Preparation of amomum tsao-ko flower tea and application of active ingredients of amomum tsao-ko flower tea in blood sugar

Abstract

The invention discloses preparation of amomum tsao-ko flower tea and application of active ingredients of the amomum tsao-ko flower tea in blood sugar reduction. The amomum tsao-ko flower tea with a potential blood sugar resisting effect is prepared by taking amomum tsao-ko flowers as a raw material through specific withering and drying steps. Three natural components capable of inhibiting the activity of alpha-glucosidase are further separated and extracted from tsaoko amomum flowers, are all diphenyl heptane compounds through identification, and can be used for reducing the peak value of postprandial blood sugar concentration so as to achieve the purpose of controlling blood sugar. Therefore, the compounds can be used for developing medicines or functional products with a hypoglycemic effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the preparation of tsaoko flower tea and application of its active ingredients in lowering blood sugar. Background Art

[0002] Amomum tsaoko (Ziberaceae) is a plant of the genus Cardamom in the ginger family. Its fruit is widely used as a seasoning and in traditional medicine. The flower, or tsaoko flower, has a red corolla and white or yellow petals. It has a rich, sweet fragrance, but is not pungent. The flowering period of tsaoko is significantly affected by climate, with temperature and precipitation having the greatest influence. Tsaoko prefers a warm, humid climate and is sensitive to heat and frost. The average annual temperature is 15-20°C, and it is less likely to flower when temperatures fall below 10°C or rise above 25°C. Tsaoko is drought-tolerant and thrives in sparsely wooded areas (with approximately 40% light transmittance). It is best cultivated in cool, well-drained valley slopes, under forests at altitudes of 1,000-2,000 meters, with a shade of approximately 50-60%, or near streams, in loose, fertile, humus-rich sandy loam.

[0003] Although the flower of Amomum villosum has a unique aroma and potential pharmacological activity, it has long been underutilized and is usually left to wither naturally. In the existing technology, most products related to Amomum villosum are concentrated on the fruit, while the development and utilization of the flower of Amomum villosum is very rare.

[0004] α-glucosidase is a digestive enzyme present in the brush border of the small intestine, responsible for breaking down complex carbohydrates (such as starch and oligosaccharides) into monosaccharides (such as glucose). The specific steps include: hydrolysis of α-1,4 glycosidic bonds, breaking down disaccharides (such as maltose and sucrose) and polysaccharides into glucose. The generated glucose is absorbed into the blood by the small intestine, resulting in increased blood sugar after a meal. The activity of this enzyme directly affects the release rate and absorption efficiency of glucose, and is therefore a key regulatory point for postprandial blood sugar (postprandial hyperglycemia). Common α-glucosidase inhibitors include acarbose and voglibose, but these drugs can cause adverse reactions such as abdominal distension, diarrhea, and abdominal pain. Therefore, the development of natural active ingredients has become a research hotspot. These ingredients have mild efficacy and few toxic side effects. Summary of the Invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a diphenylheptane compound for use in the preparation of a product for inhibiting α-glucosidase activity and / or lowering blood sugar.

[0006] Another object of the present invention is to provide a method for preparing tsaoko flower tea.

[0007] Another object of the present invention is to provide tsaoko flower tea prepared by the method.

[0008] Another object of the present invention is to provide an application of the tsaoko flower tea.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A use of a diphenylheptane compound in preparing a product for inhibiting α-glucosidase activity and / or lowering blood sugar, wherein the diphenylheptane compound is at least one of (-)-1,7-bis(p-hydroxyphenyl)-5-hydroxy-3-heptanone, 3-acetoxy-1,7-bis(4-hydroxyphenyl)-3,5-heptanediol, and 3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)heptane, and their structural formulas are shown in Formula I, Formula II, and Formula III, respectively:

[0011]

[0012] The diphenylheptane compound is preferably (-)-1,7-bis(p-hydroxyphenyl)-5-hydroxy-3-heptanone.

[0013] The products for inhibiting α-glucosidase activity include drugs or functional foods that inhibit α-glucosidase activity, such as α-glucosidase inhibitors.

[0014] The blood sugar lowering products include blood sugar lowering drugs or functional foods that assist in lowering blood sugar. They can lower the blood sugar of diabetic patients by inhibiting the activity of α-glucosidase and can be used to prevent or assist in the treatment of diabetes.

[0015] The diphenylheptane compounds can be obtained by conventional methods in the art (synthesis, commercial availability, etc.), or extracted and separated from tsaoko flowers and / or tsaoko flower tea; preferably, they are extracted and separated from tsaoko flowers and / or tsaoko flower tea.

[0016] A method for separating and extracting the diphenylheptane compounds from tsaoko flowers and / or tsaoko flower tea, comprising the following specific steps:

[0017] (1) Extraction with 95% ethanol solution

[0018] ① Dry the tsaoko flowers naturally, grind them into powder, and sieve them to obtain tsaoko flower powder;

[0019] ② After screening the tsaoko flowers, pick the unopened inflorescences, freeze-dry and / or hot-air dry until the moisture content is ≤15%, grind into powder, and sieve to obtain tsaoko flower tea;

[0020] ③ Add 95% by volume ethanol solution to the tsaoko flower powder and / or tsaoko flower tea, fully soak and extract, filter, and concentrate to obtain tsaoko flower ethanol extract;

[0021] (2) Extraction with petroleum ether and ethyl acetate

[0022] The tsaoko flower ethanol extract obtained in step (1) ③ is first extracted with petroleum ether to obtain the petroleum ether layer extract, and then extracted with ethyl acetate to obtain the ethyl acetate layer extract;

