Snakegourd fruit alkaloid extract as well as extraction method and application thereof

Abstract

The invention relates to a trichosanthes kirilowii maxim alkaloid extract and an extraction method and application thereof, and belongs to the technical field of trichosanthes kirilowii maxim extract preparation. The extraction method comprises the following steps: performing ultrasonic extraction on trichosanthes peel powder for multiple times by using a sulfuric acid solution, combining extracting solutions obtained by multiple times of extraction, performing primary concentration on the combined extracting solutions, then adjusting the pH value of the extracting solutions obtained by primary concentration to a target pH value, performing extraction for multiple times by using n-butyl alcohol, combining the extracting solutions obtained by multiple times of extraction, performing secondary concentration to obtain paste, and performing spray drying to obtain the trichosanthes peel extract. The trichosanthes kirilowii maxim alkaloid extract is obtained. The content of alkaloid in the trichosanthes kirilowii maxim alkaloid extract is 60-70%, and the trichosanthes kirilowii maxim alkaloid extract contains 20 kinds of alkaloid, has a good anti-atherosclerosis pharmacological action, and can remarkably improve liver lipid deposition, liver lipid metabolism disorder and abnormal blood lipid level of atherosclerosis mice.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of trichosanthes extract, and particularly relates to a trichosanthes alkaloid extract and an extraction method and application thereof. Background Art

[0002] Atherosclerosis (AS) is a chronic inflammatory vascular disease with the formation of lipid plaques in the vascular endothelium as the main pathological feature, and is the pathological basis of many cardiovascular diseases. The increase in the content of lipids such as low-density lipoprotein cholesterol (LDL-C), triglycerides (TG) and total cholesterol (TC) in the blood causes an increase in blood viscosity, leading to abnormal blood rheology, which is one of the important factors causing AS lesions. A large amount of lipids are deposited on the arterial wall to form plaques, causing the blood vessels to harden and the lumen to narrow, promoting the occurrence and development of AS diseases. Therefore, regulating dyslipidemia occupies a core position in the clinical treatment of AS. Its ultimate goal is to delay the progression of AS or even reverse it, so as to achieve the effect of treating AS.

[0003] Modern medical research shows that the liver is one of the most vulnerable organs in the course of AS. At the same time, as an important organ in the body that participates in lipid metabolism, abnormal lipid metabolism can lead to lipid deposition in liver cells and cholesterol transport disorders, affecting the occurrence and development of AS. Very low-density lipoprotein is converted into low-density lipoprotein under the action of lipoprotein lipolytic enzymes. After combining with cholesterol synthesized in the liver, it is transported to various tissues and organs through the blood, becoming an important pathogenic factor inducing vascular lesions. After low-density lipoprotein is oxidized and modified to form oxidized low-density lipoprotein, it cannot be accepted by cell-related receptors when combined with cholesterol and is difficult to be metabolized. Therefore, it accumulates in the blood and causes abnormal blood lipids.

[0004] TrichosantheskirilowiiMaxim., also known as Trichosanthes, is a perennial climbing herb of the genus Trichosanthes in the family Cucurbitaceae. As an important component of the classic prescriptions "Trichosanthes Xiebai Banxia Decoction" and "Zhishi Xiebai Guizhi Decoction" in "Golden Chamber", it tastes sweet, slightly bitter, and is cold in nature. It has the effects of clearing heat and resolving phlegm, promoting qi and relieving chest tightness. Trichosanthes has long been used in clinical Chinese medicine to treat cardiovascular diseases, but there is no precedent for Trichosanthes alkaloid extracts in the drugs currently used to treat AS.

[0005] At present, the treatment of AS mainly focuses on reducing the intake or synthesis of cholesterol, thereby reducing blood lipid levels. At present, statins are the first choice for the clinical treatment of AS. They can significantly improve AS by inhibiting cholesterol synthesis and have significant efficacy in preventing and treating the disease. However, statins are prone to adverse reactions such as rhabdomyolysis and hepatotoxicity, so new drugs for the prevention or treatment of AS need to be developed. Summary of the invention

[0006] In view of the above problems, the present invention provides a Trichosanthes alkaloid extract and an extraction method and application thereof.

[0007] The first object of the present invention is to provide a Trichosanthes alkaloid extract, wherein the Trichosanthes alkaloid extract is obtained by:

[0008] The powder of Trichosanthes peel is ultrasonically extracted for multiple times with a sulfuric acid solution having a mass fraction of 0.5-1.5%, the extracts obtained from the multiple extractions are combined, the extracts obtained are concentrated once, the pH of the extracts after the concentration is subsequently adjusted to the target pH, and then extracted for multiple times with n-butanol, the extracts obtained from the multiple extractions are combined, and concentrated twice to obtain a paste, thereby obtaining the Trichosanthes alkaloid extract.

[0009] A second object of the present invention is to provide a method for extracting a Trichosanthes alkaloid extract, comprising:

[0010] The powder of Trichosanthes peel is ultrasonically extracted for multiple times with a sulfuric acid solution having a mass fraction of 0.5-1.5%, the extracts obtained from the multiple extractions are combined, the extracts obtained are concentrated once, the pH of the extracts after the concentration is subsequently adjusted to the target pH, and then extracted for multiple times with n-butanol, the extracts obtained from the multiple extractions are combined, and concentrated twice to obtain a paste, thereby obtaining the Trichosanthes alkaloid extract.

