyl alcohol layer extract is obtained; and carrying out two-step normal-phase column chromatography on the n-butanol layer extract to obtain specnuezhenide and nuezhenide G13. According to the preparation method, the specnuezhenide and the nuezhenide G13 with the purity reaching up to 95% or above are prepared by taking the sweet osmanthus seeds as the raw material through operations such as percolation extraction, step-by-step extraction and two-step normal-phase column chromatography separation, the operation is convenient, large-scale production is easy, and when the specnuezhenide and the nuezhenide G13 are used for preparing the heart-arthralgia-resistant medicine or health-care product, the specnuezhenide and the nuezhenide G13 have the advantages that the specnuezhenide and the the rabbit platelet aggregation can be obviously inhibited; the anti-inflammatory activity can be exerted.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a method for preparing privet glycoside and privet glycoside G13 based on osmanthus seeds. Background Art

[0002] Heart Bi, Tongxin Bi, means obstruction. In the Neijing, heart Bi refers to a disease with heart pain as the main symptom caused by obstruction of the heart vessels and poor circulation of qi and blood. Heart Bi and heart pain are related to rheumatic heart disease and angina pectoris due to coronary heart disease described in modern medicine. The main cause of the disease is myocardial ischemia and damage caused by sclerosis of the coronary arteries, which narrows the lumen and causes imbalance between myocardial oxygen demand and oxygen supply. Clinical manifestations include angina pectoris and myocardial infarction.

[0003] Sweet Osmanthus fragrans (Thunb.) Lour., also known as sweet osmanthus, is one of the ten traditional famous flowers in my country. It is not only a famous spice plant, but also an excellent gardening tree species. It is mainly distributed in the north of Nanling Mountains to the south of Qinling Mountains and Huaihe River, and in the north to Shandong Peninsula. At present, most reports on sweet osmanthus resources involve the analysis and development of sweet osmanthus essential oil and extract components. Related research mainly focuses on traditional fields such as sweet osmanthus resources, varieties, cultivation, development, and aroma. However, there are few reports on other parts of sweet osmanthus, and the information on the chemistry and pharmacological activity of non-aromatic components is very limited.

[0004] Sweet osmanthus seeds are the mature fruits of sweet osmanthus. At present, the five major sweet osmanthus producing areas in my country (Guilin, Guangxi, Xianning, Hubei, Chengdu, Sichuan, Suzhou, Jiangsu, Hangzhou, Zhejiang) can achieve an annual output of 1 million kilograms of dried flowers, and a large amount of sweet osmanthus seed resources will be produced every year in the mature season. And the current application of sweet osmanthus seeds, except for a few as germplasm resources for sweet osmanthus seedlings and cultivation, most of them are treated as agricultural waste, causing a great waste. Sweet osmanthus seeds, as the fruits of sweet osmanthus, are harvested when ripe, soaked in warm water, and dried for medicine. "Plant Names and Realities Illustrated Long Compilation" records that sweet osmanthus seeds are used to treat heart pain. However, its material basis is still unclear, and modern pharmacy has very limited research reports on the chemical composition and cardiovascular activity of sweet osmanthus seeds. Therefore, it is of great significance to analyze the main chemical components in sweet osmanthus seeds and carry out their activity evaluation.