[0023] (3) Silica gel column separation

[0024] ④ The ethyl acetate layer extract obtained in step (2) was separated by silica gel chromatography column, dry-loaded, and then gradient eluted with dichloromethane and methanol, with the methanol concentration ranging from 0% to 100% (100:0→0:100). Each gradient was taken as a fraction of 2 L, and all fractions were subjected to TLC spot plate. The same fractions were combined and concentrated under reduced pressure to obtain 21 fractions Fr.A1 to Fr.A21;

[0025] ⑤ The Fr.A10 fraction with an elution concentration of dichloromethane:methanol = 100:10 was eluted through a gel column with dichloromethane and methanol in a volume ratio of 1:1. TLC spot plate analysis was performed and similar components were combined to obtain a total of 8 components, Fr.A10B1 to Fr.A10B8. Component Fr.A10B6 was then preparatively separated by HPLC (G1311A-1100HP, Agilent, USA) using methanol / water as the mobile phase with methanol concentrations ranging from 30% to 100%. (-)-1,7-bis(p-hydroxyphenyl)-5-hydroxy-3-heptanone and 3-acetoxy-1,7-bis(4-hydroxyphenyl)-3,5-heptanediol were collected.

[0026] ⑥ The Fr.A11 fraction with an elution concentration of dichloromethane:methanol = 100:15 was eluted through a gel column with the eluent being dichloromethane and methanol in a volume ratio of 1:1. Similar components were combined by TLC spot plate analysis to obtain a total of 15 components Fr.A11B1 to Fr.A11B15; then component Fr.A11B13 was preparatively separated by HPLC (G1311A-1100HP, Agilent, USA) using methanol / water as the mobile phase and a methanol concentration ranging from 30% to 100%, and 3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)heptane was collected.

[0027] The sieving in step (1) ① is through a 40-120 mesh sieve; preferably through a 60 mesh sieve.

[0028] The sieving in step (1) ② is through an 80-120 mesh sieve; preferably through a 100 mesh sieve.

[0029] In step (1) ③, the material-liquid ratio of tsaoko flower powder (or tsaoko flower tea) to ethanol solution is 1 g: 5-10 mL; preferably 1 g: 10 mL.

[0030] The soaking time in step (1) ③ is 2 to 4 days, preferably 3 days.

[0031] The concentration in step (1) ③ is carried out by rotary evaporation; preferably, rotary evaporation is carried out at 40-45°C (preferably 40°C).

[0032] The number of petroleum ether extractions in step (2) is preferably 4 or more.

[0033] The number of ethyl acetate extractions in step (2) is preferably 3 or more.

[0034] In step (3) ④, during the silica gel chromatography column separation, the weight ratio of the ethyl acetate layer extract to the 100-200 mesh sample mixing silica gel is 1:1, and the weight ratio to the 300-400 mesh normal phase separation silica gel is 1:10.

[0035] In step (3) ⑤, HPLC separation is achieved by the following steps: chromatographic column: semi-preparative column; model: 9.4 mm*25 cm, 4 um; HPLC absorption wavelength: 280 nm; methanol / water is used as the mobile phase, and the elution gradient is: 0-5 min, 30% methanol; 5-30 min, 30-100% methanol, 30-40 min, 100% methanol (all volume percentages); flow rate 3 mL / min; the peak collected at 25 min is concentrated by nitrogen blow to obtain compound 1, i.e., (-)-1,7-bis(p-hydroxyphenyl)-5-hydroxy-3-heptanone; the peak collected at 35 min is concentrated by nitrogen blow to obtain compound 2, i.e., 3-acetoxy-1,7-bis(4-hydroxyphenyl)-3,5-heptanediol.

[0036] In step (3) ⑥, HPLC separation is achieved by the following steps: chromatographic column: semi-preparative column; model: 9.4 mm*25 cm, 4 um; HPLC absorption wavelength: 280 nm; methanol / water as mobile phase, elution gradient: 0-5 min, 30% methanol; 5-30 min, 30-100% methanol; flow rate 3 mL / min; the peak collected at 20 min was concentrated by nitrogen blowdown to obtain compound 3, i.e., 3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)heptane.

[0037] The method for separating diphenylheptane compounds from tsaoko further comprises, after step (3), a step of further identifying the compounds using mass spectrometry, hydrogen spectrum and / or carbon spectrum.

[0038] A method for preparing tsaoko flower tea is provided, which is prepared through specific withering and drying steps (to make the tsaoko flower tea dehydrated evenly); the method specifically comprises the following steps:

[0039] Tsaoko flowers picked on a sunny day are selected, and after screening, unopened inflorescences are picked, freeze-dried and / or hot-air dried to a moisture content of ≤15%, and then ground into powder, sieved, and thus tsaoko flower tea is obtained.

[0040] The picking is preferably done manually (to avoid mechanical damage), and the whole flower buds of fresh tsaoko are collected. The time for picking tsaoko flowers is 9 a.m. or after 9 a.m.

[0041] The flower buds of the tsaoko flower can be put into a ziplock bag and stored in dry ice at -40±5°C to maintain the color and active ingredients.

[0042] The screening is to remove the mixed tsaoko branches, leaves, stems, blackened tsaoko flowers and other impurities in the tsaoko flowers.

[0043] The freeze-drying is achieved by the following steps: pre-cooling at -40±5°C, and then vacuum drying to a moisture content of ≤15%; preferably, it is achieved by the following steps: spreading the tsaoko flowers evenly on a drying tray with a thickness of less than 3 cm, then placing them in a freeze dryer, pre-cooling at -40±5°C, and then vacuum drying them to dry the tsaoko flowers to a moisture content of ≤15%.