[0011] Furthermore, the mass of the sulfuric acid solution is 4-7 times the mass of the Trichosanthes peel powder.

[0012] The mass fraction of the sulfuric acid solution is 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4% or 1.5%.

[0013] The mass of the sulfuric acid solution is 4 times, 5 times, 6 times or 7 times the mass of the Trichosanthes peel powder.

[0014] Furthermore, the ultrasonic extraction is performed 3-6 times, and the time for each ultrasonic extraction is 0.5-2h.

[0015] The ultrasonic extraction is performed 3 times, 4 times, 5 times or 6 times.

[0016] The time of each ultrasonic extraction is 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1h, 1.1h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h, 1.6h, 1.7h, 1.8h, 1.9h or 2h.

[0017] Furthermore, when the extract obtained by the first concentration is combined, the volume of the extract after the first concentration is 0.08-0.15 of the volume of the extract obtained by the first concentration.

[0018] The volume of the extract after the first concentration is 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 of the volume of the extracts combined before the first concentration.

[0019] Furthermore, the target pH is 9-10.

[0020] The target pH is 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.

[0021] Furthermore, the extraction times are 3-6 times.

[0022] The extraction times are 3 times, 4 times, 5 times or 6 times.

[0023] The third object of the present invention is to provide a method for measuring the alkaloid content in the above-mentioned Trichosanthes alkaloid extract, comprising:

[0024] The extract of Trichosanthes kirilowii alkaloids was dissolved and diluted in dichloromethane to obtain a test solution;

[0025] Prepare trigonelline reference solution;

[0026] Add buffer solution and acid dye to the test solution to mix and dye. After dyeing, add dichloromethane, mix evenly, let stand to separate layers, take the dichloromethane layer, and dilute to the test solution to be tested;

[0027] Adding a buffer solution and an acid dye solution to the reference solution for mixed dyeing, adding dichloromethane after dyeing, mixing evenly, standing to separate layers, taking the dichloromethane layer, and diluting to different concentrations of the reference solution to be tested;

[0028] Conduct absorbance tests on the reference substance solutions with different concentrations;

[0029] Perform absorbance test on the test solution;

[0030] The absorbance values ​​of the reference solution of different concentrations and the absorbance values ​​of the test solution of different concentrations are obtained according to the absorbance test.

[0031] Furthermore, the mass concentration of the Trichosanthes alkaloid extract in the test solution is 0.1 g / mL.

[0032] Furthermore, the mass concentration of the trigonelline reference solution is 1-2 mg / mL.

[0033] Furthermore, the volume ratio of the test solution, the buffer solution, the acid dye solution and the dichloromethane is 0.1:7:5:6.

[0034] Furthermore, the volume ratio of the reference solution, the buffer solution, the acid dye solution and the dichloromethane is 0.1:7:5:6.

[0035] Furthermore, the pH of the buffer solution is 2-6.

[0036] Preferably, the pH of the buffer solution is 5.5.

[0037] Furthermore, the buffer solution is one of a citrate-disodium hydrogen phosphate buffer solution, a hydrogen phthalate buffer solution, and an acetic acid-sodium acetate buffer solution.

[0038] Preferably, the buffer solution is an acetic acid-sodium acetate buffer solution.

[0039] Furthermore, the acid dye solution is prepared by mixing acid dye, sodium hydroxide and water.

[0040] Furthermore, the acid dye is bromocresol green or bromothymol blue.

[0041] Furthermore, the mass concentration of the acid dye in the acid dye solution is 0.5 mg / mL.

[0042] Furthermore, the mass concentration of the Trichosanthes alkaloid extract in the test solution to be tested is 1-2 mg / mL.

[0043] Furthermore, the mass concentration of trigonelline in the test reference solution is 1-2 mg / mL.

[0044] Furthermore, in the absorbance test, the wavelength of the scanning light is 410-440 nm.

[0045] Preferably, in the absorbance test, the wavelength of the scanning light is 419 nm.

[0046] Further, the absorbance values ​​of the reference solution of different concentrations to be tested and the absorbance values ​​of the test solution to be tested obtained according to the absorbance test include:

[0047] According to the absorbance values ​​of the reference solution with different concentrations, determine the relationship between the alkaloid concentration and the absorbance;

[0048] According to the relationship between alkaloid concentration and absorbance, and the absorbance value of the test solution to be tested, the concentration of alkaloid in the test solution to be tested is determined;

[0049] The content of alkaloids in the test solution to be tested is determined based on the concentration of alkaloids in the test solution to be tested, the volume of the test solution to be tested and the mass of the Trichosanthes alkaloid extract used to prepare the test solution to be tested.

[0050] The fourth object of the present invention is to provide the use of the Trichosanthes alkaloid extract in the preparation of a drug for preventing or treating atherosclerosis.

[0051] The fifth object of the present invention is to provide the use of the Trichosanthes alkaloid extract in the preparation of a medicament for improving liver lipid deposition.

[0052] The sixth object of the present invention is to provide the use of the Trichosanthes alkaloid extract in the preparation of lipid-lowering drugs.

