CN109810154B - Alkaloid compound, preparation method, application and composition of qingfengteng - Google Patents

Abstract

The present invention relates to pharmaceutical technology fields, and in particular to Sabia parviflora Wall.ex Roxb alkaloid compound, preparation method and its application.The structure of Sabia parviflora Wall.ex Roxb alkaloid compound of the present invention is shown in specification；The present invention also provides the preparation methods of the QFTA compound and the QFTA compound to prevent and treat the application in fatty liver injury medicament or health care product in preparation.Show that QFTA compound provided by the invention can significantly reduce fat drips content in LO2 Human normal hepatocyte caused by fatty acid and increase through pharmacological research.LDL-C/HDL-C, ALT, AST level for reducing mouse high fat diet (HFD) induction increase, and illustrating to accumulate liver cell inner lipid has obvious inhibiting effect.It is expected to develop into the new drug or health care product with the fatty liver damage disease of prevention and treatment.

Description

Alkaloid compound, preparation method, application and composition of qingfengteng

technical field

The invention relates to the technical field of medicines, in particular to the alkaloid compound, preparation method, application and composition of the alkaloid compound of fenugreek.

Background technique

Sabia parviflora Wall.ex Roxb. is the dry stem and leaf of the vine of the genus Parviflora Sabia parviflora Wall.ex Roxb. The small flower Qingfengteng produced in Guizhou is a folk medicine of the Buyi and Miao nationalities. It is mainly distributed in Xingyi City, Anlong County, Ceheng County, Wangmo County and other places in Guizhou. Its rhizomes and leaves can be used as medicine. Anti-inflammatory and analgesic effects, the treatment of viral hepatitis A and B has remarkable curative effect, and the side effects are small. Its related varieties are mostly used clinically for diseases related to the liver. However, the main active ingredients in Qingfengteng are unknown at present, and the active substances of its liver diseases are still unclear.

Nonalcoholic fatty liver disease (NAFLD) refers to a clinicopathological syndrome in which excessive fat deposition in liver cells caused by alcohol and other established liver damage factors is the main symptom, and it is closely related to the acquisition of insulin resistance and genetic susceptibility. Metabolic stress-induced liver injury. With the global prevalence of obesity and related metabolic syndrome, non-alcoholic fatty liver disease has become an important cause of chronic liver disease in developed countries such as Europe and the United States and in wealthy areas of my country. The prevalence of NAFLD in ordinary adults ranges from 10% to 30%. Among them, the incidence rate of non-alcoholic steatohepatitis within 10 years is as high as 25%. Non-alcoholic fatty liver disease can not only directly lead to decompensated cirrhosis, hepatocellular carcinoma and liver transplantation recurrence, but also affect the progress of other chronic liver diseases, and participate in the pathogenesis of type Ⅱ diabetes and atherosclerosis. Metabolic syndrome-related malignant tumors, arteriosclerotic cardiovascular and cerebrovascular diseases, and liver cirrhosis are important factors affecting the quality of life and life expectancy of patients with nonalcoholic fatty liver disease. For this reason, non-alcoholic fatty liver disease is a new challenge in the field of contemporary medicine, and the research on its therapeutic drugs is still a necessary direction and task for the cause of human health.

Contents of the invention

The present invention conducts in-depth research on the chemical components of Qingfeng teng, extracts a new alkaloid compound (QFTA for short), and proves that the QFTA compound has the effect of treating fatty liver damage through drug efficacy tests.

The object of the present invention is to provide a kind of fenugreek alkaloid compound or its pharmaceutically acceptable salt, its chemical structure is as follows:

Chemical name: 1-(N,N-dimethylethylamino-2-)-3-hydroxyphenanthrene-4-O-glucoside; white amorphous powder; molecular formula is C ₂₄ H ₂₉ NO ₇ .

The pharmaceutically acceptable salt of the present invention refers to certain salts that the above-mentioned compound can maintain the original biological activity and is suitable for medical use. The pharmaceutically acceptable salt of the compound of the present invention can be hydrochloride, sulfate, phosphoric acid salt, hydrobromide, nitrate, acetate, maleate, fumarate or succinate, etc.