[0044] The vacuum drying time is 20 to 30 hours, preferably 24 hours.

[0045] The hot air drying is achieved by the following steps: using 85±5℃ hot air circulation to dry until the moisture content is ≤15%; preferably, it is achieved by the following steps: spreading the tsaoko flowers evenly on a drying tray, controlling the paving thickness to be less than 3 cm, and then placing them in a hot air circulation drying oven, using 85±5℃ hot air circulation for 2 hours, and drying the tsaoko flowers to a moisture content of ≤15%.

[0046] The hot air circulation drying time is 1 to 3 hours, preferably 2 hours.

[0047] The water content of the tsaoko flower tea is preferably 9-15%, and more preferably 9-12%.

[0048] The sieving is through an 80-120 mesh sieve, preferably through a 100 mesh sieve.

[0049] A tsaoko flower tea is prepared by any of the methods described above.

[0050] The application of the tsaoko flower tea in preparing products for inhibiting α-glucosidase activity and / or lowering blood sugar.

[0051] The tsaoko flower can be used to separate a natural component capable of inhibiting α-glucosidase activity, thus having a blood sugar lowering effect.

[0052] The present invention has the following advantages and effects compared to the prior art:

[0053] 1. The present invention provides a preparation method of tsaoko flower tea, which uses tsaoko flowers as raw materials, allows the tsaoko flowers to maintain a relatively stable temperature development, and undergoes relatively stable physical changes and chemical reactions. The specific cooling process allows the tsaoko flowers to have a more sufficient reaction time and dehydrate evenly and completely, which is conducive to forming tsaoko flower tea with a specific flavor, pleasant fragrance and pure taste.

[0054] 2. The tsaoko flower tea provided by the present invention can still maintain the fragrance close to that of fresh tsaoko flowers after long-term storage. When brewed and drunk, the tea soup is light yellow and clear, with a mellow taste and a sweet aftertaste.

[0055] 3. After the tsaoko flowers are picked, the present invention quickly stores them in dry ice, which can reduce the moisture of the hard and brittle petals and reduce the mechanical damage of the tsaoko petals in the subsequent process. There is less broken flower tea, and the enzymes in the tsaoko flowers drive the oxidation of various components. The contents can produce slow and orderly changes, which is conducive to the formation of various flavor substances.

[0056] 4. The present invention combines dry ice preservation with freeze drying using a freeze dryer, and strictly controls the water content of the tsaoko flowers in each step, thereby reducing the astringency of the tsaoko flowers and making the fragrance purer.

[0057] 5. The present invention found that the Amomum villosum flower is rich in special aroma substances and has potential anti-glycemic effects, and has the value of being developed into a health tea.

[0058] 6. The present invention found that the active ingredient extract of Amomum villosum flower tea can significantly inhibit the effect of α-glucosidase, which is beneficial to delaying the digestion and absorption of carbohydrates, thereby effectively reducing the peak blood sugar concentration after meals in diabetic patients, achieving the purpose of controlling blood sugar, and having certain preventive and auxiliary therapeutic effects on lowering blood sugar.

[0059] 7. The present invention further explores new natural ingredients that can inhibit the activity of α-glucosidase, including the tsaoko flower monomer compounds (-)-1,7-bis(p-hydroxyphenyl)-5-hydroxy-3-heptanone, 3-acetoxy-1,7-bis(4-hydroxyphenyl)-3,5-heptane and 3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)heptane. The half-inhibitory concentration for α-glucosidase is 7.034 to 44.577 μg / mL, which is better than the positive drug acarbose. Among them, (-)-1,7-bis(p-hydroxyphenyl)-5-hydroxy-3-heptanone has the best effect and can be used to develop drugs or functional products with hypoglycemic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1This is a graph showing the inhibition of α-glucosidase activity by extracts from tsaoko flower, mulberry leaf, chrysanthemum, honeysuckle and green tea; wherein A is the sample to be tested (in the figure, 1-10 are extracts of tsaoko flower dry sample, tsaoko pollen, mulberry leaf dry sample, mulberry leaf powder, chrysanthemum dry sample, chrysanthemum powder, honeysuckle dry sample, honeysuckle powder, green tea dry sample and green tea powder, respectively); B is a fresh tsaoko flower sample; and C is an enzyme activity verification experiment.

[0061] Figure 2 1-3 and the positive control acarbose on α-glucosidase inhibition results; wherein A is the positive control acarbose inhibition results on α-glucosidase; B is the compound 1-3 inhibition results on α-glucosidase (1-3 in the figure correspond to compounds 1-3, respectively).

[0062] Figure 3 is the mass spectrum of compound 1.

[0063] Figure 4 Compound 1 is compound 1 1 H-NMR spectrum.

[0064] Figure 5 Compound 1 is compound 1 13 C-NMR spectrum.

[0065] Figure 6 Compound 1 is compound 2 1 H-NMR spectrum.

[0066] Figure 7 Compound 1 is compound 3 1 H-NMR spectrum.

[0067] Figure 8 Compound 1 is compound 3 13 C-NMR spectrum.

[0068] Figure 9 This is a molecular weight diagram of compounds 1 to 3; where A is compound 1; B is compound 2; and C is compound 3.

[0069] Figure 10 This is the position map of compounds 1 to 3 in the total ion map of Amomum villosum flower. DETAILED DESCRIPTION

[0070] The present invention will be described in further detail below in conjunction with the examples, but embodiments of the present invention are not limited thereto. Unless otherwise stated, the reagents, methods and equipment used in the present invention are all conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually obtained according to conventional experimental conditions or according to conventional separation methods. Unless otherwise stated, the experimental reagents used in the following examples can be purchased from conventional biochemical reagent manufacturers.