[0053] The seventh object of the present invention is to provide the use of the Trichosanthes alkaloid extract in the preparation of cholesterol-lowering drugs.

[0054] Beneficial effects of the present invention:

[0055] The invention discloses a trichosanthes alkaloid extract and an extraction method and application thereof. The trichosanthes alkaloid extract provided by the invention contains 20 alkaloids, including 3-hydroxy-2-methylpyridine, pyridine-3-carboxylic acid (commonly known as nicotinic acid), 1-(4-aminobutyl)guanidine (commonly known as agmatine), 3-pyridinecarboxamide (commonly known as nicotinamide), linalool, flaxseed, 4-methyl-5-hydroxyethylthiazole, L-pyroglutamic acid, aminosalicylic acid, 4-guanidinobutyric acid, (-)-3,4-dihydroxynorephedrine, 6-azidohexanoic acid, pseudoephedrine, theophylline, L-norephedrine, fenugreek, halmanine, L-hydroquinone, and the like. Threonine, neonicotinoids and betaine, and the alkaloid content in the Trichosanthes alkaloid extract obtained by the present invention is 60-70%, indicating that the alkaloid content in the obtained Trichosanthes alkaloid extract is relatively high, and the alkaloids cooperate with each other to give the Trichosanthes alkaloid extract a good anti-AS pharmacological effect (this Trichosanthes alkaloid extract containing 20 kinds of Trichosanthes alkaloids can reduce the area of ​​aortic plaques and alleviate aortic lesions), and also give the obtained Trichosanthes alkaloid extract the effect of inhibiting liver lipid deposition, improving liver lipid metabolism, and reducing the blood lipid level of AS mice, and the Trichosanthes alkaloid extract obtained by the present invention has small side effects, low cost, and small oral dosage;

[0056] The extraction method of Trichosanthes alkaloid extract is simple and efficient, and the raw materials are easily available and low in cost.

[0057] Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0059] Figure 1 The total ion diagram of the Trichosanthes alkaloid extract in positive ion mode according to Example 2 of the present invention is shown;

[0060] Figure 2 The total ion diagram of the Trichosanthes alkaloid extract under negative ion mode according to Example 2 of the present invention is shown;

[0061] Figure 3 The absorbance diagram of the test solution and the Trichosanthes alkaloid extract at all wavelengths in Example 3 of the present invention is shown;

[0062] Figure 4 The absorbance distribution diagram of the trigonelline reference substance solution with different concentrations in Example 3 of the present invention is shown;

[0063] Figure 5 The weight change trend of mice in different feeding groups from week 1 to week 16 in Example 4 of the present invention is shown;

[0064] Figure 6 The diagram shows the changes of plaques on the aortic arch of mice in different feeding groups according to Example 4 of the present invention;

[0065] Figure 7 The HE staining images of the aortic sinus of mice in different feeding groups according to Example 4 of the present invention are shown;

[0066] Figure 8 A bar graph showing the relative aortic sinus plaque area of ​​mice in different feeding groups according to Example 4 of the present invention is shown;

[0067] Fig. 9 The graph showing the changes in TC, TG, HDL-C, and LDL-C levels in the serum of mice in different feeding groups according to Example 4 of the present invention;

[0068] Fig.10 The appearance of the liver of mice in different feeding groups according to Example 4 of the present invention is shown;

[0069] Fig.11 The graph showing the changes in liver index of mice in different feeding groups according to Example 4 of the present invention is shown;

[0070] Fig.12 The Oil Red O staining of the liver of mice in different feeding groups according to Example 4 of the present invention is shown;

[0071] Fig.13 The HE staining images of liver lipids of mice in different feeding groups according to Example 4 of the present invention are shown. DETAILED DESCRIPTION

[0072] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0073] The present invention uses the herbal medicine Trichosanthes kirilowii purchased from Beijing Tong Ren Tang Hefei Pharmacy Co., Ltd.

[0074] Example 1

[0075] Preparation of Trichosanthes alkaloids extract:

[0076] (1) Weigh an appropriate amount of Trichosanthes peel, dry it at 50°C, crush it, and pass it through a 30-mesh sieve;

[0077] (2) ultrasonic extraction was performed four times with 6 times the amount of 1% sulfuric acid, each time for 1 h, and the sulfuric acid extracts were filtered and combined;

[0078] (3) Concentrate the sulfuric acid extract under reduced pressure to 1 / 10 of its original volume, and adjust the pH to 9-10 with concentrated NaOH solution;

[0079] (4) Extract with an equal volume of n-butanol for 4 times, combine the n-butanol extracts, and concentrate under reduced pressure to obtain the Trichosanthes peel alkaloid extract, which is refrigerated and stored for later use.