[0005] At present, the preparation reports of privet glycoside and privet glycoside G13 are mainly from Ligustrum lucidum fruit. There are also a few literature reports on the separation and purification of privet glycoside and privet glycoside G13 from Osmanthus fragrans seeds. Related patent literature reports methods for preparing special ligustrum lucidum from Ligustrum lucidum fruit, such as patent (CN102372754) discloses a method for preparing crude special ligustrum lucidum and high-purity special ligustrum lucidum from Ligustrum lucidum fruit, the method sequentially adopts macroporous resin, normal phase silica gel and reverse phase silica gel technology to prepare special ligustrum lucidum with a purity of 98%; patent (CN101704857A) discloses a method for obtaining special ligustrum lucidum from Ligustrum lucidum fruit using extraction, macroporous resin and reverse phase silica gel technology; patent (CN101955504A) discloses a method for separating and preparing high-purity special ligustrum lucidum, ligustrum lucidum and oleanolic acid from Ligustrum lucidum fruit using high-speed countercurrent chromatography; patent (CN106632544A) discloses a method for preparing a special ligustrum lucidum reference substance, in which the special ligustrum lucidum alcohol extract is subjected to medium-pressure preparation column combined with pure water crystallization technology to obtain the special ligustrum lucidum reference substance. Although the above-mentioned related methods for obtaining special ligustrin from Ligustrum lucidum fruit have obtained high-purity special ligustrin, there are more or less problems such as more operation steps, long preparation process, and expensive preparation instruments; at the same time, although the patent (CN106632544A) obtains special ligustrin monomers through two steps, the first step of the medium-pressure preparation step requires a medium-pressure preparation chromatograph, which is expensive and not easy to scale up for pilot production. In addition, the method for separating and preparing special ligustrin and ligustrin G13 from osmanthus seeds also has the following problems: the extraction methods such as heating reflux adopted are easy to destroy the effective ingredients special ligustrin and ligustrin G13, which is not conducive to improving the purity and yield of special ligustrin; the separation technology adopted includes three steps of MCI column, normal phase silica gel column and reverse phase silica gel column, which are complicated to operate and not easy to industrialize. At the same time, the eluent in the separation technology adopted includes dichloromethane, wherein dichloromethane has the problems of high toxicity and high cost. The reported preparation technology is only for one component, privet glycoside or privet glycoside G13, and has low efficiency. It does not involve a technology that can simultaneously prepare both privet glycoside and privet glycoside G13.

[0006] Therefore, it is of great practical significance to develop a method for preparing high-purity privet glycoside and privet glycoside G13 based on osmanthus seeds, which is easy to operate and facilitate large-scale production. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing privet glycoside and privet glycoside G13 based on osmanthus seeds, aiming to solve the technical problems in the prior art that the privet glycoside and privet glycoside G13 extracted are of low purity and the operation process is too complicated and inconvenient for production.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for preparing privet glycoside and privet glycoside G13 based on osmanthus seeds, comprising the following steps:

[0010] Performing a first step of extracting osmanthus seeds to obtain osmanthus seed extract;

[0011] dispersing the osmanthus fragrans seed extract in water to obtain an osmanthus fragrans seed extract dispersion;

[0012] The osmanthus fragrans seed extract dispersion is extracted with petroleum ether, dichloromethane and n-butanol in sequence to obtain an n-butanol layer extract;

[0013] The n-butanol layer extract was subjected to normal phase column chromatography to obtain privet glycoside and privet glycoside G13;

[0014] Wherein, the first step of extraction is percolation extraction; the extractant of the first step of extraction is methanol, ethanol, acetone or water.

[0015] Preferably, the percolation extraction time is 8 to 16 hours per time, and the number of percolation extractions is 1 to 4 times.

[0016] Preferably, the ratio of osmanthus seeds to the extractant in the first step of extraction is 1kg:5-10L; the ratio of osmanthus seed extract to water in the osmanthus seed extract dispersion is 1kg:1L.

[0017] Preferably, the extraction is specifically:

[0018] Using petroleum ether to perform a first step extraction on the osmanthus seed extract dispersion to obtain a first step extract;

[0019] Performing a second step extraction on the first step extract with dichloromethane to obtain a second step extract;

[0020] The second step extract is subjected to a third step of extraction using n-butanol to obtain the n-butanol layer extract.

[0021] Preferably, in the first step extraction, the volume ratio of the osmanthus seed extract dispersion to petroleum ether is 1-2:2-3; in the second step extraction, the volume ratio of the first step extract to dichloromethane is 1-2:1-2; in the third step extraction, the volume ratio of the second step extract to n-butanol is 2-3:2-2.5.

[0022] Preferably, the normal phase column chromatography is performed twice.

[0023] Preferably, the eluent of the normal phase column chromatography is a mixture of ethyl acetate, methanol and water; the volume ratio of ethyl acetate, methanol and water in the eluent is 20:1:1 to 2:1:1 or 5:1:1 to 1:1:1.

[0024] Preferably, the mass ratio of the n-butanol layer extract to the normal phase silica gel is 1:5 to 1:30; and the mesh size of the normal phase silica gel in the normal phase silica gel column is 300 to 400 meshes.

[0025] The present invention also provides the use of the special ligustroglucosides and ligustroglucosides G13 described in the above technical solution in the preparation of anti-cardiac paralysis drugs or health products.