[0071] The α-glucosidase (CAS: 9001-42-7) and substrate p-nitrophenyl-α-D-glucopyranose (also known as p-nitrophenyl-α-D-glucopyranoside; PNPG) (CAS: 3767-28-0) involved in the examples of the present invention were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0072] The tsaoko flowers involved in the embodiments of the present invention were collected from Sanhe Village, Luzhang Town, Lushui City, Nujiang Lisu Autonomous Prefecture, Yunnan Province. The tsaoko flowers bloom from April to June.

[0073] The organic solvents involved in the embodiments of the present invention are specifically analytical grade methanol, dichloromethane, petroleum ether, ethanol and ethyl acetate produced by Tianjin Damao Chemical Reagent Factory.

[0074] Example 1

[0075] A method for preparing tsaoko flower tea comprises the following steps:

[0076] (1) Picking: Choose the grass fruit flowers that are picked by hand at 5 pm on a sunny day ( Figure 1 B) Collect the entire flower buds of fresh tsaoko. Use a basket or mesh bag to hold the flowers during the picking process, and keep the environment well ventilated to avoid squeezing the tsaoko flowers.

[0077] (2) Storage: Put the whole tsaoko flower buds into a ziplock bag and store them in dry ice at -40℃.

[0078] (3) Screening: Remove the mixed tsaoko branches, leaves, stems, blackened tsaoko flowers and other impurities.

[0079] (4) Drying: Then pick the unopened inflorescences and evenly spread the obtained tsaoko flowers on a drying tray. The thickness of the tsaoko flowers should be controlled to be less than 3 cm. Then place the drying tray with the tsaoko flowers in a freeze dryer, pre-cool at -40°C, and vacuum dry for 24 hours. Dry the tsaoko flowers to a moisture content of 10-15%, grind them into powder, and pass through a 100-mesh sieve to obtain the finished tsaoko flower tea.

[0080] Example 2

[0081] A method for preparing tsaoko flower tea comprises the following steps:

[0082] (1) Picking: Choose to pick the tsaoko flowers manually at 9 am on a sunny day, and collect the whole buds of fresh tsaoko. Use a basket or a mesh bag to hold the flowers during the picking process, and keep the environment well ventilated and heat-dissipated to avoid squeezing the tsaoko flowers.

[0083] (2) Storage: Put the whole tsaoko flower buds into a ziplock bag and store them in dry ice at -40℃.

[0084] (3) Screening: Remove the mixed tsaoko branches, leaves, stems, blackened tsaoko flowers and other impurities.

[0085] (4) Drying: Then pick the unopened inflorescences and evenly spread the obtained tsaoko flowers on a drying tray. The thickness of the tsaoko flowers should be controlled to be less than 3 cm. Then place the drying tray with the tsaoko flowers in a freeze dryer, pre-cool it at -40°C, and vacuum dry it for 24 hours. Dry the tsaoko flowers to a moisture content of 10-15%. Grind them into powder and pass through a 100-mesh sieve to obtain the finished tsaoko flower tea.

[0086] Example 3

[0087] A method for preparing tsaoko flower tea comprises the following steps:

[0088] (1) Picking: Choose to pick the tsaoko flowers manually at 9 o'clock on a sunny day, and collect the whole buds of fresh tsaoko. Use a basket or a mesh bag to hold the flowers during the picking process, and keep the environment well ventilated and heat-dissipated to avoid squeezing the tsaoko flowers.

[0089] (2) Storage: Put the whole tsaoko flower buds into a ziplock bag and store them in dry ice at -40℃.

[0090] (3) Screening: Remove the mixed tsaoko branches, leaves, stems, blackened tsaoko flowers and other impurities.

[0091] (4) Drying: Then pick the unopened inflorescences and evenly spread the obtained tsaoko flowers on a drying tray. Then place the drying tray with the tsaoko flowers in a hot air circulation drying oven. The thickness of the tsaoko flowers is controlled to be less than 3 cm. The temperature of the drying oven is set to 85°C and dried for 2 hours. Dry the tsaoko flowers to a moisture content of 9-12%. Grind them into powder and pass through a 100-mesh sieve to obtain the finished tsaoko flower tea.

[0092] Example 4

[0093] The inhibition rate of the tsaoko flower tea prepared in Example 1 and other functional plant extracts (mulberry leaves, chrysanthemums, honeysuckle, and green tea) on α-glucosidase activity was determined to explore their hypoglycemic effects. The specific steps are as follows:

[0094] 1. Experimental materials and reagents:

[0095] 1.1 Experimental Materials: The dried samples of Amomum villosum flowers dried to a moisture content of 10-15% in Example 1 and the powdered Amomum villosum powder further ground into powder were selected. Meanwhile, mulberry leaves (brand: Beijing Tong Ren Tang), chrysanthemums (Taiju, brand: Qilixiang), honeysuckle (brand: Qilixiang), and green tea (Anji Baicha) were used as controls, among which the complete leaves or flowers were selected (a total of 10 different samples) ( Figure 1 A).

[0096] 1.2 Reagent preparation:

[0097] (1) Substrate solution: p-Nitrophenyl-α-D-glucopyran (PNPG) was dissolved in 0.2 M phosphate buffered saline (PBS), pH 7.0, at a concentration of 5 mg / ml.

[0098] (2) Enzyme solution: α-glucosidase was dissolved in PBS buffer (0.2 M, pH = 7.0) at a concentration of 0.2 U / mL.

