Subcritical preparation method of Moringa leaf extract for improving intestinal butyrate abundance

Abstract

This invention discloses a subcritical extraction method for Moringa leaf extract, a method for increasing intestinal butyric acid abundance, and its application. The Moringa leaf extract prepared by subcritical extraction has the advantages of being non-toxic, pollution-free, and easy to separate from the product. It was then applied to the intervention of type 2 diabetic rats. When the gavage dose was ≥150 mg / kg∙d, the abundance of intestinal butyric acid-producing bacteria and butyric acid was significantly increased, which is of great significance for the treatment of diabetes, obesity, and enteritis metabolic diseases.

Description

Technical Field

[0001] The present invention relates to the field of medical and health technology, and more specifically, to a subcritical preparation method of a Moringa leaf extract for improving the abundance of intestinal butyric acid. Background Art

[0002] Intestinal flora plays an important role in the occurrence and development of many human diseases and has attracted widespread attention. It is an important component for maintaining the homeostasis of the human ecosystem. Short-chain fatty acids (SCFAs) are produced by anaerobic bacteria in the colon to ferment undigested carbohydrates in the intestine, mainly including acetic acid, propionic acid, butyric acid, etc. SCFAs play an important role in maintaining intestinal function. As a special nutrient and energy component of the intestinal epithelium, they have the functions of protecting the intestinal mucosal barrier, reducing the body's inflammation level and enhancing gastrointestinal motility. Among them, butyrate is a key regulator for maintaining the integrity and health of the colon and is related to strengthening the intestinal barrier. As the main and preferred energy substrate for colonocytes, the cellular metabolism of butyrate takes precedence over glucose or glutamine, accounting for 70% of the total energy demand of the colon mucosa. Butyrate can stimulate the production of epidermal growth factor and anti-inflammatory cytokines, reduce the production of inflammatory cytokines, and has the ability to inhibit NF-κB activation. In addition, butyrate can enhance the immunogenicity of tumor cells, regulate the activity of apoptosis-related proteins, increase the body's antioxidant activity, stimulate the secretion of antimicrobial peptides, and inhibit histone deacetylation and COX-2 activation. Imbalanced intestinal flora leads to immune responses caused by reduced SCFAs such as butyrate, which plays a major role in the development of metabolic diseases such as diabetes mellitus (DM). Therefore, the impact of intestinal butyrate abundance on human health has received increasing attention and has the potential to become an important potential target for the prevention and treatment of metabolic diseases such as DM.

[0003] Moringa oleifera Lam. is a perennial deciduous tree of the Moringaceae family and Moringa genus. It has been introduced and cultivated in Yunnan, Guangxi, Guangdong, Fujian, Hainan and Taiwan in China. Moringa leaves are rich in vitamins, proteins, amino acids, folic acid, and nutrients such as calcium, potassium, iron and selenium that are beneficial to the human body. Therefore, the former Ministry of Health of my country approved it as a new resource food in 2012. Modern research shows that Moringa leaves have good effects in lowering blood sugar, lowering blood lipids and reducing weight. They are also widely used in food, health care products and other industries. However, there are few reports on its ability to relieve DM by regulating intestinal flora. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0005] Another object of the present invention is to provide a subcritical preparation method of Moringa leaf extract for improving intestinal butyric acid abundance, so as to solve the complex technical problems of the prior art in the preparation method of Moringa leaf extract, and to innovatively apply the obtained Moringa leaf extract to improve intestinal butyric acid abundance and targeted screening of active ingredients with potential hypoglycemic function, so as to contribute to the prevention and treatment of DM and obesity.

[0006] In order to achieve these purposes and other advantages according to the present invention, the present invention provides a subcritical preparation method of Moringa leaf extract for improving intestinal butyric acid abundance, comprising the following preparation steps:

[0007] A1, take the dried leaves of Moringa oleifera, crush them, add solvent and carry out subcritical extraction;

[0008] A2, concentrating the extract filtered in step A1 under reduced pressure in a rotary evaporator;

[0009] A3, pre-freezing the extract obtained by decompression concentration in step A2 at ultra-low temperature, and then freeze-drying to obtain Moringa leaf extract.

[0010] Preferably, the solvent used in the subcritical extraction is 80%-90% aqueous ethanol, the extraction time is 1 hour, and the extraction is performed twice in total.

