Moringa oleifera leaf extract, process for its preparation and use thereof

Abstract

This invention discloses a Moringa leaf extract, its preparation method, and its applications. The preparation method includes the following steps: S1, Moringa leaves are sequentially subjected to extraction treatment, followed by a first solid-liquid separation to obtain a first filtrate and a first residue; wherein, the extraction treatment includes an extraction step using an ethanol solution; the volume concentration of ethanol in the ethanol solution is higher than 50%; S2, the first residue is sequentially subjected to enzymatic hydrolysis treatment, followed by a second solid-liquid separation to obtain a second filtrate and a second residue; wherein, the enzymatic hydrolysis treatment includes an enzymatic hydrolysis step using a protease; S3, the first filtrate and the second filtrate are mixed to obtain the Moringa leaf extract. The Moringa leaf extract prepared by this method is rich in polyphenols and exhibits excellent antidepressant effects in practical applications.

Description

Technical Field

[0001] This invention relates to a Moringa leaf extract, its preparation method, and its application. Background Technology

[0002] Depression is an affective disorder characterized by chronic, recurrent, and life-threatening symptoms, and has become a serious global health problem. Statistics show that depression is the third leading cause of disease burden worldwide, and it is projected to become the leading cause of disease burden globally by 2030. The pathogenesis of depression is quite complex; genetic factors and childhood experiences are closely related to the risk of depression, and environmental factors such as trauma and stress in life can trigger it. The pathological mechanisms of depression are also very complex. Although significant progress has been made, a single mechanism is still insufficient to explain the cause of the disease; different mechanisms may be the basis for its heterogeneity.

[0003] Currently used antidepressants in clinical practice generally exert their effects by affecting the reuptake of monoamine neurotransmitters, inhibiting monoamine metabolism, and blocking presynaptic inhibitory autonomous or non-autonomous receptors. Most of these drugs are chemically synthesized, and while they are effective, they also have drawbacks such as a narrow antidepressant spectrum, significant side effects, and a high relapse rate. Furthermore, a single drug may only have a limited effect on a portion of patients, providing no relief in 60%–70% of patients and no significant response in 30%–40% of patients. They also have significant side effects and are prone to relapse upon discontinuation.

[0004] Therefore, the current trend in the development of antidepressant drugs is to find natural, highly effective, low-toxicity, and safe antidepressant active ingredients from a wide variety of plants.

[0005] Moringa oleifera is a perennial tropical deciduous tree belonging to the genus Moringa in the family Moringaceae. Also known as "drumstick tree," "horse radish," "foreign toon tree," "miracle tree," "horse root," and "wasabi tree," it is renowned for its nutritional and medicinal properties, and is considered one of the "Three Treasures of the World" alongside Ganoderma lucidum (reishi mushroom) from my country and American ginseng. In 2012, it was listed as a new resource food ingredient in my country. Moringa leaves mainly contain polyphenols, flavonoids, phenylpropanoids, terpenes, steroids, alkaloids, isothiocyanates, and various organic acids. These components exhibit good pharmacological activities such as lowering blood sugar, lowering uric acid, anti-tumor, regulating blood lipids, anti-oxidation, and liver protection.

[0006] Polyphenols are a collective term for a class of chemical substances derived from plants, named for the multiple phenolic hydroxyl groups they contain. Based on the number of aromatic rings in their structure, they can be classified into flavonoids, phenolic acids, phenolic alcohols, arbutin compounds, and lignins. Current research primarily focuses on the therapeutic effects of polyphenols on anxiety and sleep improvement. Pharmacological studies on Moringa leaves mainly focus on their effects on lowering blood sugar and uric acid, anti-tumor activity, lipid regulation, antioxidant properties, and liver protection. However, there is currently no research on the antidepressant effects of Moringa leaf extract.

[0007] To further study the pharmacology of the effective active ingredients (especially polyphenolic compounds) in Moringa leaves, effective extraction is still required.

[0008] Existing literature (Zhao Mouming, Li Qiaolin, Lin Lianzhu, et al. Preparation and antioxidant activity of Moringa leaf extract [J]. Food Science, 2018, 39(21):6.) used 16 extraction methods to prepare Moringa leaf extract, and investigated the effects of different extraction methods on the extraction rate of Moringa leaf protein, sugar, polyphenols and the dissolution rate of antioxidants. Furthermore, the interaction between protein (hydrolyzed protein), sugar and polyphenols in the extract on the antioxidant activity of Moringa leaf extract was studied. Among the 16 extraction methods, the enzymatic method, the enzymatic-coupled ethanol extraction method and the ultrasound-assisted ethanol extraction method had the best effect, but the extraction effect of polyphenols was still limited.

[0009] Therefore, there is an urgent need for a method to prepare Moringa leaf extract rich in polyphenols. Summary of the Invention

[0010] To address the aforementioned deficiencies in existing technologies, this invention proposes a Moringa leaf extract, its preparation method, and its applications. The Moringa leaf extract prepared by this method is rich in polyphenols, and this invention is the first to discover that the Moringa leaf extract possesses excellent antidepressant effects in practical applications.

[0011] To achieve the above objectives, the present invention adopts the following technical solution.