[0080] Example 2

[0081] The alkaloid components in the Trichosanthes peel alkaloid extract obtained in Example 1 were identified:

[0082] Add 2 times the amount of methanol to the alkaloid extract, vortex mix, and then perform ultrasonic extraction for 20 minutes. Centrifuge at 12000r / min for 10 minutes at 4°C, take 500μL of the supernatant, add 500μL of pure water, vortex oscillate for 3 minutes, centrifuge at 12000r / min for 10 minutes at 4°C, and take the supernatant to obtain the test solution. The test solution was diluted 5 times with 50% methanol for mass spectrometry analysis. Chromatographic conditions: ThermoVanquish (ThermoFisherScientific, USA) ultra-high performance liquid system, using HSST3 (2.1×100 mm, 1.8 μm) (Waters, Milford, MA, USA) column, flow rate of 0.3 mL / min, column temperature of 40°C, injection volume of 2 μL. The mobile phase was 0.1% formic acid acetonitrile (B) and 0.1% formic acid water (A), and the gradient elution program was: 0-2 min, 8-17% B; 2-3 min, 17%-25% B; 3-5 min, 25-28% B; 5-6 min, 28-30% B; 6-7 min, 30-60% B; 7-8 min, 60% B; 8-12 min, 60-100% B. Mass spectrometry conditions: Thermo Q Exactive mass spectrometer (Thermo Fisher Scientific, USA), electrospray ionization source (ESI), data were collected in positive and negative ion modes respectively. The positive ion spray voltage was 3.50 kV, the negative ion spray voltage was -2.50 kV, the sheath gas was 40 arb, and the auxiliary gas was 10 arb. The capillary temperature was 325 °C, and the first-level full scan was performed at a resolution of 70000. The first-level ion scan range was m / z100-1000, and HCD was used for secondary fragmentation. The collision energy was 30 eV, the second-level resolution was 17500, and the first 10 ions of the acquisition signal were fragmented. At the same time, dynamic exclusion was used to remove unnecessary MS / MS information.

[0083] Experimental results:

[0084] The total ion pattern of Trichosanthes alkaloid extract obtained by UPLC-Q-Exactive-MS in positive ion mode is shown in the figure Figure 1 As shown, the total ion diagram in negative ion mode is as follows Figure 2 As shown in Figure 1, the original mass spectrometry file was converted into mzXML file format by the MSConvert tool in the Proteowizard software package (v3.0.8789). The RXCMS (v3.12.0) software package was used for peak detection, peak filtering, and peak alignment to obtain a quantitative table of substances. The data acquisition results were attributed by molecular weight, secondary mass spectrometry information, etc. obtained from references and existing databases. The error between the measured value and the theoretical value was <10ppm as the standard. Some compounds were compared with the standard. A total of 20 alkaloid components were identified from the alkaloid extract of Trichosanthes kirilowii, and the results are shown in Table 1.

[0085] Table 1

[0086]

[0087]

[0088] In Table 1, “[]+” indicates positive ion mode, and “[]-” indicates negative ion mode.

[0089] It can be seen from the data in Table 1 that the Trichosanthes alkaloid extract obtained in Example 1 of the present invention contains 20 alkaloids, namely 3-hydroxy-2-methylpyridine, nicotinic acid, agmatine, nicotinamide, linalool, linalool (English name is Linatine), 4-methyl-5-hydroxyethylthiazole, L-pyroglutamic acid, aminosalicylic acid, 4-guanidinobutyric acid, (-)-3,4-dihydroxynorephedrine, 6-azidohexanoic acid, pseudoephedrine, theophylline, L-norephedrine, fenugreek, halmanine, L-stachydrine, anabasine and betaine.

[0090] The Chinese names of the alkaloids in Table 1 and their corresponding English names are shown in Table 2.

[0091] Table 2

[0092]

[0093]

[0094] Example 3

[0095] Determination of alkaloids in Trichosanthes kirilowii by acid dye colorimetry

[0096] 1. Solution preparation

[0097] Accurately weigh 2 mg of the cucurbitacin reference substance, place it in a 1 mL volumetric flask, and dilute to the mark with dichloromethane to obtain a 2 mg / mL cucurbitacin reference substance solution.

[0098] Take 1 g of the extract prepared in Example 1 (i.e., the Trichosanthes alkaloid extract) in a test tube, add 10 mL of dichloromethane to dissolve and dilute, and obtain a test solution.

[0099] Weigh 0.05 g of bromothymol blue powder into a 100 mL volumetric flask, add 1.6 mL of freshly prepared 0.05 mol / L sodium hydroxide solution, then dilute to the mark with ultrapure water and shake well to obtain bromothymol blue solution.

[0100] Weigh 0.05 g of bromocresol green powder into a 100 ml volumetric flask, add 1.4 ml of freshly prepared 0.05 mol / L sodium hydroxide solution, and dilute to the mark with ultrapure water. Shake well to obtain bromocresol green solution.

[0101] 2 Types of acid dyes and selection of measurement wavelength

[0102] Take two 0.1mL portions of the test solution under item 1 and place them in different stoppered centrifuge tubes. Add 7mL of acetic acid-sodium acetate buffer solution with a pH of 5.5 to both tubes. Then add 5mL of bromothymol blue solution to one tube and 5mL of bromocresol green solution to the other tube. Seal the tubes and vortex to mix them. Add 6mL of dichloromethane solution to both tubes, seal the tubes, vortex for 1min, let stand for a while to separate the layers, take the dichloromethane layer (lower layer) in a 10mL volumetric flask, dilute with dichloromethane and make up to the scale. In addition, take two 0.1mL portions of the reference solution under item 2.1 and repeat the above operation. Take another 0.1mL of dichloromethane to make a blank solution for comparison. Scan the above-prepared solution at all wavelengths, and determine the best acid dye and detection wavelength based on the maximum absorption.