[0099] (3) Stop solution: 0.1 M NaOH solution.

[0100] 2. Experimental testing:

[0101] (1) Comparison of the α-glucosidase inhibition rates of hot water extracts of each powder and dry sample (whole leaves / flowers);

[0102] (2) Compare the extraction efficiency differences between each powder and dry sample (whole leaves / flowers).

[0103] 3. Experimental steps:

[0104] 3.1 Extraction method

[0105] Using dried samples and powders of Amomum villosum and other functional plants (mulberry leaves, chrysanthemums, honeysuckle, and green tea) as raw materials, they were extracted with hot water (0.1g of dried sample and powder were dissolved in 20ml of hot water, respectively) to obtain hot water extracts as the test solutions. The extraction conditions were as follows:

[0106] Solvent: distilled water (simulating daily tea brewing);

[0107] Temperature: 90°C (close to boiling water to avoid excessive loss of volatile oil);

[0108] Time: Soak for 30 minutes (cover to prevent evaporation);

[0109] Centrifuge: 8000 rpm, 10 minutes, take the supernatant and filter (0.22 μm filter membrane).

[0110] 3.2 Determination of α-glucosidase inhibition rate

[0111] a. Sample addition and incubation: Add 20 μL of α-glucosidase solution (0.2 U / mL) to the above-extracted test solution (30 μL), mix gently, and incubate at 37°C for 5 minutes; use acarbose (Bayer, purity ≥99%) dissolved in PBS buffer (pH 7.4) as a positive control at a concentration of 5 mg / ml. Use PBS buffer (0.2 M, pH = 7.0) as a negative control, and use a blank control without adding α-glucosidase (see Table 1 and Table 2 for details). Figure 1 C);

[0112] b. Initiate the reaction: After incubation, add 20 μL of PNPG substrate solution, mix well, and continue incubation at 37°C for 15 minutes;

[0113] c. Stop the reaction: add 40 μL 0.1 M NaOH to stop the reaction;

[0114] d. Absorbance measurement: The absorbance of each well at OD405nm was measured using a multifunctional microplate reader (405nm wavelength), and the inhibition rate was calculated as follows: Inhibition rate (%) = (1-absorbance of negative control group / absorbance of experimental group) × 100%. The experiment was repeated five times.

[0115] Table 1

[0116] Hole Type Sample content illustrate Sample hole 20ul enzyme + 30ul test solution Test compound Positive control 20ul enzyme + 30ul acarbose solution Verify the effectiveness of the experiment Negative control 20ul enzyme + 30ul PBS Eliminate solvent influence Blank control 20ulPBS+30ul test solution Subtract background absorbance

[0117] In the α-glucosidase inhibition experiment, using acarbose dissolved in PBS buffer (pH 7.4) as a positive control, the inhibition rates of the tested samples (powders) were ranked from highest to lowest: green tea > tsaoko flower > chrysanthemum > mulberry leaf > acarbose > honeysuckle. This indicates that tsaoko flower has a significant blood sugar-lowering effect (Table 2).

[0118] Table 2 α-glucosidase inhibition rate

[0119]

[0120]

[0121] Example 5 Identification of compounds inhibiting α-glucosidase activity in Amomum villosum flowers

[0122] According to the above experimental results, the Amomum villosum flower contains ingredients that can inhibit the activity of α-glucosidase. In order to further determine its active ingredients, the following experiments were conducted:

[0123] 1. Extraction and Isolation of Active Compounds

[0124] (1) 95v / v% ethanol extraction

[0125] The tsaoko flower was naturally dried, ground into powder, sieved (60 mesh), and then added to a 95v / v% ethanol solution and fully soaked for 3 days for extraction. The solid-liquid ratio was 1g:10ml. During the soaking period, the ethanol was shaken once every 8 hours to allow the ethanol to fully contact with the tsaoko flower powder. After extraction, the tsaoko flower was filtered and concentrated by rotary evaporation at 40°C to obtain the tsaoko flower ethanol extract.

[0126] (2) Extraction

[0127] The obtained Amomum villosum flower ethanol extract was sequentially added with water-insoluble organic reagents petroleum ether and ethyl acetate for extraction. The petroleum ether was first extracted four times to obtain a petroleum ether layer extract and an aqueous layer extract. The obtained petroleum ether layer extract was then extracted three times with ethyl acetate to obtain an ethyl acetate layer extract and an aqueous layer extract.

[0128] (3) Separation

[0129] The ethyl acetate extract of Amomum villosum flower (176.8 g) was separated using a silica gel column chromatography column. The weight ratio of sample mixing silica gel (100-200 mesh) to sample was 1:1, and the weight ratio of normal phase separation silica gel (300-400 mesh) to sample was 10:1. The column was dry packed (14×100 cm) and dry loaded with sample. Gradient elution was performed with dichloromethane / methanol in different ratios (100:0-0:100, v / v). 2 L was used as a fraction. All fractions were then subjected to TLC spotting. The same fractions were combined and concentrated under reduced pressure to obtain a total of 21 components, named Fr.A1 to Fr.A21.