The present invention also provides a preparation method for the alkaloid compound of the genus chinensis, comprising the following steps:

(1) Take the dried medicinal material of Qingfengteng as a raw material, add an ethanol solution with 10 to 14 times the quality of the medicinal material for multiple extractions, filter, combine the extracts, concentrate under reduced pressure until there is no alcohol smell, and obtain the extract;

(2) Take the extract of step (1) and add ethanol to solubilize until the ethanol concentration is 15% to 25%, filter, remove insoluble matter, and obtain a supernatant;

(3) Take the supernatant obtained in step (2) and put it on a macroporous resin column, and then elute with water, 25% to 35% ethanol, 45% to 55% ethanol, 65% to 75% ethanol, and more than 95% ethanol , 4 to 6 column volumes for each gradient elution; take 25% to 35% ethanol eluted parts and go through LH-20 gel column chromatography, and obtain 20 fractions by eluting with 15% to 25% methanol. Fraction 20 was subjected to preparative high performance liquid chromatography to finally isolate the compound.

In an embodiment of the present invention, the extraction method in step (1) includes cold soaking method, percolation method, microwave extraction method, ultrasonic extraction method, reflux extraction method and continuous reflux extraction method.

Preferably, the extraction method in step (1) is a reflux extraction method, the extraction times are 3 times, and the extraction times are 2.5-3.5 hours, 1.5-2.5 hours and 1.5-3.5 hours respectively.

Preferably, the ethanol concentration in step (1) is 50%-80%.

Preferably, the ethanol concentration in step (2) is 50%-95%.

Preferably, the macroporous resin column in step (3) adopts one of the following types of resin columns: HP-20, D101, AB-8, 732 cation exchange resin, HPD400, HPD100.

Preferably, the high performance liquid chromatography method described in step (3) is that the chromatographic column type is a _C18 chromatographic column, and the mobile phase is 10% to 80% methanol in water or 10% to 40% acetonitrile in water, and the flow rate is 4 to 40%. 50ml/min; more preferably, the mobile phase is an aqueous solution of 25%-35% methanol, the flow rate is 10-20ml/min, and the retention time is 35min.

The present invention also provides the application of the alkaloid compound of the fenugreek vine in the preparation of health food and medicament for preventing and treating fatty liver injury.

Pharmacological studies show that the QFTA compound provided by the invention can significantly reduce the increase of lipid droplet content in LO2 human normal liver cells caused by fatty acids. Reduce the increase of LDL-C/HDL-C, ALT, and AST levels induced by high-fat diet (HFD) in mice, indicating that it has a significant inhibitory effect on lipid accumulation in liver cells. Therefore, the QFTA compound can be developed into a new medicine or health care product for preventing and treating fatty liver injury.

The present invention also provides a pharmaceutical composition, which contains a therapeutically effective amount of the alkaloid compound of the fenugreek plant described in the present invention and a pharmaceutically acceptable carrier. The present invention also provides the application of this pharmaceutical composition in the preparation of tablets, capsules, soft capsules, suppositories, granules, transdermal absorption preparations, dropping pills, oral rapid disintegrating preparations, sustained and controlled release preparations, freeze-dried powder injections, etc. .

The QFTA compound provided by the invention can significantly reduce the increase of lipid droplet content in LO2 human normal liver cells caused by fatty acids. Reduce the increase of LDL-C/HDL-C, ALT, and AST levels induced by high-fat diet (HFD) in mice, indicating that it has a significant inhibitory effect on lipid accumulation in liver cells. It is expected to be developed into a new medicine or health care product capable of preventing and treating fatty liver injury.

Description of drawings

Figure 1: Quantitative analysis of the effect of QFTA compounds on lipid droplet content in LO2 cells

Figure 2: Quantitative analysis of the effect of QFTA compounds on the triglyceride content in LO2 cells

Detailed ways

The present invention is further described below in conjunction with examples, it should be understood that these examples do not constitute a limitation to the present invention.

For the experimental methods in the following examples, unless otherwise specified, the raw materials and reagents involved are all commercially available, and all of them can be purchased from the market. Unless otherwise specified, the solutions involved are all aqueous solutions.