[0103] 3. Selection of buffer type, pH and dosage

[0104] Citrate-disodium hydrogen phosphate buffer solution, hydrogen phthalate buffer solution, and acetic acid-sodium acetate buffer solution were selected to investigate the effect of buffer type on UV absorption, and the optimal buffer solution was determined based on the maximum absorption. After determining the optimal buffer solution, the effect of pH values ​​of 2.0, 3.0, 4.0, 5.0, and 6.0 on UV absorption was investigated. In addition, the effect of the amount of buffer solution used on UV absorption was investigated at 4, 5, 6, 7, and 8 mL, respectively. Both conditions were determined based on the maximum absorption.

[0105] 4 Methodological investigation

[0106] 4.1 Linear Relationship

[0107] According to the method under item 1, a 2 mg / mL trigonelline reference solution was prepared, and then diluted with dichloromethane to obtain a series of reference solutions with concentration gradients such as 1.75 mg / mL, 1.5 mg / mL, 1.25 mg / mL, 1 mg / mL, 0.75 mg / mL, etc. The above six reference solutions of different concentrations were treated with the optimal acid dye colorimetry conditions obtained above, and dichloromethane reagent was used as a blank control. The measurement was carried out at the optimal detection wavelength, and the standard curve was plotted with the reference concentration (in mg / mL) as the abscissa and the absorbance value (A) as the ordinate to obtain the linear regression equation.

[0108] 4.2 Precision test

[0109] The optimal acid dye colorimetric conditions obtained in the early stage were substituted into item 2, and a sample with a concentration of 1.5 mg / mL was prepared according to this method. The measurement was then repeated six times at the optimal detection wavelength, and the relative standard deviation (RSD) of these six absorbance values ​​was used to determine whether the instrument precision was good.

[0110] 4.3 Stability test

[0111] Accurately pipette 200 μL of the test solution under Item 2.1 and prepare it into a 2 mg / mL sample according to the optimal conditions. Measure the absorbance at the optimal detection wavelength at seven time points: 0 min, 15 min, 30 min, 45 min, 60 min, 90 min, and 120 min, and calculate the RSD values ​​of these seven absorbances.

[0112] 4.4 Repeatability test

[0113] Take an appropriate amount of medicinal materials from the same batch, crush them, weigh six portions of powder, 100 g each, and prepare alkaloid extracts and test sample solutions in turn according to the methods under 1 and 2.1. Then, accurately pipette 200 μL of each test sample and prepare it into a sample with a concentration of 2 mg / mL according to the optimal conditions. Finally, test it at the optimal detection wavelength, record the absorbance values ​​six times, and calculate its RSD value.

[0114] 4.5 Sample recovery test

[0115] Take 70 mg of alkaloid extract with known content and prepare it into a test solution with a concentration of 7 mg / mL according to the method under item 1. Accurately pipette 1 mL of the solution into a stoppered centrifuge tube, add trigonelline (the amount of trigonelline added is detailed in Table 4), then prepare the sample according to the method under item 2 and combined with the optimal acid dye colorimetry conditions, repeat the above operation, prepare a total of 6 test solution for detection, finally record the corresponding absorbance, and calculate the sample recovery rate.

[0116] 5. Sample content determination

[0117] Take a certain amount of medicinal materials and crush them, weigh 100g of powder, prepare alkaloid extract and test solution (concentration of 100mg / mL) according to the above method, prepare six batches in parallel, then accurately draw 200μL from each, and prepare six batches of 2mg / mL test solution under the methods of 2.2 and 2.3 using the optimal conditions. At the same time, use the corresponding reagent as blank, measure the absorbance of the sample at the optimal detection wavelength, and substitute the measured value into the linear regression equation to calculate the content of the target component.

[0118] The above experimental results are shown in Tables 3-5 and Figure 3 , Figure 4 As shown;

[0119] Among them, Table 3 is a table showing the relationship between the concentrations of different trigonelline reference solutions and absorbance;

[0120] Table 4 is the recovery rate table of the sample recovery test;

[0121] Table 5 is a table showing the alkaloid contents in the medicinal materials of Trichosanthes kirilowii in six batches of samples in the sample content determination;

[0122] Among them, a1=C2×V1 / M1×100%, a2=a1×M1 / M o ×100%, M0 is the mass of the Trichosanthes kirilowii medicinal material used to generate the Trichosanthes kirilowii alkaloid extract of mass M1, M1 is the mass of the Trichosanthes kirilowii alkaloid extract (the mass of the Trichosanthes kirilowii alkaloid extract used to make the test solution to be tested), V1 is the volume of the test solution to be tested, and C2 is the alkaloid concentration in the test solution to be tested, which is calculated by absorbance and linear regression equation.

[0123] Table 3

[0124]

[0125] Table 4

[0126]

[0127] In Table 4, the sample content is the mass of Trichosanthes powder.

[0128] Table 5

[0129]

[0130] Figure 3 It is the absorbance diagram of the test solution and the extract of Trichosanthes alkaloids at all wavelengths;

[0131] Figure 4 This is the absorbance distribution diagram of trigonelline reference solution with different concentrations.

