CN100427112C - A kind of mulberry active extract with hypotensive effect and its preparation method and application - Google Patents

Abstract

The invention relates to a mulberry with antihypertensive effect, which belongs to the technical fields of food, health food and medicine, in particular to a mulberry or mulberry juice and mulberry active extract prepared from the mulberry and its preparation method and application. The present invention shows that mulberry fruit, mulberry fruit juice or mulberry fruit active extract has obvious antihypertensive effect on spontaneously hypertensive rats, and the mulberry fruit series of pharmaceutical preparations or series of functional foods obtained by the method of the present invention can be used to prepare prevention and treatment of hypertension And related cardiovascular and cerebrovascular diseases, as well as dizziness, tinnitus, palpitations and insomnia caused by high blood pressure, the invention has a wide range of sources of raw materials, the preparation method is easy to industrialize, and the prevention and treatment of high blood pressure and other related cardiovascular and cerebrovascular diseases have good effects.

Description

A kind of mulberry active extract with hypotensive effect and its preparation method and application

technical field

The invention belongs to the technical fields of medicine, food, and health products, and specifically relates to mulberry fruit, mulberry juice, a mulberry fruit active extract, a preparation method thereof, and applications in food, health food, and medical medicine.

technical background

Mulberry, also known as mulberry, mulberry, mulberry, etc., is the ripe fruit ears of plants such as Morus alba L., M. cathayana Hemsl., and M. nigra Linn. of the family Moraceae, which can be eaten immediately. Also used as medicine, it has the effects of nourishing blood and nourishing yin, promoting body fluid and moistening dryness, and is used for dizziness, tinnitus, palpitation, insomnia, premature graying of beard and hair, thirsty wound, blood deficiency and constipation. Mulberry contains a large amount of reducing sugars, organic acids, vitamins, amino acids, morin and phospholipids and other nutrients, as well as bioactive molecules such as flavonoids and alkaloids. Modern pharmacological studies have shown that mulberry and its preparations have obvious pharmacological activities such as regulating immunity, anti-aging, promoting hematopoietic cell growth, lowering blood sugar, lowering blood fat, and protecting the liver. However, there are no reports in the literature about the antihypertensive pharmacological activity of mulberry fruit, the effect of active parts and active ingredients, and the extraction method.

Contents of the invention

The object of the present invention is to provide mulberry, mulberry juice, mulberry active extract and preparation method thereof.

The purpose of the present invention is to provide the application of mulberry, mulberry juice and mulberry active extract in food, health food and medical medicine.

The mulberry fruit provided by the present invention is derived from the fruit of any one or variant plant of the genus Morus in the family Moraceae. For the scientific definition and category of Moraceae Morus, see Vol. 1, Vol. 1 of Flora of China (edited by the Editorial Committee of Flora of China, Beijing: Science Press, 1998) or Chronicle of Mulberry Species in China (Mulberry Industry of Chinese Academy of Agricultural Sciences Editor-in-Chief of the Research Institute, Beijing: Agricultural Press, 1993). Among them, the fruit of any one or variety of the Morus group is preferred.

The active extract of mulberry fruit provided by the present invention is the active part prepared from the above-mentioned fresh or dried mulberry fruit through the steps of extraction, purification, concentration, drying and the like.

The active substances extracted from the above active parts mainly contain oxyresveratrol (1), morin (2), dihydromorin (3), genistein (4), kaempferol (5), bergamot lactone (6) and Several kinds of flavonoid components such as anthocyanins (7-11), its structural formula is as follows (see embodiment 4,5):

The present invention also provides an optimal preparation method of the active part of mulberry fruit, the specific steps of which are: extract fresh or dried mulberry fruit multiple times with 2-20 times the weight of ethanol, methanol or acetone, combine the extracts, centrifuge, Filter and purify.

In the present invention, the mature mulberries are extracted 1-4 times with 20-100 wt% ethanol, methanol or acetone, and the effect is better for 1.5-3.0 hours each time.

The filtrate in the above extraction is adsorbed by a low-polarity or non-polar macroporous adsorption resin (such as D101, Diaion HP-20, etc.), and after all the extracts pass through the resin column, first use 2-3 times the column bed volume of the pure Rinse with water, then rinse with 20wt% ethanol of 2-3 times the volume of the column bed, then elute with 50-75wt% ethanol, collect the eluate, and the content in the eluate is the active extract of mulberry , can be made into various preparations or compound preparations.

In the present invention, the eluent obtained from the macroporous resin is vacuum-recovered to obtain a concentrated solution after ethanol is recovered. After the eluate is concentrated, it is directly formulated into liquid preparations such as oral liquid, or is prepared into powder by spraying, vacuum or freeze-drying, for the preparation of solid preparations such as tablets, capsules, and mixtures (see Examples 3, 8 ,9).

In the present invention, the obtained concentrate is prepared into powder by spraying, vacuum or freeze-drying.

In the present invention, fresh mulberries can be brewed to prepare mulberry preserves, which can be eaten directly.