[0011] Preferably, the negative pressure of the reduced pressure concentration is 0.1 MPa, and the pre-freezing temperature of the freeze drying is -80 o C.

[0012] The present invention also provides a method for improving intestinal butyric acid abundance by using a Moringa leaf extract, comprising the following steps:

[0013] S1. Establishment of type 2 diabetic rat model;

[0014] S2, using the Moringa leaf extract to intervene in type 2 diabetes model rats;

[0015] S3, detection of short-chain fatty acids in feces of rats with type 2 diabetes model;

[0016] Preferably, after adaptive feeding, the rats are fed with a high-fat diet; then chemical drugs are used to destroy the pancreatic islet function to establish a type 2 diabetes rat model; and then the rats are given an intragastric administration intervention, and the intervention period is 30 days.

[0017] The present invention also provides an application of the Moringa leaf extract, using the Moringa leaf extract to analyze the in vitro activity inhibition of α-glucosidase, and to perform targeted, high-throughput screening of potential glucose-lowering active ligands in the Moringa leaf extract.

[0018] Preferably, the method specifically comprises the following steps:

[0019] S41, testing the inhibition degree of α-glucosidase activity by different concentrations of Moringa leaf extract, and selecting nitro-α-D-pyranoglucoside as the reaction substrate;

[0020] S42, using α-glucosidase as the target, affinity ultrafiltration mass spectrometry technology was used to screen potential hypoglycemic active ligands in Moringa leaf extract, with an ultrafiltration tube cutoff of 30Kd;

[0021] S43. The degree of binding between enzyme and ligand is used to evaluate the activity of different ligands.

[0022] The present invention also provides another application of the Moringa leaf extract, which is used to prepare a health adjuvant for increasing the content of the beneficial short-chain fatty acid butyrate in the intestinal tract of animals.

[0023] Preferably, the health adjuvant is a functional food or a medicine.

[0024] Preferably, the health adjuvant is in the form of tablets, capsules, oral liquid or syrup.

[0025] The present invention has at least the following beneficial effects:

[0026] (1) The subcritical extraction method used in the present invention to prepare the Moringa leaf extract is based on the principle that like dissolves like in organic matter. Through molecular diffusion between the extraction material and the extractant during the soaking process, the material is subjected to countercurrent extraction with a liquefied subcritical solvent. This is a new extraction and separation technology for ultimately obtaining the target product. It has the advantages of being non-toxic, pollution-free, and easy to separate from the product.

[0027] (2) Butyric acid plays an important role in inhibiting harmful bacteria, maintaining the balance of intestinal microecology, protecting the intestinal mucosal barrier and reducing inflammatory response. The Moringa leaf extract prepared by the present invention can significantly increase the abundance of butyric acid in the intestine of animals, and therefore has important significance for the treatment of diabetes, obesity, enteritis and metabolic diseases.

[0028] (3) The targeted ultrafiltration mass spectrometry screening technology adopted in the present invention can quickly and effectively identify potential α-glucosidase ligands in Moringa leaf extract. It has the characteristics of high throughput, high sensitivity and strong specificity, and is also suitable for the targeted screening of functional active ingredients in other complex natural products.

[0029] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1The present invention is a flowchart of the overall steps of the Moringa leaf extract from subcritical preparation to increasing the abundance of butyric acid in the intestinal tract of animals and applying it to the targeted screening of potential hypoglycemic functional active ingredients.

[0031] Figure 2 This is a comparison chart of the effects of the Moringa leaf extract on the abundance of total short-chain fatty acids (SCFAs) in the intestine of type 2 DM rats in Example 3 of the present invention.

[0032] Figure 3 This is a comparison chart of the effects of the Moringa leaf extract on the abundance of butyric acid in the intestine of type 2 DM rats in Example 3 of the present invention.

[0033] Figure 4 The graph is a variation graph of the inhibitory activity of the Moringa leaf extract on α-glucosidase in Example 4 of the present invention.

[0034] Figure 5 This is a comparative chromatogram of the targeted screening of α-glucosidase ligands in Moringa leaf extract using affinity ultrafiltration mass spectrometry technology in Example 4 of the present invention.

[0035] Figure 6 Schematic diagram of the structures of five representative α-glucosidase ligands screened in Example 4 of the present invention. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0037] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified, and should not be construed as limitations on the present invention.