[0026] Preferably, the special ligustroglucoside, ligustroglucoside G13 and a pharmaceutically acceptable carrier are made into a medicine or health product in the form of tablets, capsules, granules, pills, pellets, powders, pellets, decoctions, syrups, mixtures, decoctions or extracts.

[0027] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0028] (1) The present invention adopts the method of percolation to extract osmanthus seeds, which can avoid the destruction of the effective ingredients of lisu glycoside and lisu glycoside G13 in the osmanthus seeds; petroleum ether, dichloromethane and n-butanol are used for extraction in sequence, which can obtain more n-butanol extracts on the basis of removing low-polarity impurities as much as possible; two-step normal phase column chromatography separation technology is used to simultaneously prepare lisu glycoside and lisu glycoside G13, which can improve the preparation efficiency of obtaining the two components, reduce their loss in the separation process, and improve the yield; and ethyl acetate, methanol and water system are used in the separation process, which has the characteristics of less toxicity and lower cost compared with the existing dichloromethane, methanol and water system.

[0029] (2) In the two-step normal phase column chromatography separation technology adopted in the preparation method of the present invention, the required silica gel filler and column chromatography material are very common equipment in the field of pharmaceutical production, which are low-priced and easy to use on a large scale; and in the preparation method of the present invention, a mixed solution of ethyl acetate-methanol-water is used to perform gradient elution on the normal phase silica gel column, which is beneficial to quickly remove impurities, separate special privet glucoside and privet glucoside G13, and save separation time and cost.

[0030] (3) The present invention also provides the use of Ligustrum lucidum and Ligustrum lucidum G13 in the preparation of anti-cardiac paralysis drugs or health products. When used in the preparation of anti-cardiac paralysis drugs or health products, they can significantly inhibit rabbit platelet aggregation and have anti-inflammatory activity, which is of great significance for the prevention and treatment of cardia paralysis diseases and the development of new drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The flowchart of the preparation process of Ligustrum lucidum and Ligustrum lucidum G13 in Example 1 of the present invention is as follows;

[0032] Figure 2 The HPLC spectrum of the purity test of the special privet glycosides prepared in Example 1 of the present invention;

[0033] Figure 3 The HPLC spectrum of the purity test of Ligustrum lucidum G13 prepared in Example 1 of the present invention;

[0034] Figure 4 This is a schematic diagram of the effect of the terepril glycosides prepared in Example 1 of the present invention on the aggregation of zebrafish macrophages in the inflammatory lesions of the lateral line neuromasts;

[0035] Figure 5 This is a schematic diagram of the effect of Ligustrum lucidum G13 prepared in Example 1 of the present invention on the aggregation of zebrafish macrophages in inflammatory lesions of the lateral line neuromasts. DETAILED DESCRIPTION

[0036] The present invention provides a method for preparing privet glycoside and privet glycoside G13 based on osmanthus seeds, comprising the following steps:

[0037] Performing a first step of extraction on osmanthus seeds to obtain osmanthus seed extract;

[0038] dispersing the osmanthus fragrans seed extract in water to obtain an osmanthus fragrans seed extract dispersion;

[0039] The osmanthus fragrans seed extract dispersion is extracted with petroleum ether, dichloromethane and n-butanol in sequence to obtain an n-butanol layer extract;

[0040] The n-butanol layer extract was subjected to normal phase column chromatography to obtain privet glycoside and privet glycoside G13;

[0041] Wherein, the first step of extraction is percolation extraction; the extractant of the first step of extraction is methanol, ethanol, acetone or water.

[0042] The structural formula of the special ligustrin obtained by the present invention is:

[0043]

[0044] The structural formula of the Ligustrum lucidum glycoside G13 obtained by the present invention is:

[0045]

[0046] The invention mixes osmanthus seeds with an extractant before percolation, and then performs percolation. The volume concentrations of methanol, ethanol and acetone in the extractant are 50% to 95%, 50% to 95% and 50% to 95% respectively.

[0047] In the present invention, the percolation extraction time is preferably 8 to 16 h / time, and the number of percolation extractions is preferably 1 to 4 times.

[0048] In the present invention, the osmanthus seeds are extracted by percolation, which can avoid the destruction of the effective ingredients ligustroglucosidum and ligustroglucosidum G13 in the osmanthus seeds.

[0049] In the present invention, the dosage ratio of osmanthus seeds to the extractant in the first step of extraction is preferably 1kg:5-10L; the dosage ratio of osmanthus seed extract to water in the osmanthus seed extract dispersion is preferably 1kg:1L.