Embodiment 1: preparation embodiment

The preparation method of a kind of new alkaloid compound in Teng chrysantha, comprises the following steps:

(1) Get the dry Teng japonica medicinal material, add 12 times the concentration of 70% ethanol for reflux extraction each time, the number of extractions is three times, the three extraction times are respectively 3 hours, 2 hours, and 2 hours, filter, and combine the extracts, Concentrate under reduced pressure until there is no alcohol smell to obtain the extract;

(2) Take the extract of step (1) and add 95% ethanol to adjust the alcohol concentration to 20%, and filter to remove insoluble matter to obtain a supernatant.

(3) Take the supernatant obtained in step (2) and load it on a HP-20 macroporous resin column (sample: resin is filled according to the volume of 1:3), followed by water, 30% ethanol, 50% ethanol, 70% Ethanol, 95% ethanol solution elution, 5 column volumes for each gradient elution. The fraction eluted with 30% ethanol was subjected to LH-20 gel column chromatography and eluted with 20% methanol-water to obtain 20 fractions. Fraction 20 was prepared by high-performance liquid chromatography. The chromatographic conditions were: C ₁₈ -ODS chromatographic column (30mm×250mm, 10μm), methanol-water 30:70 as mobile phase, flow rate 16ml/min, retention time 35min, and finally The QFTA alkaloid compound (QFTA) was isolated and obtained with a purity of 99.6%.

Embodiment 2: preparation embodiment

The preparation method of a kind of new alkaloid compound in Teng chrysantha, comprises the following steps:

(1) Take the dried herb of Chrysanthemum japonica, add 14 times the concentration of 50% ethanol to extract three times under ultrasonic conditions, the extraction time is 2.5 hours, 1.5 hours, and 1.5 hours respectively, filter, combine the extracts, and concentrate under reduced pressure To no alcohol smell, the extract is obtained;

(2) Take the extract of step (1) and add 50% ethanol to adjust the alcohol concentration to 15%, and filter to remove insoluble matter to obtain a supernatant.

(3) Take the supernatant obtained in step (2) and load it on a D101 type macroporous resin column (sample: resin is filled according to the volume of 1:3), followed by water, 25% ethanol, 45% ethanol, 65% ethanol, 95% ethanol solution elution, 4 column volumes for each gradient elution. The fraction eluted with 25% ethanol was subjected to LH-20 gel column chromatography, and eluted with 15% methanol-water to obtain 20 fractions. Fraction 20 was prepared by high-performance liquid chromatography. The chromatographic conditions were: C ₁₈ -ODS chromatographic column (30mm×250mm, 10μm), methanol-water 25:75 as mobile phase, flow rate 16ml/min, retention time 35min, and finally The QFTA alkaloid compound (QFTA) was isolated and obtained with a purity of 99.3%.

Embodiment 3: preparation embodiment

The preparation method of a kind of new alkaloid compound in Teng chrysantha, comprises the following steps:

(1) Take the dried herb of Chrysanthemum japonica, add 10 times the concentration of 50% ethanol to extract three times under ultrasonic conditions, the extraction time is 3.5 hours, 2.5 hours, and 2.5 hours respectively, filter, combine the extracts, and concentrate under reduced pressure To no alcohol smell, the extract is obtained;

(2) Take the extract of step (1) and add 75% ethanol to adjust the alcohol concentration to 25%, and filter to remove insoluble matter to obtain a supernatant.

(3) Take the supernatant obtained in step (2) and load it on a HP-20 macroporous resin column (sample: resin is filled according to the volume of 1:3), followed by water, 35% ethanol, 55% ethanol, 75% Ethanol, absolute ethanol elution, 6 column volumes for each gradient elution. The fraction eluted with 35% ethanol was subjected to LH-20 gel column chromatography and eluted with 25% methanol-water to obtain 20 fractions. Fraction 20 was prepared by high-performance liquid chromatography. The chromatographic conditions were: C ₁₈ -ODS chromatographic column (30mm×250mm, 10μm), methanol-water 35:65 as mobile phase, flow rate 25ml/min, retention time 35min, and finally The QFTA alkaloid compound (QFTA) was isolated and obtained with a purity of 99.5%.