[0038] The present invention provides a subcritical preparation method of Moringa leaf extract and a method and application thereof for improving intestinal butyric acid abundance. The complete process of Moringa leaf extract from subcritical preparation to improving intestinal butyric acid abundance of animals and applying to targeted screening of potential hypoglycemic functional active ingredients is combined. Figure 1 As shown. That is, the following steps are included in sequence according to the step flow:

[0039] A1, take the dried leaves of Moringa oleifera, crush them, add solvent and carry out subcritical extraction;

[0040] A2, concentrating the extract filtered in step A1 under reduced pressure in a rotary evaporator;

[0041] A3, pre-freezing the extract obtained by decompression concentration in step A2 at ultra-low temperature, and then freeze-drying to obtain Moringa leaf extract;

[0042] S1. Establishment of type 2 diabetic rat model;

[0043] S2, using the Moringa leaf extract to intervene in type 2 diabetes model rats;

[0044] S3. Detection of short-chain fatty acids in feces of rats with type 2 diabetes model by GC-MS coupling technology;

[0045] S4. Analyze the inhibition of α-glucosidase activity in vitro using the Moringa leaf extract, and perform targeted, high-throughput screening of potential glucose-lowering active ligands in the Moringa leaf extract.

[0046] Embodiment 1:

[0047] The subcritical preparation method of Moringa leaf extract comprises the following steps:

[0048] A1. Take Moringa oleifera ( Moringa oleifera Lam.) leaves, dried and crushed, soaked in 5-10 times the amount of 80%-90% aqueous ethanol, and then subjected to subcritical extraction twice, each time for 1 hour;

[0049] The specific experimental process is as follows:

[0050] Weigh and measure the dried Moringa leaf material and the extractant according to the above material ratio, and put them into the extraction tank;

[0051] ②Start the electric heater and set the oil temperature to 90 o C-100 o C;

[0052] ③ Start the vacuum pump and draw the extraction tank to a vacuum state;

[0053] ④ Close all valves of the extraction tank, start the thermal oil pump, and circulate and heat the extraction tank; extract twice in total;

[0054] Open the cooling water inlet valve of the condenser and let in cooling water; the extract passes through the filter and condenser under negative pressure and enters the extract temporary storage tank;

[0055] A2, the extracts were combined, filtered under reduced pressure, and the filtrate was concentrated under reduced pressure on a rotary evaporator at a negative pressure of 0.1 MP;

[0056] A3. Heat the above concentrated extract at -80 o C for 6-8 h, and then freeze-dried for 24-48 h to obtain Moringa leaf extract dry powder.

[0057] The subcritical extraction method adopted by the present invention for preparing the moringa leaf extract is based on the principle that like dissolves like in organic matter, and through molecular diffusion between the extraction material and the extraction agent during the soaking process, the material is countercurrently extracted with a liquefied subcritical solvent. This is a new extraction and separation technology for finally obtaining the target product, and has the advantages of being non-toxic, pollution-free, and easy to separate from the product, etc.

[0058] Embodiment 2:

[0059] Modeling of type 2 DM rat model. The rats are SD rats or Wistar rats, preferably male; the chemical drug used in the modeling of type 2 DM model in this embodiment is preferably streptozotocin (STZ). Modeling of type 2 DM rat model includes the following steps:

[0060] (1) Rats and breeding conditions:

[0061] Forty 6-week-old SPF male SD rats weighing 180-200 g were purchased from Beijing Weitonglihua Animal Experiment Technology Co., Ltd.

[0062] The rats were kept in an SPF animal room under the following conditions: room temperature 22 ± 2 o C, humidity 50 ± 10%; light / dark cycle 12h / 12h; adaptive feeding for 3 days, during which time the animals were free to eat and drink.

[0063] (2) Modeling of type 2 DM rats

[0064] After 3 days of adaptive feeding, 7 rats were randomly selected as the normal group (NC). The rats in the normal control group were fed with conventional feed, and the rats in the other groups were fed with high-fat feed for 4 weeks.

[0065] After the high-fat diet-induced modeling, the rats were fasted but not watered for 12 h, and intraperitoneally injected with 1% STZ solution (35 mg / kg, dissolved in 0.1 mol / L sodium citrate buffer solution, injection completed within 30 min). The rats in the normal control group were injected with the same dose of sodium citrate buffer solution.