[0130] Component Fr.A10 (dichloromethane:methanol = 100:10) was subjected to isocratic elution with a dichloromethane:methanol (v / v) ratio of 1:1. TLC spot plate analysis and pooling of similar fractions yielded eight components, designated Fr.A10B1 to Fr.A10B8. Component Fr.A10B6 was then separated by HPLC using a semi-preparative column (G1311A-1100HP, Agilent, USA). The column was 9.4 mm x 25 cm, 4 μm; the HPLC absorption wavelength was 280 nm; the mobile phase was methanol / water; the elution gradient was: 30% methanol (0-5 min); 30-100% methanol (5-30 min); 100% methanol (30-40 min); the flow rate was 3 mL / min. The peak collected at 25 min was concentrated by nitrogen purge to obtain compound 1 (105.6 mg); the peak collected at 35 min was concentrated by nitrogen purge to obtain compound 2 (33.9 mg);

[0131] Component Fr.A11 (dichloromethane:methanol = 100:15) was subjected to isocratic elution with a dichloromethane:methanol ratio of 1:1 (v / v). Similar fractions were combined by TLC spot plate analysis to yield 15 components, designated Fr.A11B1 to Fr.A11B15. Component Fr.A11B13 was then separated by preparative HPLC (G1311A-1100HP, Agilent, USA) using a semi-preparative column (9.4 mm x 25 cm, 4 μm) at a wavelength of 280 nm. The mobile phase was methanol / water with an elution gradient of: 30% methanol (0-5 min); 30-100% methanol (5-30 min); and 100% methanol (30-40 min) at a flow rate of 3 mL / min. The peak collected at the 20th minute was concentrated with nitrogen purge to yield compound 3 (56.1 mg).

[0132] Compound 1: Yellow oil, easily soluble in methanol and chloroform, with characteristic UV absorption at 280 nm. High-resolution mass spectrometry (HR-ESI-MS) data showed that the quasi-molecular ion peak of the compound was m / z 313.1443 [MH]-

[0133] (calculated C19H21O4, m / z 313.1440[MH]-), its molecular formula is inferred to be C 19 H 22 O4, unsaturation is 9. 1H NMR (600MHz, MeOD) δ 6.99 (m, 4H), 6.69 (m, 4H), 4.00 (dq, J = 8.1, 6.3Hz, 1H), 3.78 (m, 1H), 2.72 (m, 4H), 2.63 (dt, J = 15.1, 6.5Hz, 2H), 2.55 (tt, J = 12.4, 6.3Hz, 2H), 2.50 (dd, J = 15.8, 4.5Hz, 2H), 1.66 (m, 2H); 13C-NMR (151MHz, MeOD): δ 210.02 (C-5), 154.40 (C-11, C-11'), 132.28 (C-8, C-8'), 131.27 (C-13, C-13'), 128.31 (C-9, C-9'), 114.16 (C-10, C-12, C-10', C-12'), 68.96 (C-3), 47.04 (C-4), 44.37 (C-6), 38.71 (C-2), 30.17 (C-1), 21.37 (C-7). By MS ( Figure 3 ), 1 H-NMR ( Figure 4 )and 13 C-NMR ( Figure 5) data confirmed the structure to be: (-)-1,7-bis(p-hydroxyphenyl)-5-hydroxy-3-heptanone ((-)-1,7-bis(p-hydroxyphenyl)-5-hydroxy-3-heptanone). Its structural formula is as follows:

[0134]

[0135] Compound 2: Pale yellow oil, easily soluble in methanol and chloroform, with characteristic UV absorption at 280 nm. High-resolution mass spectrometry (HR-ESI-MS) data showed that the quasi-molecular ion peak of the compound was m / z 357.1711[MH]-

[0136] (calculated C21H25O5, m / z 357.1702[MH]-), its molecular formula is inferred to be C 21 H 26 O5, the unsaturation degree is 9. 1H NMR (600MHz, MeOD) δ 6.96 (m, 4H), 6.70 (m, 4H), 5.07 (m, 1H), 3.52 (ddd, J = 12.5, 7.6, 3.1Hz, 1H), 2.76 (m, 1H), 2.77 (m, 1H), 2.64 (m, 3H), 2.51 (m, 1H), 1.96 (d, J = 18.9Hz, 3H), 1.81 (m, 2H), 1.72 (tt, J = 16.3, 5.0Hz, 1H), 1.65 (m, 2H), 1.60 (m, 1H). 1 H-NMR data ( Figure 6 ) was identified as 3-acetoxy-1,7-bis(4-hydroxyphenyl)-3,5-heptanediol. Its structural formula is as follows:

[0137]

[0138] Compound 3: Pale yellow oil, easily soluble in methanol and chloroform, with characteristic UV absorption at 280 nm. High-resolution mass spectrometry (HR-ESI-MS) data showed that the quasi-molecular ion peak of the compound was m / z 315.1664 [MH]-

[0139] (calculated C19H23O4, m / z 315.1675[MH]-), its molecular formula is inferred to be C 19 H 24O4, unsaturation degree is 8. 1H NMR (600 MHz, MeOD) δ 6.99 (m, 4H), 6.66 (m, 4H), 3.73 (m, 2H), 2.53 (m, 2H), 1.68 (m, 2H), 1.53 (m, 1H); 13C NMR (151 MHz, MeOD) δ 156.61 (C-11, C-11'), 134.70 (C-8, C-8'), 130.59 (C-9, C-13, C-9', C-13'), 116.39 (C-10, C-12, C-10', C-12'), 71.22 (C-3, C-5), 45.10 (C-2, C-6), 41.14 (C-4), 32.12 (C-1, C-7). 1 H-NMR ( Figure 7 )and 13 C-NMR ( Figure 8 ) data, identified as 3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)heptane. Its structural formula is as follows:

[0140]

[0141] 2. UPLC-QTOF-MS analysis of different parts of Amomum villosum

[0142] (1) Preparation of test solution

[0143] Freeze-dried samples of tsaoko flowers, kernels, peels, roots, stems, leaves, and tubers were placed in a mortar, added with liquid nitrogen, and ground into powder. 250 mg of each sample was weighed into a capped vial, 5 mL of chromatographic-grade methanol (containing internal standard: 6,7-dihydroxycoumarin, 10 ppm) was added, and the sample was ultrasonicated in an ice bath for 60 min. After centrifugation, the supernatant was aspirated and diluted to a final concentration of 50 mg / mL (internal standard 10 ppm). The sample was filtered through a 0.22 μm organic phase filter membrane and placed in a 2 mL liquid phase sample vial for LC-MS analysis.