[0012] This invention provides a method for preparing Moringa leaf extract, which includes the following steps:

[0013] S1. Moringa leaves are extracted sequentially, followed by a first solid-liquid separation to obtain a first filtrate and a first residue.

[0014] The extraction process includes an extraction step using an ethanol solution; the volume concentration of ethanol in the ethanol solution is higher than 50%.

[0015] S2. The first residue is sequentially subjected to enzymatic hydrolysis and solid-liquid separation to obtain the second filtrate and the second residue.

[0016] The enzymatic hydrolysis process includes a step of enzymatic hydrolysis with protease;

[0017] S3. The first filtrate and the second filtrate are mixed to obtain the Moringa leaf extract.

[0018] In this invention, the first solid-liquid separation, the first filtrate, the first residue, the second solid-liquid separation, the second filtrate, and the second residue are only used to distinguish different steps and materials.

[0019] In this invention, the volume concentration of ethanol in the ethanol solution can be 100%, and the ethanol solution in this case can be understood as pure ethanol, the specific meaning of which can be understood by those skilled in the art.

[0020] In some implementations, in step S1, the volume concentration of ethanol in the ethanol solution is 60%-80%, for example, 70%.

[0021] In some implementations, in step S1, the weight of the ethanol solution is 10-30 times, for example, 20 times, the weight of the moringa leaves.

[0022] In some implementations, in step S1, the extraction temperature is 60-80°C, preferably 60-70°C.

[0023] In some implementations, the extraction time in step S1 is 1-3 hours, for example, 2 hours.

[0024] In some implementations, the extraction is performed twice in step S1; preferably, the conditions for the two extractions are consistent.

[0025] In some implementations, step S1 includes the steps of filtration, concentration, and re-filtration.

[0026] The filtration is preferably performed using a filter screen; the filter screen is, for example, a 120-mesh filter screen.

[0027] The concentration is preferably carried out under reduced pressure; the temperature of the reduced pressure concentration is preferably 50-70°C, for example 60°C; and the pressure of the reduced pressure concentration is preferably -0.070 to -0.090 MPa.

[0028] Preferably, the re-filtration includes the steps of first coating filtration and then fine filtration; the coating filtration is, for example, laterite coating filtration; and the fine filtration is, for example, sand core fine filtration.

[0029] In some embodiments, in step S2, the protease includes one or more of acidic protease, cellulase, bromelain, papain, and alkaline protease, preferably alkaline protease.

[0030] In some embodiments, in step S2, the weight of the protease is 0.05%-6% of the weight of the moringa leaves, preferably 3%-6%, for example 5%.

[0031] In some specific implementations, in step S2, the protease is an alkaline protease, and its weight is 5% of the weight of the moringa leaf.

[0032] In some implementations, the enzymatic hydrolysis temperature in step S2 is 50-60°C, for example, 55°C.

[0033] In some implementations, the enzymatic hydrolysis time in step S2 is 3-5 hours, for example, 4 hours.

[0034] In some implementations, step S2 includes a step of mixing with pure water before the enzymatic hydrolysis step.

[0035] Preferably, the weight of the pure water is 40-60 times, for example, 50 times, the weight of the moringa leaves.

[0036] In some implementations, step S2 further includes enzyme inactivation and concentration steps after the enzymatic hydrolysis step.

[0037] The enzyme inactivation temperature is preferably 80-95℃, for example 85℃; the enzyme inactivation time is preferably 10-20 min, for example 15 min.

[0038] Preferably, the concentration is concentrated to 1 / 2 to 1 / 4 of the original volume, for example, 1 / 3; the concentration temperature is preferably 55-65°C, for example, 60°C; and the concentration pressure is preferably -0.070 to -0.090 MPa.

[0039] In some implementations, step S2, the second solid-liquid separation includes a process of coating filtration followed by fine filtration.

[0040] The coating filter is, for example, a red clay coating filter.

[0041] The fine filtration process is, for example, a sand core filtration process.

[0042] In some implementations, a drying step is included after step S2.

[0043] The drying process is preferably spray drying.

[0044] The present invention also provides a moringa leaf extract, which is prepared by the method for preparing moringa leaf extract as described above.

[0045] In some embodiments, the total polyphenol content in the moringa leaf extract is not less than 15%, preferably not less than 25%, for example 29.8%.

[0046] The term "total polyphenols" can refer to the collective name of multiple phenolic hydroxyl compounds in Moringa leaf extract, and those skilled in the art will understand its specific meaning.

[0047] In some embodiments, the total polyphenols in the Moringa leaf extract include the following components: cryptochlorogenic acid, chlorogenic acid isomers, kaempferol-3-O-glucoside, quercetin-3-O-galactoside, and isorhamnetin-3-O-glucoside.

[0048] In some preferred embodiments, the Moringa leaf extract comprises the following components by weight percentage:

[0049] Cryptochlorogenic acid 4%-6%, chlorogenic acid isomers 4%-6%, kaempferol-3-O-glucoside 1%-3%, quercetin-3-O-galactoside 1%-3%, isorhamnetin-3-O-glucoside 0.05%-1%.