[0132] like Figure 3 As shown, the absorbance of the samples using bromocresol green as the acid dye is greater than that of the samples using bromothymol blue as the dye, so the type of acid dye can be determined to be bromocresol green; and when the wavelength is between 200nm and 250nm, although the absorbance can reach the maximum, the peak is too sharp. In order to reduce the error of the experimental results, it is generally considered that the best measurement wavelength is 419nm.

[0133] The results of the test on the selection of buffer type, pH and dosage showed that:

[0134] A1>A2>A3, so the buffer solution should be acetic acid-sodium acetate buffer solution, A1 is the absorbance corresponding to acetic acid-sodium acetate buffer solution, A2 is the absorbance corresponding to citric acid-sodium dihydrogen phosphate buffer solution, and A3 is the absorbance corresponding to hydrogen phthalate buffer solution;

[0135] Acetic acid-sodium acetate was used as buffer to investigate the effect of different pH values ​​on the experimental results. The results showed that A pH＝3.0 >A pH＝2.0 >A pH＝4.0 >ApH＝5.0 >A pH＝6.0 , so the pH of the buffer solution is 3.0. Then the effect of the amount of buffer solution on the experimental results was investigated. The results showed that A 5mL >A 4mL >A 6mL >A 7mL >A 8mL , based on which the amount of buffer solution can be determined to be 5 mL.

[0136] The results of the linear relationship investigation are shown in Table 3 and Figure 4 As shown, the obtained linear regression equation is: Y=0.2088X-0.0086, R2=0.9992, which has a good linear relationship with the absorbance of the test solution in the range of 0.75 mg / mL to 2 mg / mL; therefore, in subsequent experiments, the absorbance obtained from the test of the test solution is substituted into the above linear regression equation to obtain the concentration of the alkaloid in the test solution.

[0137] According to the experimental results, the absorbance RSD value in the precision test is 1.04%, indicating that the instrument has good precision. In the stability test, the RSD between the absorbances measured at different time points of the same sample is 2.29%, indicating that the sample has good stability within 120min. In the repeatability test, it is calculated that the average content of Trichosanthes alkaloids is about 2.45%, and the RSD of absorbance is 1.92%, indicating that the method has good repeatability. The results are shown in Table 4, and the average value of the six sample recovery rates is 99.75%, and the RSD value is 2.77%, indicating that the accuracy of the determination method is good. The absorbance values ​​of the six samples are brought into the linear regression equation to obtain the concentration of each sample, and then the alkaloid content of Trichosanthes medicinal materials is calculated. The results are shown in Table 5, and the average content of alkaloids in Trichosanthes medicinal materials is 2.56%.

[0138] This part of alkaloids is the alkaloids in the Trichosanthes alkaloid extract (ie, extract) obtained in this experiment. After calculation, the alkaloid content in the Trichosanthes alkaloid extract obtained in this experiment is 63.4%.

[0139] Example 4

[0140] Example 1 Acute toxicity test of Trichosanthes alkaloid extract

[0141] 1. Preliminary Experiment

[0142] ICR mice were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd., with production license number: SCXK (Zhejiang) 2019-0004. Twelve mice were randomly divided into six groups, which were set as normal group and five test groups, with 2 mice in each group. According to the method of equal volume and different concentration, the normal group was gavaged with 0.8 ml of normal saline per mouse, and the test group was gavaged with the same volume of different concentrations of Trichosanthes alkaloid extract solution, with the dosage of 60 g / kg, 50 g / kg, 40 g / kg, 30 g / kg, and 20 g / kg, respectively. As a result, all mice died at the two doses of 60 g / kg and 50 g / kg, while there was no death in the 20 g / kg dose group. Therefore, it was preliminarily judged that the "minimum lethal dose" of Trichosanthes alkaloid extract was 50 g / kg, and the "maximum tolerable dose" was 20 g / kg.

[0143] 2. Dosage and grouping

[0144] According to the results of the preliminary experiment and the principle of geometric progression, 5 gradient doses (50kg / g, 40kg / g, 32kg / g, 25.6kg / g, and 20.48kg / g mice) were designed between the "minimum lethal dose" and the "maximum non-lethal dose" for the experiment. 60 healthy ICR mice were adaptively raised for one week and randomly divided into 6 groups by completely random grouping, with 10 mice in each group, namely the normal group and the group administered with the extract of Trichosanthes alkaloids.

[0145] 3. Medication, observation and testing indicators

[0146] The Trichosanthes alkaloid extract groups were gavaged with different doses of Trichosanthes alkaloid extracts, with the doses being 50kg / g, 40kg / g, 32kg / g, 25.6kg / g, and 20.48kg / g, respectively. The mice were fasted for 12 hours before gavage but not water. The poisoning symptoms, appearance, behavior, defecation, etc. of the mice in each group were observed in time after gavage, and continued for 14 days after administration. The appearance, behavior, defecation, body weight, etc. of the mice were observed and recorded every day. Another control group was set up, and the solvent was gavaged in the same way and observed simultaneously.

[0147] The experimental results are shown in Table 3. The death of mice on day 14 is shown in Table 3. According to the modified Koch method, the LD50 is 25.6 g / kg, and the 95% confidence limit of LD50 is 23.12-28.35 g / kg. 1 / 5 LD50, 1 / 10 LD50, and 1 / 20 LD50 are used as experimental doses for subsequent experimental studies of drug efficacy.