[0144] (2) Liquid chromatography-mass spectrometry conditions

[0145] Chromatographic conditions: Waters ACQUITY BEHC18 chromatographic column (2.1 x 100 mm, 1.70 μm); mobile phase: A: 0.1% (v / v) formic acid-water solution, D: 0.1% (v / v) formic acid-acetonitrile solution, gradient elution program: 0-1.0 min, 10%-15% D; 1.0-2.0 min, 15%-30% D; 2.0-4.5 min, 30%-50% D; 4.5-7.0 min, 50%-80% D; 7.0-9.0 min, 80%-98% D; 9.0-11.5 min, 98% D; 11.5-12.0 min, 98%-10% D; 12.0-14.0 min, 10% D; flow rate, 0.3 mL / min; column temperature, 40°C; injection volume, 2 μL.

[0146] Mass spectrometry conditions: Mass spectrometry detection consisted of a Xevo G2 QTOF equipped with an electrospray ionization (ESI) source, and the mass spectrometry software was MassLynx V4.1. The mass scan range was 100–1000 m / z in both positive and negative ion modes. The capillary voltage was set to 3000 V in positive ion mode and 1500 V in negative ion mode. Argon was used as the collision gas, and nitrogen was used as the desolvation gas and cone gas, with flow rates set to 600 L / h and 20 L / h, respectively. The ion source temperature and desolvation temperature were maintained at 120°C and 400°C, respectively. The scan time was set to 0.5 s, and positive and negative ion data were collected simultaneously in MSE mode.

[0147] The relative peak area contents of the three compounds in different tissues of Tsaoko are shown in Table 3. It can be seen that the contents of these three compounds in Tsaoko flowers are significant. Figure 9 As shown, the total ion map is as follows Figure 10 shown.

[0148] Table 3 Peak areas of three compounds in different tissues of Amomum villosum

[0149] compounds 1 2 3 Retention time / molecular weight 3.25_313.144 3.82_357.171 3.05_315.162 flowers 1021567 1644642 2148819 leaf 2820 2945 / pericarp 56331 9795 358773 Tuber 9609 744 14328 stem 80818 1700 201680 Nuts (seed) 5520 / 25395 root / 642 /

[0150] 2. Determination of the inhibition rate of the diphenylheptane compounds of the present invention on α-glucosidase

[0151] The above-extracted compounds 1, 2, and 3 are all diphenylheptane compounds. To determine their inhibitory effects on α-glucosidase, the following test was performed:

[0152] (1) p-Nitrophenyl-α-D-glucopyran was dissolved as a substrate in phosphate buffered saline (PBS) (pH = 7.0, 0.2 M) to obtain a 5 mg / mL PNPG solution; α-glucosidase was dissolved in PBS (pH = 7.0, 0.2 M) to a final concentration of 0.2 U / mL; the test compound was dissolved in 1 mL of methanol (MeOH) solution to prepare a concentration gradient of the test compound solution.

[0153] (2) Acarbose (Bayer) dissolved in PBS (pH = 7.0, 0.2 M) was used as a positive control. 20 μL of 0.2 U / mL α-glucosidase and 30 μL of the test compound (concentrations of 50, 25, 12.5, 6.25, and 3.175 μg / mL, respectively) were sequentially added to a 96-well plate and mixed, with three replicates for each sample. The reaction was incubated at 37°C for 5 min, and 20 μL of PNPG was added to initiate the reaction. The reaction was incubated at 37°C for 15 min, and 40 μL of 0.1 M NaOH was added to stop the reaction.

[0154] (3) The absorbance was recorded at 405 nm using a multifunctional microplate reader (Epoch, Bio Tek, USA). A negative control was prepared using the same method as the experimental method, except that a mixture of MeOH and PBS (50:50, v / v) was added instead of the sample. PBS phosphate buffer was used instead of α-glucosidase, and the blank was prepared using the same method. Inhibition rate (%) = (D blank - D sample) / D blank × 100%. All data were analyzed for variance using SPSS 27.0.1, and graphs were plotted using Graphpad software.

[0155] The experiment found that (-)-1,7-bis(p-hydroxyphenyl)-5-hydroxy-3-heptanone, 3-acetoxy-1,7-bis(4-hydroxyphenyl)-3,5-heptane and 3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)heptane in the tsaoko flower can significantly inhibit the activity of α-glucosidase, among which (-)-1,7-bis(p-hydroxyphenyl)-5-hydroxy-3-heptanone has the best inhibitory effect. Figure 2 and Table 4 ).

[0156] Table 4 Half Inhibitory Concentration for α-glucosidase

[0157]

[0158]

[0159] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of a diphenylheptane compound in the preparation of a product for inhibiting α-glucosidase activity and / or lowering blood sugar, characterized in that: The diphenylheptane compound is at least one of (-)-1,7-bis(p-hydroxyphenyl)-5-hydroxy-3-heptanone, 3-acetoxy-1,7-bis(4-hydroxyphenyl)-3,5-heptanediol and 3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)heptane, and their structural formulas are shown in Formula I, Formula II and Formula III, respectively:

2. The use according to claim 1, characterized in that: The product for inhibiting α-glucosidase activity includes drugs or functional foods that inhibit α-glucosidase activity; The blood sugar lowering products include blood sugar lowering drugs or functional foods that assist in lowering blood sugar.