[0050] In some specific embodiments, the Moringa leaf extract comprises the following components by weight percentage:

[0051] Cryptochlorogenic acid 4.96%, chlorogenic acid isomer 4.61%, kaempferol-3-O-glucoside 2.15%, quercetin-3-O-galactoside 1.26%, isorhamnetin-3-O-glucoside 0.08%.

[0052] The present invention also provides an application of Moringa leaf extract in food, the food having antidepressant effects.

[0053] In some embodiments, the Moringa leaf extract is as defined above.

[0054] This invention also provides the application of Moringa leaf extract in the preparation of antidepressant drugs.

[0055] In some embodiments, the Moringa leaf extract is as defined above.

[0056] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0057] The reagents and raw materials used in this invention are all commercially available.

[0058] The positive and progressive effects of this invention are as follows:

[0059] The preparation method of the present invention can further improve the polyphenol content in Moringa leaf extract based on the existing preparation method of Moringa leaf extract, and ensure a high polyphenol extraction rate. Moreover, the entire preparation is green and environmentally friendly, simple and easy to implement, and can be easily mass-produced.

[0060] Further, relevant in vivo experiments of the present invention show for the first time that the Moringa oleifera leaf extract has excellent antidepressant efficacy, and the high content of polyphenols in the Moringa oleifera leaf extract prepared by the present invention can significantly inhibit the actual toxic effect caused by impurities in the extract. Brief Description of the Drawings

[0061] Figure 1 is the LC-MS ion flow result diagram (base peak chromatogram in positive ion mode) of the Moringa oleifera leaf extract in Effect Example 1 of the present invention.

[0062] Figure 2 is the LC-MS ion flow result diagram (base peak chromatogram in negative ion mode) of the Moringa oleifera leaf extract in Effect Example 1 of the present invention.

[0063] Figure 3 is the result diagram of the effect of the Moringa oleifera leaf extract in Effect Example 2 of the present invention on hippocampal histopathology of depression model mice. Detailed Description of the Embodiments

[0064] The present invention is further described below by way of examples, which however are not intended to limit the present invention to the scope of the described examples. For the experimental methods without specifying specific conditions in the following examples, conventional methods and conditions are followed, or selection is made according to the product specification.

[0065] Example 1

[0066] The Moringa oleifera leaf extract of this example is prepared by the following preparation steps:

[0067] S1,

[0068] 1. Extraction: Take 100g of Moringa oleifera leaves, add 20 times the weight of 70% ethanol for stirring extraction, the extraction temperature is 60°C, keep warm for extraction for 2h, and repeat the extraction once under the same conditions;

[0069] 2. Filtration: Filter with a 120-mesh filter screen, combine the filtrates, and set the material residue aside for later use;

[0070] 3. Concentration: Concentrate the filtrate under reduced pressure until no ethanol remains to obtain concentrated liquid 1, under the conditions of 60°C and -0.070~-0.090MPa;

[0071] 4. Filtration: Concentrated liquid 1 is filtered by red earth coating and then finely filtered with a sand core to obtain a first filtrate and a first material residue;

[0072] S2,

[0073] 5. Enzymolysis: Add pure water (the dosage is 50 times the weight of the Moringa oleifera leaf raw material) to the first material residue, stir evenly and heat up to 55°C, add alkaline protease (SUNSON, article number / model: FDG-2202) (the dosage is 5% of the weight of the raw material), and keep warm for enzymolysis for 4h;

[0074] 6. Enzyme deactivation: After the enzymatic hydrolysis reaction is completed, the temperature is raised to 85°C to deactivate the enzyme for 15 min;

[0075] 7. Concentration: The filtrate is concentrated under reduced pressure to 1 / 3 of the original volume to obtain concentrated liquid 2, under the conditions of 60°C and -0.070~-0.090 MPa;

[0076] 8. Filtration: The concentrated liquid 2 is subjected to red earth coating filtration and sand core fine filtration to obtain a second filtrate and a second filter residue;

[0077] S3,

[0078] 9. Spray drying: Combine the first filtrate and the second filtrate, and perform spray drying to obtain the Moringa oleifera leaf extract.

[0079] Example 2

[0080] Compared with Example 1, the only difference is that in step S1, the extraction temperature is 80°C.

[0081] Example 3

[0082] Compared with Example 1, the only difference is that in step S2, the enzymatic hydrolysis time is 2 h.

[0083] Example 4

[0084] Compared with Example 1, the only difference is that in step S2, the amount of protease used during enzymatic hydrolysis is 2.5% of the weight of the Moringa oleifera leaf raw material.

[0085] Comparative Example 1

[0086] Compared with Example 1, the only difference is that in step S1, the ethanol concentration is 50%.