[0050] In the present invention, the extraction is specifically:

[0051] Using petroleum ether to perform a first step extraction on the osmanthus seed extract dispersion to obtain a first step extract;

[0052] Performing a second step extraction on the first step extract with dichloromethane to obtain a second step extract;

[0053] The second step extract is subjected to a third step of extraction using n-butanol to obtain the n-butanol layer extract.

[0054] The invention adopts petroleum ether, dichloromethane and n-butanol to extract in sequence, and can obtain more n-butanol extract on the basis of removing low-polarity impurities as much as possible.

[0055] In the present invention, before the normal phase column chromatography, the n-butanol layer extract is preferably mixed with a solvent, and then normal phase silica gel is added for normal phase column chromatography to obtain special privet glycosides and privet glycoside G13; wherein the mesh number of the normal phase silica gel is preferably 100-200 meshes; the dosage ratio of the n-butanol layer extract to the solvent is 1kg:1L-1kg:5L; the solvent is methanol and / or ethanol.

[0056] In the present invention, the volume ratio of the osmanthus seed extract dispersion to petroleum ether in the first step extraction is preferably 1:1; the volume ratio of the first step extract to dichloromethane in the second step extraction is preferably 1:1; and the volume ratio of the second step extract to n-butanol in the third step extraction is preferably 1:1.

[0057] In the present invention, the normal phase column chromatography is preferably performed twice.

[0058] The invention adopts a two-step normal phase column chromatography separation technology to reduce the loss of privet glycoside and privet glycoside G13 in the separation process, thereby improving the yield; at the same time, an ethyl acetate, methanol and water system is adopted in the separation process, which has the characteristics of less toxicity and lower cost compared with the existing dichloromethane, methanol and water system.

[0059] In the present invention, the eluent of the normal phase column chromatography is preferably a mixture of ethyl acetate, methanol and water; the volume ratio of ethyl acetate, methanol and water in the eluent is preferably 20:1:1 to 2:1:1 or 5:1:1 to 1:1:1.

[0060] In the present invention, the mass ratio of the n-butanol layer extract to the normal phase silica gel is preferably 1:5 to 1:30; the mesh size of the normal phase silica gel in the normal phase silica gel column is preferably 300 to 400 meshes.

[0061] The present invention also provides the use of the special ligustroglucosides and ligustroglucosides G13 described in the above technical solution in the preparation of anti-cardiac paralysis drugs or health products.

[0062] In the present invention, the special ligustroglucoside, ligustroglucoside G13 and a pharmaceutically acceptable carrier are made into a medicine or health product in the form of tablets, capsules, granules, pills, pellets, powders, pellets, decoctions, syrups, mixtures, decoctions or extracts.

[0063] In the present invention, unless otherwise specified, the required raw materials for preparation are all commercially available products well known to those skilled in the art.

[0064] 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.

[0065] Example 1

[0066] S1. Preparation of Osmanthus fragrans seed extract

[0067] S1-1, take 5 kg of osmanthus seeds, peel them, crush them, add 50 L of 95% ethanol solution by volume, mix well, and obtain a mixture;

[0068] S1-2, subjecting the mixture obtained in step S1-1 to percolation extraction, specifically, continuously adding a fresh ethanol solution with a volume concentration of 95% from the top, and collecting the percolation liquid at the outlet below the percolator, collecting 25L each time; in this step, the number of percolation extractions is 3 times, and the time for a single percolation extraction is 12 hours.

[0069] S1-3. After each percolation extraction is completed, the ethanol in the extract is recovered under reduced pressure at 40° C. using a rotary evaporator, and the obtained concentrated solutions are combined to obtain an osmanthus seed extract.

[0070] S2. Preparation of n-butanol extract

[0071] S2-1. Disperse the osmanthus fragrans seed extract obtained in step S1 in water at a mass volume ratio of 1 kg:1 L to obtain an osmanthus fragrans seed extract dispersion.

[0072] S2-2, adding petroleum ether to the osmanthus seed extract dispersion at a volume ratio of 1:1 to extract the low-polarity fat-soluble impurities therein, and after the extraction, removing the petroleum ether layer, and performing the second step extraction on the remaining osmanthus seed extract dispersion.