3. The use according to claim 2, characterized in that: The product for inhibiting α-glucosidase activity is an α-glucosidase inhibitor.

4. A method for separating and extracting the diphenylheptane compounds according to claim 1 from tsaoko flowers and / or tsaoko flower tea, characterized in that: The specific steps are as follows: (1) Extraction with 95% ethanol solution ① Dry the tsaoko flowers naturally, grind them into powder, and sieve them to obtain tsaoko flower powder; ② After screening the tsaoko flowers, pick the unopened inflorescences, freeze-dry and / or hot-air dry until the moisture content is ≤15%, grind into powder, and sieve to obtain tsaoko flower tea; ③ Add 95% by volume ethanol solution to the tsaoko flower powder and / or tsaoko flower tea, fully soak and extract, filter, and concentrate to obtain tsaoko flower ethanol extract; (2) Extraction with petroleum ether and ethyl acetate The tsaoko flower ethanol extract obtained in step (1) ③ is first extracted with petroleum ether to obtain the petroleum ether layer extract, and then extracted with ethyl acetate to obtain the ethyl acetate layer extract; (3) Silica gel column separation ④ The ethyl acetate extract obtained in step (2) was separated by a silica gel column chromatography, dry-loaded, and then gradient eluted with dichloromethane and methanol, with the methanol concentration ranging from 0% to 100%. Each gradient was taken as a fraction of 2 L, and all fractions were subjected to TLC spotting. The same fractions were combined and concentrated under reduced pressure to obtain a total of 21 fractions Fr.A1 to Fr.A21; ⑤ The Fr.A10 fraction with an elution concentration of dichloromethane:methanol = 100:10 was eluted through a gel column with dichloromethane and methanol in a volume ratio of 1:

1. Similar components were combined by TLC spot plate analysis to obtain a total of 8 components Fr.A10B1 to Fr.A10B8. Component Fr.A10B6 was then preparatively separated by HPLC using methanol / water as the mobile phase with a methanol concentration ranging from 30% to 100%, and (-)-1,7-bis(p-hydroxyphenyl)-5-hydroxy-3-heptanone and 3-acetoxy-1,7-bis(4-hydroxyphenyl)-3,5-heptanediol were collected. ⑥ The Fr.A11 fraction with an elution concentration of dichloromethane:methanol = 100:15 was eluted through a gel column with the eluent being dichloromethane and methanol in a volume ratio of 1:

1. Similar components were combined by TLC spot plate analysis to obtain a total of 15 components Fr.A11B1 to Fr.A11B15; component Fr.A11B13 was then preparatively separated by HPLC using methanol / water as the mobile phase with a methanol concentration ranging from 30% to 100%, and 3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)heptane was collected.

5. The method according to claim 4, characterized in that: In step (3) ⑤, HPLC separation is achieved by the following steps: chromatographic column: semi-preparative column; model: 9.4 mm*25 cm, 4 um; HPLC absorption wavelength: 280 nm; methanol / water as mobile phase, elution gradient: 0-5 min, 30% methanol; 5-30 min, 30-100% methanol, 30-40 min, 100% methanol; flow rate 3 mL / min; the peak collected at 25 min was concentrated by nitrogen blow to obtain compound 1, i.e., (-)-1,7-bis(p-hydroxyphenyl)-5-hydroxy-3-heptanone; the peak collected at 35 min was concentrated by nitrogen blow to obtain compound 2, i.e., 3-acetoxy-1,7-bis(4-hydroxyphenyl)-3,5-heptanediol; In step (3) ⑥, HPLC separation is achieved by the following steps: chromatographic column: semi-preparative column; model: 9.4 mm*25 cm, 4 um; HPLC absorption wavelength: 280 nm; methanol / water as mobile phase, elution gradient: 0-5 min, 30% methanol; 5-30 min, 30-100% methanol; flow rate 3 mL / min; the peak collected at 20 min was concentrated by nitrogen blowdown to obtain compound 3, i.e., 3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)heptane.

6. The method according to claim 4, characterized in that: In step (1) ③, the material-liquid ratio of tsaoko flower powder or tsaoko flower tea to ethanol solution is 1 g: 5-10 mL; The soaking time in step (1) ③ is 2 to 4 days; In step (3) ④, during the silica gel chromatography column separation, the weight ratio of the ethyl acetate layer extract to the 100-200 mesh sample mixing silica gel is 1:1, and the weight ratio to the 300-400 mesh normal phase separation silica gel is 1:

10.

7. A method for preparing tsaoko flower tea, characterized in that: The method specifically comprises the following steps: selecting tsaoko flowers picked on a sunny day, screening them, picking their unopened inflorescences, freeze-drying them and / or hot-air drying them to a moisture content of ≤15%, grinding them into powder, sieving them, and obtaining tsaoko flower tea.

8. The preparation method according to claim 7, characterized in that: The tsaoko flower buds are placed in a ziplock bag and stored in dry ice at -40±5°C; The freeze drying is achieved by the following steps: precooling at -40±5°C, and then vacuum drying to a water content of ≤15%; The hot air drying is achieved by the following steps: using 85±5°C hot air circulation drying to a moisture content of ≤15%.

9. A tsaoko flower tea, characterized by: It is prepared by the method according to any one of claims 7 or 8.

10. Use of the tsaoko flower tea according to claim 9 in the preparation of products for inhibiting α-glucosidase activity and / or lowering blood sugar.

Images