[0148] Table 6

[0149]

[0150] Example 5

[0151] The anti-atherosclerotic effect test of the Trichosanthes alkaloid extract obtained in Example 1 was carried out

[0152] Experimental protocol

[0153] 1. Establishment of Atherosclerosis Model

[0154] Fifty ApoE- / - male mice (20-22 g, 6-8 weeks) and 10 C57BL / 6J male mice (20-22 g, 6-8 weeks) were obtained from Changzhou Cavens Experimental Animal Co., Ltd., production license number: SCXK (Su) 2021-0013, and adaptive feeding was performed for 1 week. All animal experimental protocols were approved by the Ethics Committee of Anhui University of Traditional Chinese Medicine (approval number: AHUCM-mouse-2024113). C57BL / 6J mice were fed with normal feed, and ApoE- / - mice were fed with high-fat feed. After 12 weeks, 10 C57BL / 6J mice were taken as the control group, and the hfd-fed ApoE- / - mice were randomly divided into the model group, GLTA-L (0.8 g / kg) group, GLTA-M (1.6 g / kg) group, and GLXB-H (3.2 g / kg) group. Gavage once a day for 4 consecutive weeks.

[0155] 2. Experimental Plan

[0156] After successful modeling, SPF-grade C57BL / 6J mice were used as controls, and AS mice were randomly divided into 4 groups, 6 in each group, namely the model group, the low-dose group of Trichosanthes alkaloid extract, the medium-dose group of Trichosanthes alkaloid extract, and the high-dose group of Trichosanthes alkaloid extract. Trichosanthes alkaloid extract was administered by intragastric gavage once a day for 4 consecutive weeks. After 4 weeks of administration, the mice were fasted for one day, and the aorta, liver, and serum were taken to measure the relevant biochemical indicators in the serum. H&E staining to detect the size of aortic sinus plaques. The frozen sections were taken out in advance and restored to room temperature, dried for about 1 hour; rinsed with PBS 3 times, 5 minutes each time; immersed in 95% ethanol for 2 minutes; rinsed gently with running water for 5 minutes, stained the nucleus with hematoxylin for 5 minutes, and washed away the excess dye with pure water; differentiated with hydrochloric acid alcohol for a few seconds; returned to blue with 60℃ warm water for 5 minutes; restored the sections to room temperature in PBS; rinsed gently with running water for 6 minutes, immersed in 95% ethanol for 4 minutes; stained with eosin for 5 minutes, immersed in 95% ethanol for 2 minutes; dehydrated in anhydrous ethanol for 2 minutes, transparentized with xylene for 10 minutes, and sealed with neutral gum after drying. The left lobe tissue of the liver of each group of mice was taken, washed with ice water, fixed and preserved in 4% paraformaldehyde solution, embedded in paraffin, and stained with HE. The left lobe tissue of the liver of another mouse was taken, embedded with embedding agent, frozen in liquid nitrogen, and stained with frozen oil red O. The body weight of mice was recorded before they were killed; after they were killed, their livers were taken out, the liver weight was weighed, and the liver index was calculated. The levels of TC, TG, HDL-C, and LDL-C in serum were detected using kits.

[0157] The experimental results are detailed in Figure 3-Figure 10 .

[0158] exist Figure 5-Figure 12 In the table, Control represents mice in the normal diet group (fed with normal feed), Model represents mice in the model group, GLTAL-L represents mice in the low-dose group of Trichosanthes alkaloid extract, GLTAL-M represents mice in the medium-dose group of Trichosanthes alkaloid extract, and GLTAL-H represents mice in the high-dose group of Trichosanthes alkaloid extract;

[0159] like Figure 6 As shown in the figure, the weight of mice in the normal diet group gradually increased, while the weight of mice in the high-fat feeding group increased steadily from 1 to 8 weeks after the start of high-fat feeding, and then decreased significantly from 9 to 12 weeks. Compared with the high-fat feeding group, the weight of mice after intervention with Trichosanthes alkaloid extract also increased as the experimental time prolonged, and this increase may be due to individual differences.

[0160] like Figure 7 and Figure 8 As shown, after 12 weeks of modeling, a large number of plaques appeared on the aortic arch of mice in the model group. After administration of Trichosanthes alkaloid extract, the plaques on the aortic arch of AS mice were significantly reduced.

[0161] like Fig. 9 As shown, after 12 weeks of modeling, HE staining of the aortic sinus showed that the aortic sinus intima of mice in the normal diet group was smooth and complete, the vascular wall thickness was uniform, and no AS plaques were observed; compared with the normal diet group, the aortic sinus intima of mice in the model group was thickened, and the area of ​​AS plaques increased significantly. After administration of Trichosanthes alkaloid extract, the area of ​​aortic sinus lesions in AS mice gradually decreased (##P<0.01, **P<0.01). Figure 7 , Figure 8 and Fig. 9 The results show that the extract of Trichosanthes alkaloids has the effect of treating atherosclerosis.