Example 4: Structural identification

Get the QFTA compound prepared in Example 1 and utilize spectroscopic techniques, including ultraviolet, infrared, mass spectrometry, nuclear magnetic resonance (UV, IR, MS, ¹ H-NMR, ¹³ C-NMR, 2D-NMR) to identify its structure, and then through TOF High-resolution mass spectrometry accurately determines its molecular weight and molecular formula.

Its spectral data are as follows:

(1) White amorphous powder, high-resolution mass spectrometry ESI-TOF-MS: 444.2010 [M+H] ⁺ . , the molecular formula is determined to be C ₂₄ H ₂₉ NO ₇ , in the NMR spectrum δ _C : 45.0 is two azinomethyl signals, δ _C : 134.3(C-1), 119.4(C-2), 148.0(C-3) ,140.1(C-4),124.4(C-4a),127.4(C-5),129.1(C-5a),125.0(C-6),126.4(C-7),129.6(C-8), 132.3(C-8a), 124.5(C-9), 122.3(C-10), 125.3(C-10a), 30.6(C-11), 60.2(C-12) indicate that the structure is a phenanthrene alkaloid, _δC : 105.8, 77.2, 76.2, 74.3, 69.7, 61.0 indicates that there is a glucose group in the structure. Then combined with HMBC, HSQC and other two-dimensional nuclear magnetic resonance spectrum data to determine the final structure of the compound, as shown below.

¹ H NMR (600MHz, DMSO-d6) δ: 2.27 (6H, s, 2×N-CH ₃ ), 7.20 (1H, s, H-2), 9.84 (1H, d, J=8.6Hz, H- 5),7.85(1H,d,J=8.9Hz,H-6),7.56(1H,m,H-7),7.51(1H,m,H-8),7.64(1H,d,J=9.1 Hz,H-9),7.85(1H,d,J=8.9Hz,H-10),3.16(2H,m,H-11),2.58(2H,t,J=7.5Hz,H-12), 4.86(1H,d,J=7.0Hz,H-1′),3.61(1H,m,H-2′),3.29(1H,m,H-3′),3.25(1H,m,H-4 ′),3.04(1H,m,H-5′),3.41(1H,m,H-6′ _a ),3.30(1H,m,H-6′ _b );

¹³ C NMR (150MHz, DMSO-d6) δ: 134.3(C-1), 119.4(C-2), 148.0(C-3), 140.1(C-4), 125.3(C-4a), 129.6(C -5), 129.1(C-5a), 127.4(C-6), 126.4(C-7), 125.0(C-8), 132.3(C-8a), 124.4(C-9), 122.3(C- 10), 124.5(C-10a), 30.6(C-11), 60.2(C-12), 45.0(C-NCH ₃ ), 105.8(C-1′), 74.3(C-2′), 76.2( C-3'), 69.7 (C-4'), 77.2 (C-5'), 61.0 (C-6').

Embodiment 5: drug efficacy experiment research

1. Experimental materials

1. Drugs, reagents, animals, cells

Human normal liver cell line (LO2 cells, Shanghai Huiying Biotechnology Co., Ltd.), fetal bovine serum (Hangzhou Sijiqing Bioengineering Materials Co., Ltd.), RPMI Medium 1640 medium (Solarbio Company), oleic acid (OA, O7501), Palmitic acid (PA, P9767) was purchased from SIGMA Company, biochemical detection kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.), trypsin-EDTA digestion solution (Solarbio Company), alanine aminotransferase assay kit (R1, R2) (Japan Wako Pure Chemical Industry Co., Ltd.), aspartate aminotransferase assay kit (R1, R2) (Japanese Wako Pure Chemical Industry Co., Ltd.).

2. Experimental equipment

Inverted microscope (Olympus-ckx41), biological safety cabinet (Heal Force), AL204 electronic analytical balance (Mettler Toledo Instrument (Shanghai) Co., Ltd.), MTV-100 multi-tube vortex mixer (Hangzhou Aosheng Instrument Co., Ltd.), Hitachi 7180 automatic biochemical analyzer (Hitachi).