[0087] Comparative Example 2

[0088] Compared with Example 1, the only difference is that the order of step S1 and step S2 is exchanged, specifically:

[0089] The Moringa oleifera leaf extract of this comparative example is prepared through the following preparation steps:

[0090] S1,

[0091] 1. Enzymatic hydrolysis: Add pure water (the dosage is 50 times the weight of Moringa oleifera leaves) to 100 g of Moringa oleifera leaves, stir evenly, raise the temperature to 55°C, add alkaline protease (SUNSON, item number / model: FDG-2202) (the dosage is 5% of the weight of the raw material), and perform heat-preserved enzymatic hydrolysis for 4 h;

[0092] 2. Enzyme deactivation: After the enzymatic hydrolysis reaction is completed, the temperature is raised to 85°C to deactivate the enzyme for 15 min;

[0093] 3. Concentration: The filtrate is concentrated under reduced pressure to 1 / 3 of its original volume to obtain concentrate 2. The conditions are: 60℃, -0.070~-0.090MPa.

[0094] 4. Filtration: The concentrated liquid 2 is filtered through red clay coating and then finely filtered through sand core to obtain the first filtrate and the first residue.

[0095] S2,

[0096] 5. Extraction: Take the first residue, add 20 times its weight of 70% ethanol and stir to extract. The extraction temperature is 60℃ and the extraction is kept at this temperature for 2 hours. Repeat the extraction once under the same conditions.

[0097] 6. Filtration: Filter using a 120-mesh filter, combine the filtrates, and set aside the residue;

[0098] 7. Concentration: The filtrate is concentrated under reduced pressure until no ethanol remains, to obtain concentrate 1, under the following conditions: 60℃, -0.070~-0.090MPa;

[0099] 8. Filtration: The concentrated liquid 1 is filtered through a red clay coating and then finely filtered through a sand core to obtain the second filtrate and the second residue.

[0100] S3

[0101] 9. Spray drying: Combine the first and second filtrates and spray dry to obtain Moringa leaf extract.

[0102] Comparative Example 3

[0103] The Moringa leaf extract of this comparative example was prepared using the following steps:

[0104] 1. Extraction: Take 100g of Moringa leaves, add 20 times the weight of pure water and stir to extract. The extraction temperature is 60℃ and the extraction is kept at this temperature for 2 hours. Repeat the extraction once under the same conditions.

[0105] 2. Filtration: Filter using a 120-mesh filter, combine the filtrates, and set aside the residue;

[0106] 3. Concentration: The filtrate is concentrated under reduced pressure to a high solids content (6%-7%) to obtain concentrate 1, under the following conditions: 60℃, -0.070~-0.090 MPa;

[0107] 4. Filtration: The concentrated liquid is filtered through a red clay-coated filter and then finely filtered through a sand core filter to obtain a fine filtrate;

[0108] 5. Spray drying: The fine filtrate was spray dried to obtain Moringa leaf extract.

[0109] Example 1

[0110] The total polyphenol content and polyphenol extraction rate of the Moringa leaf extracts obtained in Examples 1-4 and Comparative Examples 1-3 were determined as follows:

[0111] 1. Testing method:

[0112] (1) Determination of total polyphenol content

[0113] Referring to Method 2 in the national standard GB / T 8313-2008, gallic acid was used as an external standard, and the polyphenol content of Moringa leaf extract was quantitatively determined by the Folin-Ciocalteu method. The basic principle is that under alkaline conditions, Folin-Ciocalteu reagent can oxidize the -OH group in the polyphenol structure and turn blue, with a maximum absorption wavelength of 765 nm. Gallic acid was used as a standard to quantify the total polyphenol content in the product.

[0114] The specific steps are as follows:

[0115] S1. Reagent Preparation

[0116] S1.1, Gallic acid: content greater than 98% (HPLC), Shanghai Yuanye Biotechnology Co., Ltd.

[0117] S1.1.1 Gallic acid standard stock solution (80 μg / mL): Accurately weigh 0.0040 g of phosphorus pentoxide dried under reduced pressure and dilute to 50 mL with pure water.

[0118] S1.2, Folin & Clocalteu's phenol reagent: S30000, BR grade, Shanghai Yuanye Biotechnology Co., Ltd.

[0119] S1.2.1 Folin-Ciocalteu reagent (10%): Take 25 mL of Folin-Ciocalteu and place it in a 250 mL volumetric flask. Dilute to volume with distilled water, shake well, and store in a brown reagent bottle for later use.

[0120] S1.3, Sodium carbonate (Na2CO3): AR grade, Shanghai Guoyao Reagent.

[0121] S1.3.1, Na2CO3 (7.5% mass concentration): Accurately weigh 37.5g Na2CO3 (AR grade), dissolve in an appropriate amount of water, transfer to a 500mL volumetric flask, dilute to volume with distilled water, shake well, and set aside.

[0122] S2, Operating Method

[0123] S2.1 Preparation of Standard Curve

[0124] Take 0.2, 0.4, 0.6, 0.8, and 1.0 μL of gallic acid standard stock solution respectively and place them in 10 mL reaction tubes. Add the solution according to the requirements in Table 1. Use the reaction system without gallic acid standard as a blank control. Adjust the zero point of the spectrophotometer and measure the absorbance A value at 765 nm. Plot the absorbance value on the ordinate and the standard content (μg) on ​​the abscissa to obtain the linear equation of the standard curve as Y = bx + a.