[0073] S2-3, adding dichloromethane to the remaining osmanthus seed extract dispersion obtained in step S2-2 at a volume ratio of 1:1, extracting the medium polar fat-soluble impurities therein, removing the dichloromethane layer after completion, and performing the third step of extraction on the remaining osmanthus seed extract dispersion.

[0074] S2-4. Add n-butanol to the remaining osmanthus seed extract dispersion obtained in step S2-3 at a volume ratio of 1:1 to extract the special privet glycosides and privet glycoside G13 therein, and concentrate the obtained n-butanol extract under reduced pressure at 40° C. using a rotary evaporator to recover the n-butanol to obtain a n-butanol extract.

[0075] S3. Preparation of Ligustrum lucidum and Ligustrum lucidum G13

[0076] S3-1. Dissolve the n-butanol layer extract obtained in step S2 with methanol at a dosage ratio of 1kg:2L, i.e., dissolve it in methanol; add 100-200 mesh normal phase silica gel at a mass ratio of n-butanol layer extract to normal phase silica gel of 1:15 to obtain a n-butanol layer extract sample.

[0077] S3-2, using a 300-400 mesh (normal phase silica gel) normal phase silica gel column to perform the first step normal phase column chromatography on the n-butanol layer extract sample obtained in step S3-1, using a mixed solution of ethyl acetate-methanol-water as an eluent for gradient elution on the normal phase silica gel column, and collecting the eluent, specifically: using a mixed solution of ethyl acetate-methanol-water with a volume ratio of ethyl acetate, methanol, and water of 20:1:1, 15:1:1, 10:1:1, 5:1:1, and 2:1:1 for gradient elution, eluting 5 column volumes at each ratio, using a rotary evaporator at 40°C, and concentrating under reduced pressure to obtain secondary fractions, and merging the eluted fractions according to the TLC thin layer spot plate, and dividing them into 10 portions in total. The fraction with the most concentrated content of special privet glycosides and privet glycoside G13 and the least impurities shown on the TLC thin layer plate is used as the sample for the next stage.

[0078] S3-3, using a 300-400 mesh (normal phase silica gel) normal phase silica gel column to perform a second step of normal phase column chromatography on the eluate obtained in step S3-2 (the fraction containing the most terebinurin and terebinurin G13 in step S3-2), using a mixed solution of ethyl acetate-methanol-water as an eluent for gradient elution on the normal phase silica gel column, and collecting the eluate, specifically: using a mixed solution of ethyl acetate-methanol-water with a volume ratio of 5:1:1, 2:1:1, and 1:1:1 of ethyl acetate, methanol, and water in turn for gradient elution, eluting 8 column volumes in each ratio, using a rotary evaporator at 40°C, and concentrating under reduced pressure to obtain each fraction. According to TLC thin layer spot plate tracking, the highest fractions containing terebinurin and terebinurin G13 are obtained respectively, and after merging and standing, terebinurin and terebinurin G13 monomers are obtained.

[0079] The ligusticum glycoside obtained in Example 1 is a white amorphous powder (methanol) with a molecular formula of C 31 H 42 O 17 The molecular weight is 686.2. The specific NMR data are shown in Table 1.

[0080] Table 1 Magnetic resonance data of the special ligustrin prepared in Example 1

[0081]

[0082]

[0083] The Ligustrum lucidum glycoside G13 obtained in Example 1 is a white amorphous powder (methanol) with a molecular formula of C 48 H 64 O 27 , molecular weight is 1072. Specific NMR data are shown in Table 2.

[0084] Table 2 Magnetic resonance data of the special ligustrin obtained in Example 1

[0085]

[0086]

[0087] Example 2

[0088] Evaluation of the anti-cardiac activity of osmanthus fragrans seed extract, n-butanol extract, ligustrin and ligustrin G13 monomer

[0089] The osmanthus seed extract, n-butanol extract, privet glycoside and privet glycoside G13 prepared in Example 1 were used as test substances to conduct the following experiments:

[0090] Effects of ADP on platelet aggregation in rabbits

[0091] Take healthy Japanese white rabbits weighing 2.5-3.0kg, take 3mL of blood from the ear vein (anticoagulant: blood = 1:9), mix it immediately, seal it, and centrifuge it at 500 rpm for 10 minutes to prepare platelet-rich plasma (PRP). After separating PRP, centrifuge it again (3000rpm / min, 10min) to prepare platelet-poor plasma (PPP). Take PRP and put it into a 300μL test cup with magnetic beads, mix the remaining blood, and centrifuge it at 3000 rpm for 10 minutes. Take PPP and put it into a 300μL test cup. After zeroing with PRP, take 200μLPRP and add 5μL of equimolar concentration of the test substance DMSO (dimethyl sulfoxide) solution. Add an equal volume of solvent DMSO to the solvent control group. After incubation for 2min, use 20μL 5mmol / LADP as an inducer to measure platelet aggregation reaction. (Preparation of ADP: Buffer solution (pH=7.4, 2 mg / mL) was prepared with disodium hydrogen phosphate and sodium dihydrogen phosphate. t-test was used to test the significance of the differences among the groups.

[0092] Effects of AA on platelet aggregation in rabbits

[0093] Take healthy Japanese white rabbits weighing 2.5-3.0kg, take 3mL of blood from the ear vein (anticoagulant: blood = 1:9), mix it immediately, seal it, and centrifuge it at 500 rpm for 10 minutes to prepare platelet-rich plasma (PRP). After separating PRP, centrifuge it again (3000rpm / min, 10min) to prepare platelet-poor plasma (PPP). Take PRP and put it into a 300μL test cup with magnetic beads, mix the remaining blood, and centrifuge it at 3000 rpm for 10 minutes. Take PPP and put it into a 300μL test cup. After zeroing with PRP, take 200μLPRP and add 5μL of DMSO solution of the test substance with equal molar concentration. Add equal volume of solvent DMSO to the solvent control group. After incubation for 2min, use 20umol / LAA as an inducer to measure platelet aggregation reaction, and t test the significance of the difference between the groups.

[0094] Effects of collagen on platelet aggregation in rabbits

[0095] Take healthy Japanese white rabbits weighing 2.5-3.0kg, take 3mL of blood from the ear vein (anticoagulant: blood = 1:9), mix it immediately, seal it, and centrifuge it at 500 rpm for 10 minutes to prepare platelet-rich plasma (PRP). After separating PRP, centrifuge it again (3000rpm / min, 10min) to prepare platelet-poor plasma (PPP). Take PRP and put it into a 300μL test cup with magnetic beads, mix the remaining blood, and centrifuge it at 3000 rpm for 10 minutes. Take PPP and put it into a 300μL test cup. After zeroing with PRP, take 200μLPRP and add 5μL of DMSO solution of the test substance with equal molar concentration. Add equal volume of solvent DMSO to the solvent control group. After incubation for 2min, use 1mg / mL collagen as an inducer to measure platelet aggregation reaction, and t test the significance of the difference between the groups.

[0096] The experimental results of the test substances under the three induction models are shown in Tables 3 to 5.

[0097] Table 3 Effects of different test substances on ADP-induced platelet aggregation

[0098] Concentration (mg / mL) Maximum aggregation rate (%) Inhibition rate (%) blank 26.02±10.15 Ligustrum lucidum 2.8 17.90±5.94* 31.21 Ligustrum lucidum 1.4 19.47±6.85 25.18 Ligustrum lucidum 0.7 22.20±16.83 14.61 Ligustrum lucidum G13 2.8 15.9±2.26* 38.89 Ligustrum lucidum G13 1.4 20.75±1.34 20.25 Ligustrum lucidum G13 0.7 20.47±3.09 21.25 Extracts 2.8 17.41±4.38* 33.13 Extracts 1.4 18.55±0.92* 28.71 Extracts 0.7 22.25±0.42 14.68 n-Butanol 2.8 16.55±3.18* 36.39 n-Butanol 1.4 18.21±1.84* 30.05 n-Butanol 0.7 19.80±5.80 23.90 Danshen injection 1.4 14.2±3.31** 37.16

[0099] Compared with the blank group, *P<0.05, **P<0.01.

[0100] Table 4 Effects of different test substances on collagen-induced platelet aggregation

[0101]

[0102]

[0103] Compared with the blank group, *P<0.05, **P<0.01.