[0066] 72 hours after STZ injection, blood was collected from the tail tip of the rats to test fasting blood glucose (FBG). FBG>11.1 mmol / L and random blood glucose value>16.7 mmol / L were determined to be a successful type 2 DM rat model.

[0067] Embodiment 3:

[0068] Experiment on the effect of Moringa leaf extract on improving the abundance of intestinal butyric acid in type 2 DM rats. The butyric acid described in the present invention is a common intestinal short-chain fatty acid, which has important functions such as inhibiting harmful bacteria, maintaining intestinal microecological balance, protecting intestinal mucosal barriers and reducing inflammatory responses. The positive control drug used in the animal experiment of this embodiment is preferably metformin hydrochloride (Met). The experiment includes the following steps:

[0069] Grouping of S1 and 2 DM rats

[0070] The 28 type 2 DM rats successfully modeled in Example 2 were randomly divided into 4 groups (n=7) according to body weight and FBG value, namely, model group (DM), positive group (Met, 150 mg / kg), low-dose group (MO-L, 150 mg / kg) and high-dose group (MO-H, 300 mg / kg).

[0071] Drug intervention in S2 and type 2 DM rats

[0072] Before use, the Moringa leaf extract prepared in Example 1 was dissolved in physiological saline to prepare a solution (suspension) with a concentration of 20-30 mg / mL. Rats in the positive group and the low / high dose groups of Moringa leaf extract were gavaged at the above doses for 30 days; rats in the normal group and the model group were gavaged with physiological saline according to their body weight. 2-3 rats in each group were allowed to drink water freely until the end of the 30th day of administration.

[0073] S3. Detection of short-chain fatty acids in feces of rats with type 2 diabetes mellitus

[0074] One day before the end of the administration, the feces of the rats in each group were collected and stored in sterile EP tubes. o C refrigerator, and sent for testing as soon as possible. GC-MS analysis of intestinal short-chain fatty acids in rat feces, set the parameters as follows:

[0075] Metabolite extraction:

[0076] Take an appropriate amount of fecal sample in a 2 mL centrifuge tube, add 50 μL of 15% phosphoric acid, then add 100 μL of 125 μg / mL internal standard (isocaproic acid) solution and 400 μL of ether to homogenize for 1 min. o C Centrifuge at 12000 rpm for 10 min and take the supernatant for testing.

[0077] Chromatographic conditions:

[0078] Chromatographic column: Agilent HP-INNOWAX capillary column (30 m × 0.25 mm × 0.25 μm); split injection, injection volume 1 μL, split ratio 10:1. Injection port temperature 250 o C; ion source temperature 300o C; transmission line temperature 250 o C. Program temperature rise starting temperature 90 o C; then 10 o C / min heating up to 120 o C; then 5 o C / min heating up to 150 o C; finally 25 o C / min heating up to 250 o C was maintained for 2 min. The carrier gas was helium with a carrier gas flow rate of 1.0 mL / min.

[0079] Mass spectrometry conditions:

[0080] Electron impact ionization (EI) source, SIM scanning mode, electron energy 70 eV.

[0081] The experimental data were processed and analyzed using SPSS 24.0 statistical software, and the measurement data were expressed as mean ± standard deviation (M ± SD). One-way ANOVA was used for comparison among multiple groups and the data with homogeneity of variance, and the LSD method was used for multiple comparisons between two groups. p <0.05 was considered statistically significant.

[0082] Analysis results of short-chain fatty acids in rat intestine:

[0083] Combination Figure 2-3 As shown, Figure 2 This is a comparison chart of the effects of Moringa leaf extract on the abundance of total short-chain fatty acids in the intestine of type 2 DM rats. Figure 3 This is a comparison chart of the effect of Moringa leaf extract on the abundance of butyric acid in the intestine of type 2 DM rats. The experimental data show that both the low-dose group (GS-L, 150 mg / kg) and the high-dose group (GS-H, 300 mg / kg) of Moringa leaf extract can significantly increase the abundance of total short-chain fatty acids in the intestine of type 2 DM rats; both the low-dose group (GS-L, 150 mg / kg) and the high-dose group (GS-H, 300 mg / kg) of Moringa leaf extract can increase the abundance of butyric acid in the intestine of type 2 DM rats. This shows that the Moringa leaf extract of the present invention can significantly increase the abundance of Clostridium butyricum in the intestine, and when the dosage is ≥150 mg / kg∙d, the abundance of the beneficial short-chain fatty acid butyric acid in the intestine is significantly increased.