[0125] Table 1. Standard Curve Construction

[0126]

[0127] S3. Determination of polyphenol content in Moringa leaf extract

[0128] Accurately weigh 2.000~2.500g of sample (i.e. Moringa leaf extract), dilute to 50mL with pure water, and take 1mL of the sample into a 10mL reaction tube for testing. Perform the test according to the preparation method of the standard curve, record the absorbance value, and calculate the total polyphenol content in the product.

[0129] The specific calculation formula is as follows:

[0130] .

[0131] In the formula, W is the total polyphenol content (%); Y is the absorbance of the sample; a is the intercept of the standard curve; b is the slope of the standard curve; m is the mass of the sample (g); V1 is the volume of the sample dissolved (mL); and V2 is the volume of the sample taken (mL).

[0132] (2) Determination of polyphenol extraction rate

[0133] Moringa leaf extract (i.e., moringa leaf extract before spray drying) was concentrated under reduced pressure to obtain a paste. The solid content of the paste was measured and converted to the dry weight. Based on the total polyphenol content (calculated as gallic acid) as above, the polyphenol extraction rate was further calculated using the following formula:

[0134] Polyphenol extraction rate = (weight of dried product after extraction × total polyphenol content) / amount of Moringa leaves fed.

[0135] 2. Test Results:

[0136] The details are shown in Table 2.

[0137] Table 2. Relevant test results of Moringa leaf extracts obtained in Examples 1-4 and Comparative Examples 1-3

[0138]

[0139] Example 2

[0140] The Moringa leaf extract obtained in Example 1 was analyzed by LC-MS. The specific analytical method was as follows: 1 mL of 70% methanol internal standard extract was added to the Moringa leaf extract sample, swirled for 5 min to mix, centrifuged (1200 r / min, 4℃) for 10 min, and the supernatant was filtered through a microporous membrane (0.22 µm) and stored in a sample vial for LC-MS / MS detection.

[0141] The acquisition conditions for chromatography-mass spectrometry are as follows:

[0142] 1. The liquid chromatography instrument was an UltiMate 3000 UHPLC, and the chromatogram was a Thermo Hypersil gold C18 (1.9µm, 2.1mm×100mm). The analytical flow rate was 0.3mL / min, and the injection volume was 10µL. Mobile phase A was 0.1% formic acid / water (where 0.1% means that the volume percentage of formic acid in the mixed solution of formic acid and water is 0.1%), and mobile phase B was 0.1% formic acid / acetonitrile (where 0.1% means that the volume percentage of formic acid in the mixed solution of formic acid and acetonitrile is 0.1%). The gradient elution program for the solutions is shown in Table 3.

[0143] Table 3. Mobile phase gradient elution program

[0144]

[0145] 2. The mass spectrometer used was a Q-Exactive (Thermo Fisher Scientific, CA, USA) HESI source with an ion source temperature of 310℃, a capillary temperature of 320℃, a sheath gas flow rate of 30 units, an auxiliary gas flow rate of 10 units, a spray voltage of 3 kV in positive ion mode, and a spray voltage of 2.8 kV in negative ion mode. Data-dependent scan analysis (DDA) was used, with a lop count set to 10. The HCD energy was set to step-wise normalized collision energy at 10, 28, and 35 eV. The primary mass spectrometry scan range was 70–1050 m / z, with a resolution of 70,000, an AGC target of 3E6, and an injection time of 200 ms. The secondary mass spectrometry resolution was set to 17,500, the AGC target to 1E5, and the injection time to 50 ms.

[0146] like Figure 1 and Figure 2 The image shows the LC-MS ion current results of the Moringa leaf extract obtained in Example 1; wherein, Figure 1 and Figure 2are the base peak chromatograms in positive ion mode and negative ion mode respectively. Further component analysis shows that in the positive ion mode, the main peaks in the base peak chromatogram correspond to molecular weights of 281.27 (Oleamide) and 264.15 (neochlorogenic acid derivative); in the negative ion mode, the main peak is 354.10 (chlorogenic acid-like substance), indicating that polyphenol components dominate in the Moringa oleifera leaf extract.

[0147] Specifically, the Moringa oleifera leaf extract obtained in Example 1 contains 4.96% neochlorogenic acid, 4.61% chlorogenic acid isomer, 2.15% kaempferol-3-O-glucoside, 1.26% quercetin-3-O-galactoside, and 0.08% isorhamnetin-3-O-glucoside.

[0148] Effect Example 3

[0149] To further verify the antidepressant efficacy of the polyphenol-rich Moringa oleifera leaf extract of the present invention, three types of Moringa oleifera leaf extracts prepared in Example 1, Comparative Example 1 and Comparative Example 3 (with total polyphenol contents of 29.8%, 10.3% and 1.5% respectively, designated as Moringa oleifera leaf extract 1, Moringa oleifera leaf extract 2, and Moringa oleifera leaf extract 3 respectively) were selected to carry out pharmacological experiments. The experimental methods and results are as follows.

[0150] 1 Experimental Materials

[0151] 1.1 Test Samples

[0152] Three kinds of Moringa oleifera leaf extracts

[0153] 1.2 Positive Control

[0154] Fluoxetine Hydrochloride Tablets, batch numbers: 202210311, 202210312, specification: 10mg / tablet, valid until 2024-10-30, product of Changzhou Siyao Pharmaceutical Co., Ltd.