[0104] Table 5 Effects of different test substances on AA-induced platelet aggregation

[0105] Concentration (mg / mL) Maximum aggregation rate (%) Inhibition rate (%) blank 26.02±10.15 Ligustrum lucidum 2.8 16.50±8.63* 36.59 Ligustrum lucidum 1.4 17.40±3.80* 33.13 Ligustrum lucidum 0.7 17.87±9.54* 31.33 Ligustrum lucidum G13 2.8 19.57±3.16 24.80 Ligustrum lucidum G13 1.4 19.83±4.35 23.78 Ligustrum lucidum G13 0.7 20.10±4.05 22.75 Extracts 2.8 12.77±6.16* 50.94 Extracts 1.4 19.33±7.44* 25.70 Extracts 0.7 21.53±5.91 17.24 n-Butanol 2.8 17.33±1.98* 33.38 n-Butanol 1.4 19.63±11.06 24.56 n-Butanol 0.7 21.27±6.03 18.26 Danshen injection 1.4 19.16±3.51* 26.38

[0106] Compared with the blank group, *P<0.05, **P<0.01.

[0107] It can be seen from the above results that in the ADP-induced platelet aggregation model, compared with the blank group, the ethanol extract and n-butanol layer extract of osmanthus seeds at medium and high concentrations showed significant inhibitory effects. At high concentrations, the inhibition rates of the two were close to the positive control drug Danshen injection. Special ligustrin and ligustrin G13 had significant inhibitory effects at high concentrations, among which ligustrin G13 showed an inhibitory effect stronger than the positive drug Danshen injection; in the collagen-induced model, compared with the blank group, osmanthus seed extract and n-butanol extract showed significant inhibition of rabbit platelet aggregation. At high concentrations, their platelet inhibition rate was twice that of Danshen injection. In addition, at medium and high concentrations, special ligustrin and ligustrin G13 also showed results with higher inhibition rates than the positive drug; in the arachidonic acid-induced model, the ethanol extract and n-butanol layer extract of osmanthus seeds at high concentrations showed higher inhibition rates than Danshen injection. At medium concentrations, it showed an inhibitory effect close to Danshen injection. In addition, the inhibition rate of Ligustrum lucidum glycosides at low, medium and high concentrations was higher than that of positive drug Danshen injection. Ligustrum lucidum glycosides G13 showed an inhibition effect close to that of positive drug Danshen injection at low, medium and high concentrations.

[0108] Example 3

[0109] The high-purity ligustrin and ligustrin G13 prepared in Example 1 were used as test substances to investigate their anti-inflammatory activity on zebrafish Specific implementation method:

[0111] Methods: Transgenic zebrafish (Lyz:EGFP JS7) were used as an experimental model to study the effects of two sample concentrations on anti-inflammatory activity.

[0112] Zebrafish embryo acquisition

[0113] Male and female zebrafish were fed separately, with alternating lighting for 14h / darkness for 10h, and feeding was done regularly. When collecting eggs, healthy and sexually mature zebrafish were placed in a mating tank at a ratio of ♀:♂1:1, and fertilized eggs were obtained at 9-10 am the next day. After disinfection and washing, the fertilized eggs were transferred into zebrafish embryo culture water (containing 5.0mM NaCl, 0.17mM KCl, 0.4mM CaCl2, 0.16mMMgSO4) and cultured at 28°C with controlled light.

[0114] Effects of two monomers on the inflammatory response in zebrafish

[0115] Zebrafish was used as an animal model. When the zebrafish embryos developed to 3dpf (days post fertilization), healthy zebrafish were selected and carefully aspirated into each well of a 24-well plate, with 10 or 12 embryos per well. Different doses of samples were added to ensure that the DMSO limit was controlled within 0.5%. At the same time, a solvent control group (0.5% DMSO), an ibuprofen (20μM) group, and a CuSO4 (40μM) group were set up. Six concentration gradient administration groups were set for each sample. After 3 hours of treatment, the zebrafish in the above groups were treated with 40μM CuSO4 respectively. The zebrafish were treated with CuSO4 in the dark for 1 hour and then observed. The aggregation of macrophages in the inflammatory lesions of the lateral line neuromasts was observed under an Olympus fluorescence microscope and photographed to clarify the subsequent administration concentration. The experimental data were expressed as mean±SD, and SPSS software was used for analysis of variance between the groups. P<0.05 was considered to be significantly different, and P<0.01 was considered to be extremely significantly different.