2. Experimental method

1. Effect of QFTA on lipid levels in LO2 cells

1.1 Cell culture

L-02 cells were cultured in 1640 medium containing 5% fetal bovine serum for 48 hours at 37°C in an incubator containing 5% CO ₂ . When the cells were 70%-80% confluent, they were used for experiments. Oleic acid or palmitic acid was dissolved in isopropanol to prepare a 500 mM stock solution. The cells were planted in a 96-well plate, about 10,000 cells per well, cultured for 24 hours, the old medium was sucked off, and then the cells were randomly divided into 5 groups: blank group, model group (200mM FFAs, oleic acid: palmitic acid=2 : 1), administration group (high, middle, and low doses, respectively 2, 4, and 8 μg/ml of QFTA culture fluid), each group had 6 duplicate wells, the blank group added 100 μl of new culture fluid, and the model group and the administration group added 100 μl of culture medium containing 200 mM FFAs, continue to culture for 24 hours, absorb the culture medium, add 100 μl of new culture medium to the blank group and model group, add 100 μl of QFTA culture medium containing 2, 4, and 8 μg/ml to the high, middle and low dose groups respectively , continue to cultivate for 24h.

1.2 Lipid Droplet Oil Red O Staining Detection

LO2 cells were intracellularly washed twice with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 30 minutes, and then stained with Oil Red O solution for 1 hour at room temperature. Aspirate the staining solution, rinse twice with pure water, add 150 μL dimethyl sulfoxide to each well, shake, and measure the absorbance with a microplate reader at a wavelength of 510 nm.

1.3 Determination of triglyceride (TG) content

Cells in each treatment group were collected, and the content of triglyceride in hepatocytes was determined by tissue cell enzyme method. The specific operation steps were carried out according to the instructions of the kit.

2. Animal experiments and serum analysis

5-week-old ICR mice were placed in a temperature (22±2°C) and humidity-controlled (50%±5%) room. Regular diet (RD; 10% kcal% fat) and HFD (60% kcal% fat;) Experimental diets were purchased from Research Diets (NJ, USA) and fed with food excess and drinking water. After acclimatization for 1 week, the mice were randomly divided into the following groups: RD feeding group (n=7) was the blank control group, HFD feeding group (n=7) was the model group and three treatment groups (n=7, HFD plus QFTA 250mg/kg (QFTA250) group, HFD plus QFTA 500mg/kg (QFTA500) group and HFD plus fenofibrate (FEN) 30mg/kg group as the positive control group). The vehicle or the corresponding drug was orally administered to the mice once a day for 12 weeks, and the body weight was measured every day. After 12 weeks of treatment, mice were fasted overnight and bled. After collecting the blood sample, the blood was allowed to coagulate at room temperature, and then centrifuged at 2,000 r/min for 10 minutes at 4°C to prepare serum. Serum was stored at -75°C for later use. Serum high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT) and asparagus were measured using a kit (StanbioLaboratory, Boerne, USA) and an automatic biochemical analyzer. Amino acid aminotransferase (AST) levels.

3. Experimental results

1. Effect of QFTA on lipid levels in LO2 cells

Oil red O staining detection result is as shown in Figure 1, and the implication of ordinate among Fig. 1 is the percentage (unit: %) of the lipid content of model group and each administration group and blank group lipid content; The test results are shown in Figure 2. The ordinate in Figure 2 is the content of triglycerides. Compared with the blank group, after the free fatty acid was applied to LO2 cells for 24 hours, the content of lipid droplets in the cells increased significantly, indicating that the model was established obviously. Compared with the model group, the administration group significantly reduced the content of intracellular lipid droplets (P<0.05). The triglyceride content in the liver cells was significantly lower in the administration group than in the model group (P<0.05).

Note: * in the figure represents a significant difference compared with the model group (*P<0.05).

2. Animal experiment results

The experimental results showed that QFTA significantly increased HDLC levels while preventing the increase in LDL-C levels induced by HFD feeding. Compared with the HFD group, the ratio of LDL-C to HDL-C was significantly lower in the QFTA group. Compared with HFD group, QFTA250 and QFTA500 groups had significantly lower serum ALT and AST levels. The specific experimental results are shown in Table 1.

Table 1: Effect of QFTA on high-fat diet (HFD)-induced changes in ICR mice. (mean±SD, n=7)

Note: ^* means that compared with the model group, the difference is significant ( ^* P<0.05), ^** means that compared with the model group, the difference is extremely significant ( ^** P<0.01).