[0155] 1.3 Experimental Animals

[0156] 22 SPF-grade male C57 mice, weighing 18~22g, were purchased from Hunan Silaike Jingda Experimental Animal Co., Ltd. The experimental animal production license number is SCXK(Xiang)2019-0004, and the experimental animal quality certificate number is 430727231101501437. The animals were raised in Zone D of the barrier environment animal experiment facility of Hunan Prema Pharmaceutical Research Center, with the experimental animal use license number SYXK(Xiang)2020-0015.

[0157] 1.4 Main Reagents

[0158] Purified water, prepared in-house. Neutral resin, batch number: AWI0238a, product of abiowell1; hematoxylin, batch number: AWI0001a, product of abiowell; PBS (7.2-7.6), batch number: AWIO129a, product of abiowell1; eosin, batch number: AWI0029a, product of abiowell1.

[0159] 1.5 Main Instruments

[0160] Behav animal behavior analysis system (Shanghai Jiliang Technology Co., Ltd.); ME2002E electronic balance (Mettler AG); HistoCore BIOCUT paraffin microtome (Leica, Germany); TP1020 fully automatic dehydrator (Leica, Germany); HBM-200LF tissue embedding machine (Wuhan Hairuit Technology Co., Ltd.); DFC 420C pathological imaging system (Leica, Germany); SORVALL Stratos high-speed refrigerated centrifuge (Thermo Science); JY88-IIN ultrasonic cell disruptor (Ningbo Xinzhi Biotechnology Co., Ltd.).

[0161] 2 Research Content

[0162] 2.1 Modeling and Grouping

[0163] Twenty-two qualified SPF-grade male C57 mice, weighing 18.0–22.0 g, were selected. Three mice were randomly selected as the normal control group based on their weight. Except for the normal control group, the remaining mice were randomly subjected to various stressors daily, including: restraint for 12 hours, wet cage for 12 hours, swimming in ice water (4℃) for 10 minutes, strobe light for 12 hours, 45-degree inclined cage for 12 hours, noise for 30 minutes, day-night reversal, fasting for 12 hours, and water deprivation for 12 hours. The stressors were randomly arranged each day, and the same stressor could not be given consecutively, so that the animals could not predict the stressors given. Two to three types of stressors were given each day, and the modeling period lasted for 28 days (as shown in Table 4). After modeling, mice underwent a sucrose preference test. Based on the sucrose preference index, 15 successfully modeled mice were selected and randomly divided into 5 groups: model control group, fluoxetine hydrochloride group, and Moringa leaf extract groups 1, 2, and 3, with 3 mice in each group. The normal control group and model control group were given pure water by gavage, while the other groups were given the corresponding drug solution (all prepared with pure water) by gavage. The drug volume was 20 mL / kg, and the drugs were administered once a day for 28 consecutive days. Modeling continued in each group during the drug administration period, and behavioral tests were conducted after the last administration.

[0164] Table 4. Timeline of Chronic Unpredictable Stress

[0165]

[0166] 2.2 Detection Indicators

[0167] 2.2.1 Sugar Water Preference Test

[0168] After modeling, testing was conducted, divided into an adaptation period, a training period, and a testing period. During the adaptation period, animals were given two bottles of 1% sugar solution to acclimate to sucrose drinking water for 24 hours. During the training period, animals were given one bottle of 1% sucrose solution and one bottle of pure water for 24 hours. During the testing period, animals were deprived of food and water for 16 hours before the test, and were again given one bottle of 1% sucrose solution and one bottle of pure water for a 12-hour testing period. To avoid the influence of positional preference, the positions of the two bottles were swapped during the testing period. At the end of the test, the sugar solution preference index was calculated.

[0169] Sugar water preference index = Sugar water consumption / (Sugar water consumption + Pure water consumption) × 100%.

[0170] 2.2.2 Three-box social interaction experiment

[0171] The three-box social interaction test is primarily used to evaluate the social behavior of mice and reflects their social avoidance status. The test consists of three identical square boxes, with a cylindrical cage in each of the two outer boxes for holding the animal. Before the experiment, the mouse is placed in the middle box and allowed to explore freely for 10 minutes. At the start of the experiment, a white circular object is placed in the cage in the right box, and a mouse of the same sex is placed in the cage in the left box. The test mouse is then placed into all three boxes along the side wall of the middle box, allowing it to move freely within each box. A camera tracks the mouse's movement and analyzes relevant parameters. After each mouse's test, feces are removed with a paper towel, and the boxes are wiped with 75% alcohol to remove odor and prevent interference with other mice.

[0172] 2.2.3 Histopathological examination

[0173] After the last dose of medication and behavioral testing, mice were sacrificed, and brain tissue was harvested. The cortex and hippocampus were separated, fixed in 10% formalin, and stained with hematoxylin and eosin (HE). Procedure:

[0174] 1) Bake the slices at 60℃ for 12 hours;

[0175] 2) Dewaxing the sections to water: First, place the sections in xylene for 20 minutes, three times. Then, place them successively in 100%, 100%, 95%, 85%, and 75% ethanol, 5 minutes each time. Finally, rinse with distilled water for 5 minutes.