[0116] The results of the study on the effect of teruncle glycosides on the accumulation of zebrafish macrophages in the inflammatory lesions of the lateral line neuromasts are as follows Figure 4 As shown; Figure 4 Middle: (A) The number of macrophages aggregated under the 10-fold concentration gradient of the sample; (B) The anti-inflammatory effect of the 10-fold concentration gradient of the sample; (C) The number of macrophages aggregated under the 2-fold concentration gradient of the sample; (D) The anti-inflammatory effect of the 2-fold concentration gradient of the sample; (E) Macrophage aggregation under different concentrations of the sample (red arrows indicate where the macrophages aggregate);

[0117] Effects of Ligustrum lucidum G13 on the accumulation of zebrafish macrophages in inflammatory lesions of lateral line neuromasts Figure 5 As shown; Figure 5 Middle: (A) The number of macrophages aggregated under 10-fold concentration gradient of the sample; (B) The anti-inflammatory effect of 10-fold concentration gradient of the sample; (C) The number of macrophages aggregated under 2-fold concentration gradient of the sample; (D) The anti-inflammatory effect of 2-fold concentration gradient of the sample; (E) Macrophage aggregation under different concentrations of the sample (red arrows indicate the location where macrophages aggregate).

[0118] based on Figure 4-5 It can be seen that both samples of privet glycoside and privet glycoside G13 have anti-inflammatory effects on zebrafish; privet glycoside has obvious anti-inflammatory effects at a concentration of 0.1μg / mL-100μg / mL, and privet glycoside G13 has obvious anti-inflammatory effects at a concentration of 5μg / mL-20μg / mL.

[0119] 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 special privet glycoside and privet glycoside G13 based on osmanthus seeds, characterized in that: The following steps are involved: Performing a first step of extracting osmanthus seeds to obtain osmanthus seed extract; dispersing the osmanthus fragrans seed extract in water to obtain an osmanthus fragrans seed extract dispersion; The osmanthus fragrans seed extract dispersion is extracted with petroleum ether, dichloromethane and n-butanol in sequence to obtain an n-butanol layer extract; The n-butanol layer extract is subjected to normal phase column chromatography to obtain privet glycoside and privet glycoside G13; Wherein, the first step of extraction is percolation extraction; the extractant of the first step of extraction is methanol, ethanol, acetone or water.

2. The preparation method according to claim 1, characterized in that: The percolation extraction time is 8 to 16 hours per time, and the number of percolation extractions is 1 to 4 times.

3. The preparation method according to claim 1, characterized in that: The dosage ratio of osmanthus seeds to the extractant in the first step of extraction is 1kg:5-10L; the dosage ratio of osmanthus seed extract to water in the osmanthus seed extract dispersion is 1kg:1L.

4. The preparation method according to claim 1, characterized in that: The extraction is specifically: Using petroleum ether to perform a first step extraction on the osmanthus seed extract dispersion to obtain a first step extract; Performing a second step extraction on the first step extract with dichloromethane to obtain a second step extract; The second step extract is subjected to a third step of extraction using n-butanol to obtain the n-butanol layer extract.

5. The preparation method according to claim 4, characterized in that: The volume ratio of the osmanthus seed extract dispersion to petroleum ether in the first step extraction is 1-2:2-3; the volume ratio of the first step extraction liquid to dichloromethane in the second step extraction is 1-2:1-2; the volume ratio of the second step extraction liquid to n-butanol in the third step extraction is 2-3:2-2.

5.

6. The preparation method according to claim 1, characterized in that: The normal phase column chromatography was performed twice.

7. The preparation method according to claim 1, characterized in that: The eluent of the normal phase column chromatography is a mixture of ethyl acetate, methanol and water; the volume ratio of ethyl acetate, methanol and water in the eluent is 20:1:1 to 2:1:1 or 5:1:1 to 1:1:

1.

8. The preparation method according to claim 1, characterized in that: The mass ratio of the n-butanol layer extract to the normal phase silica gel is 1:5-1:30; the mesh number of the normal phase silica gel in the normal phase silica gel column is 300-400 meshes.

9. Use of the special privet glycoside and privet glycoside G13 as described in any one of claims 1 to 8 in the preparation of anti-cardiac paralysis drugs or health products.

10. The use according to claim 9, characterized in that: The special ligustroglucoside, ligustroglucoside G13 and a pharmaceutically acceptable carrier are made into a medicine or health product in the form of tablets, capsules, granules, pills, pellets, powders, drop pills, decoctions, syrups, mixtures, decoctions or extracts.

Images