The above experimental results show that the compounds provided by the present invention can significantly reduce the lipid droplet content and triglyceride content in liver cells caused by fatty acids, and reduce the LDL-C/HDL-C and ALT levels induced by high-fat diet (HFD) in mice. , AST increased, and its effect is basically the same as that of the positive drug. However, considering that this compound is derived from the traditional Chinese medicine Xiaohua Qingfengteng, which has been clinically used for many years and has no serious toxic and side effects reports, it is expected to be developed into a new safe drug with the effect of treating fatty liver. Low toxicity, effective medicine or health product.

Claims (11)

1. a kind of Sabia parviflora Wall.ex Roxb alkaloid compound or its pharmaceutical salt, chemical structure are as follows:

2. a kind of Sabia parviflora Wall.ex Roxb alkaloid compound according to claim 1 or its pharmaceutical salt, it is described can medicine Salt is hydrochloride, sulfate, phosphate, hydrobromate, nitrate, acetate, maleate, fumarate or succinic acid Salt.

3. the preparation method of Sabia parviflora Wall.ex Roxb alkaloid compound described in claim 1, comprising the following steps:

(1) it takes dry Sabia parviflora Wall.ex Roxb medicinal material as raw material, 10~14 times of quality of medicinal material of ethanol solution is added and carries out repeatedly It extracts, filtering, combined extract is concentrated under reduced pressure into no alcohol taste, obtains extract；

(2) taking the extract of step (1) that ethyl alcohol solubilising to concentration of alcohol is added is 15%~25%, and filtering removes insoluble matter, obtains To supernatant；

(3) macroporous resin column on the supernatant for taking step (2) to obtain successively uses water, 25%~35% ethyl alcohol, 45%~55% second Alcohol, 65%~75% ethyl alcohol, 95% or more ethanol elution, 4~6 column volumes of each gradient elution；Take 25%~35% second Alcohol elutes position through LH-20 gel filtration chromatography, affords 20 fractions with 15%~25% methanol, takes fraction 20 through preparing High performance liquid chromatography is finally separating to obtain the compound.

4. the preparation method of Sabia parviflora Wall.ex Roxb alkaloid compound according to claim 3, which is characterized in that the step (1) method of the extraction includes cold-maceration, percolation, microwave loss mechanisms, ultrasonic extraction, reflux extraction and continuous backflow Extraction method.

5. the preparation method of Sabia parviflora Wall.ex Roxb alkaloid compound according to claim 3, which is characterized in that step (1) Described in extracting method be reflux extraction, the number of the extraction is 3 times, and extraction time is respectively 2.5~3.5 hours, 1.5 ~2.5 hours and 1.5~3.5 hours.

6. the preparation method of Sabia parviflora Wall.ex Roxb alkaloid compound according to claim 3, which is characterized in that step (1) The concentration of alcohol is 50%~80%；And/or step (2) concentration of alcohol is 50%~95%.

7. the preparation method of Sabia parviflora Wall.ex Roxb alkaloid compound according to claim 3, which is characterized in that step (3) The macroporous resin column use following models one of resin column: HP-20, D101, AB-8,732 cation exchange resins, HPD400、HPD100。

8. the preparation method of Sabia parviflora Wall.ex Roxb alkaloid compound according to claim 3, which is characterized in that step (3) The efficient liquid-phase chromatography method, the column model are C₁₈Chromatographic column, the mobile phase are 10%~80% methanol Aqueous solution or 10%~40% acetonitrile solution.

9. the preparation method of Sabia parviflora Wall.ex Roxb alkaloid compound according to claim 8, which is characterized in that the flowing It is mutually the aqueous solution of 25%~35% methanol.

10. Sabia parviflora Wall.ex Roxb alkaloid compound described in claim 1 or its pharmaceutical salt prevent and treat rouge in preparation Application in fat liver injury medicament health food and drug.

11. a kind of pharmaceutical composition, the Sabia parviflora Wall.ex Roxb alkaloids chemical combination described in claim 1 containing therapeutically effective amount Object and pharmaceutically acceptable carrier.