[0176] 3) Stain with hematoxylin for 1-10 min, rinse with distilled water, and then stain with PBS to restore blue color;

[0177] 4) Stain with eosin for 1-5 minutes, then rinse with distilled water;

[0178] 5) Dehydrate with gradient ethanol (95-100%), 5 min per stage (or directly bake the slide dry). After removal, place in xylene for 10 min, twice, then mount with neutral resin and observe under a microscope.

[0179] 2.2.4 Data Processing and Statistical Analysis

[0180] Data from this experiment were rounded to the nearest whole number and statistical analysis was performed according to relevant standard operating procedures (SOPs). SPSS 23.0 was used for statistical analysis. Quantitative data were expressed as mean ± standard deviation (X̄ ± SEM), and normality and homogeneity of variance were tested using Leven's test. If there was no statistical significance (P > 0.05), one-way ANOVA was used. If the ANOVA was statistically significant (P ≤ 0.05), the LSD test (parametric method) was used for comparative analysis. If the variances were unequal (P ≤ 0.05), the Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P ≤ 0.05), Dunnett's test (non-parametric method) was used for comparative analysis. The statistical results were compared with α = 0.05, where P ≤ 0.05 indicated statistical significance, and P ≤ 0.01 indicated a highly significant difference.

[0181] 3. Experimental Results

[0182] 3.1 Effects of Moringa Leaf Extract on Body Weight of Inhibition Model Mice

[0183] As shown in Table 5, compared with the normal control group, the body weight of mice in the model control group showed a decreasing trend, but there was no statistical difference. Compared with the model control group, the moringa leaf extract groups 1, 2, and 3 had no significant effect on mice.

[0184] Table 5. Effects of Moringa leaf extract on body weight in depressed mice (X̅±SEM, n=3)

[0185]

[0186] 3.2 Effect of Spicy Leaf Extract on Saccharide Preference Index in Depressed Mice

[0187] As shown in Table 6, compared with the normal control group, the sucrose preference index of mice in the model control group was significantly reduced (P≤0.01), while compared with the model control group, the sucrose preference index of mice in Moringa leaf extract groups 1 and 2 was significantly increased (P≤0.05 or P≤0.01).

[0188] Table 6. Effects of Moringa leaf extract on sucrose preference index in depressed mice (X̅±SEM, n=3)

[0189]

[0190] Note: Compared with the normal control group, ++P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01.

[0191] 3.3 Effects of Moringa Leaf Extract on Social Interaction in a Depression Model

[0192] As shown in Table 7, compared with the normal control group, the number of exposures of mice in the model control group was significantly reduced (P≤0.01), while compared with the model control group, the number of exposures of mice in Moringa leaf extract groups 1 and 2 was significantly increased (P≤0.05 or P≤0.01).

[0193] Table 7. Effects of Moringa leaf extract on social interaction in a depressed mouse model (X̅±SEM, n=3)

[0194]

[0195] Note: Compared with the normal control group, ++P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01.

[0196] 3.4 Effects of Moringa Leaf Extract 1 on Hippocampal Histopathology in Depressed Mice

[0197] like Figure 3 As shown, in the normal control group, no abnormalities were observed in the morphology of neurons, glial cells, and hippocampal pyramidal cells in the hippocampal tissue. In the model control group, some pyramidal cells showed sparse and disordered arrangement, some pyramidal cells showed necrosis, and interstitial edema in the hippocampal tissue. In the Moringa leaf extract group 3, some pyramidal cells showed necrosis and interstitial edema in the hippocampal tissue. In the Moringa leaf extract groups 1 and 2, there was an increase in cortical neurons, and some hippocampal pyramidal cells showed a dense arrangement; a small amount of interstitial edema was also observed. In the fluoxetine hydrochloride tablet group, the hippocampal tissue showed an increase in neurons, dense arrangement of hippocampal pyramidal cells, and a small amount of interstitial edema.

[0198] Note: A - Normal control group; B - Model control group; C - Fluoxetine hydrochloride group; D - Moringa leaf extract group 3; E - Moringa leaf extract group 2; F - Moringa leaf extract group 1.

[0199] 4. Summary of Experimental Results

[0200] In summary, the Moringa leaf extract 1, which is rich in polyphenols (with a polyphenol content as high as 29.8%) obtained in the embodiments of the present invention, significantly increases the sucrose preference index in mice, increases the frequency of social interaction in mice, significantly improves the morphological changes of nerve cells in the hippocampus of mice, and has a significant antidepressant effect.

[0201] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, material changes, equivalent variations, or alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A process for the preparation of moringa oleifera leaf extract, characterized by, The preparation method of the Moringa leaf extract includes the following steps: S1. Moringa leaves are extracted sequentially, followed by a first solid-liquid separation to obtain a first filtrate and a first residue. The extraction process includes an extraction step using an ethanol solution; the volume concentration of ethanol in the ethanol solution is higher than 50%. S2. The first residue is sequentially subjected to enzymatic hydrolysis and solid-liquid separation to obtain the second filtrate and the second residue. The enzymatic hydrolysis process includes a step of enzymatic hydrolysis with protease; S3. The first filtrate and the second filtrate are mixed to obtain the Moringa leaf extract.