[0084] Furthermore, compared with the model group (DM), *p<0.05 showed significant differences, and **p<0.01 showed extremely significant differences. The above experimental data showed that compared with the model group (DM) rats, Moringa leaf extract could increase the abundance of butyric acid in the intestine of type 2 DM rats, and the effect showed a good dose-effect relationship, and a significant difference could be achieved at a concentration of 150 mg / kg.

[0085] Embodiment 4:

[0086] This embodiment provides an application of a Moringa leaf extract, using the Moringa leaf extract to analyze the in vitro activity inhibition of α-glucosidase, and to perform targeted, high-throughput screening of potential hypoglycemic active ligands in the Moringa leaf extract. Specifically, the steps include:

[0087] S41, Analysis of the inhibitory activity of Moringa leaf extract on α-glucosidase in vitro

[0088] The Moringa leaf extract and α-glucosidase (1.0 U / mL) were dissolved in an appropriate amount of PBS buffer solution (pH 6.8), and then the two were incubated at 37 o C, then add p-nitro-α-D-pyranoglucoside (0.5 mmol / L) solution as substrate, add Na2CO3 to terminate the reaction after a period of reaction, and calculate the absorbance at 405 nm to obtain the half inhibitory concentration IC of the sample for α-glucosidase 50 .

[0089] Enzyme inhibition rate (%) = 1-(A0-A s ) / A0× 100%

[0090] In the formula, A0, A s are the absorbance values ​​of blank control and sample to be tested, respectively.

[0091] The positive control drug used in this example is preferably acarbose.

[0092] According to the calculation results of the above formula, the in vitro inhibitory activity IC of Moringa leaf extract on α-glucosidase 50 The value was 123.34 ± 3.89 µg / mL, which was comparable to the positive control acarbose (81.74 ± 2.26 µg / mL). Figure 4 shown.

[0093] This example demonstrates that the Moringa leaf extract has a high inhibitory activity against α-glucosidase, and therefore can be used for the screening of α-glucosidase inhibitors from natural sources, with great application potential.

[0094] S42, targeted, high-throughput screening of potential α-glucosidase ligands in Moringa leaves. Using α-glucosidase as the target, affinity ultrafiltration mass spectrometry technology was used to high-throughput screen potential hypoglycemic active ligands in Moringa leaf extracts. The ultrafiltration tube had a pore size of 30Kd.

[0095] The Moringa leaf extract sample was dissolved in PBS to prepare a Moringa leaf extract solution with a concentration of 10-20 mg / mL, and a certain amount of α-glucosidase (1.0 U / mL) solution was added. After mixing in an EP tube, the solution was incubated at a constant temperature for 20-30 min. The incubated solution was added to an ultrafiltration centrifuge tube, and the unbound components were washed away with PBS after centrifugation. After repeating 2-3 times, 50%-60% acetonitrile was added to the eluent, and the reaction was centrifuged after 10 min. The eluent was collected and the spectral information at 254 nm - 280 nm was collected in a liquid chromatography-mass spectrometer. The type of the compound was identified by comparing the retention time and main MS / MS fragments with those in the literature, database and corresponding standards.

[0096] S43. Evaluate the activity of different ligands based on the degree of binding between enzyme and ligand

[0097] By comparing the spectral data of Moringa leaf extract samples and activated and inactivated α-glucosidase ultrafiltration screening, the binding factors (EF) of different compounds and enzymes were obtained. The calculation formula of EF is:

[0098] EF = (A t - A c ) / A0

[0099] Among them A t , A c are the areas of each chromatographic peak after incubation with activated and inactivated α-glucosidase, respectively, and A0 is the area of ​​each chromatographic peak in the Moringa leaf extract.

[0100] The negative control chosen was inactivated α-glucosidase by boiling in boiling water for 10-20 min.

[0101] The chromatogram of the targeted screening of α-glucosidase ligands in Moringa leaf extract by ultrafiltration mass spectrometry in this embodiment is shown in the attached figure. Figure 5 As shown, the data corresponding to the active α-glucosidase are Figure 5 The data corresponding to the inactivated α-glucosidase are located on the upper side. Figure 5 The middle is located on the upper side.