[0162] like Fig.10 As shown in the figure, compared with the normal diet group, the TC, TG, and LDL-C levels of the mice in the model group were significantly increased, and the HDL-C level was significantly decreased (##P<0.01); compared with the model group, the serum TC, TG, and LDL-C levels of the mice in the low, medium, and high dose groups of Trichosanthes alkaloid extract were significantly decreased, and the HDL-C level was significantly increased (**P<0.01). This shows that the Trichosanthes alkaloid extract can reduce the cholesterol level of AS mice and improve the blood lipid level of AS mice.

[0163] like Fig.11As shown in the figure, after 12 weeks of modeling, the liver of the model group was yellow in color, with a rough and greasy surface. After the administration of the extract of Trichosanthes alkaloids, the liver was significantly improved and the color gradually returned to ruddy. Figure 8 It can be seen that compared with the normal diet group, the liver index of the model group was greatly different, but it was significantly improved after administration of Trichosanthes alkaloid extract, reducing the liver swelling of AS mice.

[0164] like Fig.12 As shown, there were no orange-red lipid droplets in the liver cells of mice in the normal diet group; compared with the normal diet group, a large number of red lipid droplets appeared in the liver cells of mice in the model group; compared with the model group, the lipid droplets in the liver cells of AS mice in the low, medium and high dose groups of Trichosanthes alkaloid extract gradually decreased.

[0165] like Fig.13 As shown, the liver cells of mice in the normal diet group were normal in size, and the cells were neatly arranged and clearly distributed; compared with those in the normal diet group, the number of lipid vacuoles in the liver of mice in the model group was significantly increased and the vacuolar area was larger; compared with the model group, the number and area of ​​lipid vacuoles in AS mice in the low and medium dose groups of Trichosanthes alkaloid extract gradually decreased, and the lipid vacuoles in AS mice in the high dose group of Trichosanthes alkaloid extract almost disappeared, and the liver cells were neatly arranged.

[0166] from Figure 10-13 The data in the literature show that Trichosanthes alkaloid extracts have an outstanding effect in improving liver lipid deposition.

[0167] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Trichosanthes alkaloid extract, characterized in that The method for obtaining the Trichosanthes alkaloid extract comprises: The powder of Trichosanthes peel is ultrasonically extracted for multiple times with a sulfuric acid solution having a mass fraction of 0.5-1.5%, the extracts obtained from the multiple extractions are combined, the extracts obtained are concentrated once, the pH of the extracts after the concentration is subsequently adjusted to the target pH, and then extracted for multiple times with n-butanol, the extracts obtained from the multiple extractions are combined, and concentrated twice to obtain a paste, thereby obtaining the Trichosanthes alkaloid extract.

2. The method for extracting the Trichosanthes alkaloid extract according to claim 1, characterized in that: include: The powder of Trichosanthes peel is ultrasonically extracted for multiple times with a sulfuric acid solution having a mass fraction of 0.5-1.5%, the extracts obtained from the multiple extractions are combined, the extracts obtained are concentrated once, the pH of the extracts after the concentration is subsequently adjusted to the target pH, and then extracted for multiple times with n-butanol, the extracts obtained from the multiple extractions are combined, and concentrated twice to obtain a paste, thereby obtaining the Trichosanthes alkaloid extract.

3. The method for extracting the Trichosanthes alkaloid extract according to claim 2, characterized in that: The mass of the sulfuric acid solution is 4-7 times the mass of the Trichosanthes peel powder.

4. The method for extracting the Trichosanthes alkaloid extract according to claim 2, characterized in that: The ultrasonic extraction is performed 3-6 times, and the time for each ultrasonic extraction is 0.5-2 hours.

5. The method for extracting the Trichosanthes alkaloid extract according to claim 2, characterized in that: When the extracts obtained by the first concentration are combined, the volume of the extracts after the first concentration is 0.08-0.15 of the volume of the extracts obtained by the first concentration.

6. The method for extracting the Trichosanthes alkaloid extract according to claim 2, characterized in that: The target pH is 9-10, and the number of extractions is 3-6 times.

7. Use of the Trichosanthes alkaloid extract according to claim 1 in the preparation of a medicament for preventing or treating atherosclerosis.

8. Use of the Trichosanthes alkaloid extract according to claim 1 in the preparation of a medicament for improving hepatic lipid deposition.

9. Use of the Trichosanthes alkaloid extract according to claim 1 in the preparation of a drug for lowering blood lipids or cholesterol.

10. The method for measuring the alkaloid content in the Trichosanthes alkaloid extract according to claim 1, characterized in that: include: The extract of Trichosanthes kirilowii alkaloids was dissolved and diluted in dichloromethane to obtain a test solution; Prepare trigonelline reference solution; Add buffer solution and acid dye solution to the test solution for mixed dyeing. After dyeing, add dichloromethane, mix evenly, let stand to separate layers, take the dichloromethane layer, and dilute to the volume of the test solution to be tested; Adding a buffer solution and an acid dye solution to the reference solution for mixed dyeing, adding dichloromethane after dyeing, mixing evenly, standing to separate layers, taking the dichloromethane layer, and diluting to different concentrations of the reference solution to be tested; Conduct absorbance tests on the reference substance solutions with different concentrations; Perform absorbance test on the test solution; The alkaloid content in the Trichosanthes alkaloid extract is obtained according to the absorbance values ​​of the reference solution of different concentrations and the absorbance values ​​of the test solution of different concentrations obtained from the absorbance test.

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