2. The method for preparing Moringa leaf extract as described in claim 1, characterized in that, The preparation method of the Moringa leaf extract satisfies one or more of the following conditions: (1) In step S1, the volume concentration of ethanol in the ethanol solution is 60%-80%, for example, 70%; (2) In step S1, the weight of the ethanol solution is 10-30 times the weight of the Moringa leaves, for example, 20 times; (3) In step S1, the extraction temperature is 60-80℃, preferably 60-70℃.

3. The method for preparing Moringa leaf extract as described in claim 1, characterized in that, The preparation method of the Moringa leaf extract meets one or more of the following conditions: (1) In step S2, the weight of the protease is 0.05%-6% of the weight of the moringa leaves, preferably 3%-6%, for example 5%; (2) In step S2, the temperature of the enzymatic hydrolysis is 50-60℃, for example 55℃; (3) In step S2, the enzymatic hydrolysis time is 3-5 hours, for example 4 hours.

4. The method for preparing Moringa leaf extract as described in claim 1, characterized in that, The preparation method of the Moringa leaf extract meets one or more of the following conditions: (1) In step S1, the extraction time is 1-3 hours, for example 2 hours; (2) In step S1, the extraction is performed twice; preferably, the conditions for the two extractions are the same. (3) In step S1, the first solid-liquid separation includes the steps of filtration, concentration, and re-filtration; The filtration is preferably performed using a filter screen; the filter screen is, for example, a 120-mesh filter screen. Preferably, the concentration is vacuum concentration; the temperature of the vacuum concentration is preferably 50-70°C, for example 60°C; and the pressure of the vacuum concentration is preferably -0.070 to -0.090 MPa. Preferably, the re-filtration includes the steps of first coating filtration and then fine filtration; the coating filtration is, for example, laterite coating filtration; and the fine filtration is, for example, sand core fine filtration.

5. The method for preparing Moringa leaf extract as described in claim 1, characterized in that, The preparation method of the Moringa leaf extract meets one or more of the following conditions: (1) In step S2, the protease includes one or more of acidic protease, cellulase, bromelain, papain and alkaline protease, preferably alkaline protease. (2) In step S2, the second solid-liquid separation includes the steps of coating and filtration followed by fine filtration; The coating filter is, for example, a laterite coating filter; The fine filtration process is, for example, a sand core filter.

6. The method for preparing Moringa leaf extract as described in claim 1, characterized in that, The preparation method of the Moringa leaf extract satisfies one or more of the following conditions: (1) In step S2, the enzymatic hydrolysis step is preceded by a step of mixing with pure water; Preferably, the weight of the pure water is 40-60 times, for example, 50 times, the weight of the moringa leaves; (2) In step S2, the enzymatic hydrolysis step is followed by an enzyme inactivation and concentration step; The enzyme inactivation temperature is preferably 80-95℃, for example 85℃; the enzyme inactivation time is preferably 10-20 min, for example 15 min. Preferably, the concentration is concentrated to 1 / 2 to 1 / 4 of the original volume, for example, 1 / 3; the concentration temperature is preferably 55-65°C, for example, 60°C; and the concentration pressure is preferably -0.070 to -0.090 MPa. (3) After step S2, a drying step is also included; The drying process is preferably spray drying.

7. A Moringa leaf extract, characterized in that, The moringa leaf extract is prepared by the method described in any one of claims 1-6.

8. The Moringa leaf extract as described in claim 7, characterized in that, The Moringa leaf extract meets one or more of the following conditions: (1) The total polyphenol content in the moringa leaf extract is not less than 15%, preferably not less than 25%, for example 29.8%; (2) The total polyphenols in the Moringa leaf extract include the following components: cryptochlorogenic acid, chlorogenic acid isomer, kaempferol-3-O-glucoside, quercetin-3-O-galactoside, and isorhamnetin-3-O-glucoside. Preferably, the Moringa leaf extract comprises the following components by weight percentage: Cryptochlorogenic acid 4%-6%, chlorogenic acid isomers 4%-6%, kaempferol-3-O-glucoside 1%-3%, quercetin-3-O-galactoside 1%-3%, isorhamnetin-3-O-glucoside 0.05%-1%; the percentages are the mass percentages of each component in the Moringa leaf extract. More preferably, the Moringa leaf extract comprises the following components in weight percentage: Cryptochlorogenic acid 4.96%, chlorogenic acid isomer 4.61%, kaempferol-3-O-glucoside 2.15%, quercetin-3-O-galactoside 1.26%, isorhamnetin-3-O-glucoside 0.08%; the percentages are the mass percentages of each component in the Moringa leaf extract.

9. The application of Moringa leaf extract in food, said food having antidepressant effects; Preferably, the moringa leaf extract is the moringa leaf extract as described in claim 7 or 8.

10. The application of a Moringa leaf extract in the preparation of antidepressant drugs; Preferably, the moringa leaf extract is the moringa leaf extract as described in claim 7 or 8.

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