[0102] Chromatography-mass spectrometry analysis of potential active ingredients in Moringa leaf extract:

[0103] High performance liquid chromatography (HPLC) analysis conditions are as follows:

[0104] High performance liquid chromatography: Thermo Accela 600; chromatographic column: Waters Symmetry RP-C18; specifications: 4.6 × 150 mm, 5 μm; mobile phase: A-0.1% formic acid-water, B-acetonitrile; flow rate: 0.6-0.8 mL / min; injection volume: 5-10 μL; detection wavelength: 254 nm - 280 nm; gradient elution program: 0-30 min, 8% -30% B; 30-40 min, 30%-95% B; 40-45 min, 95%-8% B.

[0105] The analysis conditions of triple quadrupole tandem mass spectrometry (TSQ-MS) were as follows:

[0106] Mass spectrometer: TSQ Quantum Access MAX; ion source: electrospray (ESI) ionization source; acquisition mode: negative ion mode; molecular weight scanning range: 100-1500 Da; secondary spectrum scanning mode: data-dependent scanning; spray voltage: 3000 V; cone voltage: 40 V; capillary temperature: 350 o C; auxiliary nitrogen pressure: 40 psi; collision energy 30-45 eV;

[0107] The retention time, parent ion and fragment ion information of each peak in the spectrum are compared with existing references or standards to perform structural analysis and identification of the chemical components of the chromatographic peaks.

[0108] A total of 15 major chromatographic peaks were found in the Moringa leaf extract. By comparing the retention times and major MS / MS fragments of these peaks with those of literature, databases and corresponding standards, a total of 14 compounds were identified.

[0109] After targeted ultrafiltration screening with α-glucosidase, 9 peaks with high BD values ​​were screened out from the Moringa leaf extract, namely Peak 13, Peak 15, Peak 14, Peak 10, Peak 9, Peak 12, Peak 11, Peak 2 and Peak 6. Among them, Peak 13 had the highest binding degree (18.39%), followed by Peak 15 (17.92%), Peak 14 (17.19%), Peak 10 (16.51%), Peak 9 (16.06%), and Peak 12 (15.80%). It is preliminarily speculated that these 6 compounds are potential α-glucosidase ligands in Moringa leaves. The ultrafiltration mass spectrometry technology provided by the present invention is applied to the targeted screening of potential hypoglycemic active ingredients in Moringa leaf extract, which is helpful for the research and development of targeted drugs for the prevention and treatment of metabolic diseases such as DM and obesity. The mass spectrometry information of some representative effective ingredients of Moringa leaf extract is as shown in the attached figure. Figure 6 As shown, Figure 6 The numbers below the compounds correspond to the peak numbers with higher BD values ​​in the Moringa leaf extract.

[0110] Embodiment 5:

[0111] The present embodiment provides an application of a Moringa leaf extract, wherein the Moringa leaf extract is used to prepare a health adjuvant that increases the beneficial short-chain fatty acid butyrate in the intestine of an animal. For example, when preparing a solid tablet, the dry powdered Moringa leaf extract is evenly mixed with other ingredients and pressed into shape; further, the health adjuvant is a functional food or medicine, and the animal is a mammal; further, the dosage form of the health adjuvant is a tablet, capsule, oral liquid or syrup.

[0112] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A subcritical preparation method of a Moringa leaf extract for improving intestinal butyric acid abundance, characterized in that: The method comprises the following preparation steps: A1, take the dried leaves of Moringa oleifera, crush them, add solvent and carry out subcritical extraction; The solvent used in the subcritical extraction is 80%-90% aqueous ethanol, the extraction electric heating oil temperature is 90°C-100°C, the extraction time is 1h, and the extraction is performed twice in total; A2, the extract filtered in step A1 is concentrated under reduced pressure in a rotary evaporator, wherein the negative pressure of the reduced pressure concentration is 0.1 MP; A3. Pre-freeze the extract obtained by decompressing and concentrating in step A2 at -80°C for 6-8 hours, and then freeze-dry for 24-48 hours to obtain Moringa leaf extract.

2. The subcritical preparation method of the Moringa leaf extract for improving intestinal butyric acid abundance as claimed in claim 1, characterized in that, The Moringa leaf extract obtained in step A3 was dissolved in physiological saline to prepare a solution with a concentration of 20-30 mg / mL, and the dosage was ≥150 mg / kg∙d.

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