The present invention also provides an optimal preparation process of mulberry juice. The specific method and steps are as follows: take the fresh mulberry that has just been picked, first use a press to squeeze the fruit juice 1, and disperse the residue with 3 times the weight of pure water and fully stir After pressing and filtering, collect the juice 2. Combine Juice 1 and Juice 2, stir well and add a certain amount of clarifier, such as 101 juice clarifier, ZTC1+1 natural clarifier, etc., continue to stir well, let it stand for more than 4 hours to make it fully clarified, and then use pressing method , suction filtration or centrifugal filtration, and the filtrate is sterilized to obtain clarified mulberry juice, which can be stored at low temperature for more than 6 months. The raw mulberry juice can be further prepared into beverage or oral liquid (see Examples 2, 6, and 7).

In the present invention, the clarifier is added after the squeezed mulberry juice is fully stirred evenly, continued to be fully stirred evenly, and left to stand for 2-4 hours to make it fully clarified. Clarifiers such as 101 fruit juice clarifier, ZTC1+1 natural clarifier, etc.

In the present invention, the clarified liquid is filtered by pressing method, suction filtration method or centrifugal filtration method, and the filtrate is sterilized to obtain clarified mulberry fruit juice.

In the present invention, mulberry fruit, mulberry fruit juice or mulberry fruit active extract can be used alone or in combination with medicine or food.

In the present invention, if the compatible compound preparation is any one of decoction, wine, granule, granule, capsule, tablet, oral liquid, syrup, and ointment, it is suitable for the needs of different groups of people.

In the present invention, the application of mulberry fruit, mulberry fruit juice and mulberry fruit active extract as preparation of medicine for treating hypertension has good effect and no side effects.

The present invention proposes the use of the mulberry fruit, mulberry juice and mulberry active extract, all of which can be used alone to prepare food, functional health food or medical medicine, and can also be combined with any other Chinese and Western medicine or food, especially with a certain It is compatible with some drugs or foods that have the functions of lowering blood pressure, lowering blood fat, nourishing blood and nourishing yin, calming liver and suppressing yang, and is used for preparing drugs, functional health foods or foods.

Drugs, functional health food and food prepared from mulberry, mulberry juice or mulberry active extract alone, or compound drugs, functional health food and food composed of mulberry, mulberry juice and mulberry active extract respectively with other Chinese and Western medicines or foods Foods, all have obvious blood pressure lowering activity, and can be used to prevent and treat hypertension and related cardiovascular and cerebrovascular diseases, as well as dizziness, tinnitus, palpitations and insomnia caused by high blood pressure.

Mulberry is used as medicine, functional health food and food. Generally, fresh fruit can be eaten directly, or it can be dried, air-dried, freeze-dried into dried mulberry, or candied into mulberry preserves, or fresh mulberry or dried mulberry can be eaten with water. After decocting and soaking in wine, take the juice and drink it. In addition, mulberry can also be used as the main ingredient, combined with other Chinese and Western medicines or foods, such as apocynum, uncaria, hawthorn, ginkgo biloba, salvia miltiorrhiza, celery, etc., to make oral liquids, capsules, tablets, granules, etc.

When preparing medicine, functional health food and food from mulberry juice, generally the raw mulberry juice can be directly diluted with purified water, wine, or edible ethanol, and a certain amount of flavoring agents such as sugar, honey, essence, etc. can be added, and then filtered, Filling, sterilizing, and preparing into simple mulberry juice or other liquid preparations such as oral liquid, syrup, etc. It can also be made into compound mulberry juice by mixing natural mulberry juice with mountain residue juice, celery juice, pear juice, horseshoe juice, orange juice, etc., or adding specific vitamins, minerals, amino acids, fatty acids, etc. in a certain proportion. Or other liquid preparations such as oral liquids, syrups, etc.

When the active extract of mulberry fruit, or the drug and food combination containing the extract, is used as food, medicine or health food, it can be made into capsules directly and with the addition of necessary auxiliary materials by methods and techniques well known to those skilled in the art , tablets, injections, granules, oral liquids, syrups, ointments, liquors, beverages, fruit juices, instant teas, granules and other preparations. When active extract of mulberry fruit of the present invention or its composition are made into tablet, the excipient that it contains has: diluent, as starch, dextrin, lactose etc.; Wetting agent or binding agent, as: water , ethanol, starch slurry, dextrin, gelatin slurry, low-substituted hydroxypropyl cellulose, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants, such as: dry starch, effervescent disintegrants, surfactants, etc.; Lubricants, such as talc, magnesium stearate, liquid paraffin, polyethylene glycol 6000 or 4000, etc. When the mulberry active extract of the present invention or its composition is made into capsules, the excipients it contains include: diluents, such as: starch, dextrin, lactose, magnesium oxide, magnesium carbonate, etc.; wetting agents or Binders, such as: water, ethanol, starch paste, dextrin paste, gelatin paste, low-substituted hydroxypropyl cellulose, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants, such as: dry starch, effervescent disintegration Antilytic agents, surfactants, etc.; and gelatin hard capsule shells or soft capsule shells. When the mulberry active extract of the present invention or its composition is made into oral liquid or beverage, the excipients it contains are: sucrose aqueous solution, flavoring agent; Suspending agent, such as hydroxymethylcellulose, polyvinylpyrrolidone , methyl cellulose, etc.; preservatives, such as paraben ethyl or methyl paraben, propylene glycol, benzoic acid, sorbitol, etc.

The product of the invention has wide sources, low cost and low toxicity. Since the mulberry fruit is edible, it can be used in combination with other foods and medicines to improve the effects of health care products and medicines. Taking animal experiments as an example, it has been shown that it has an obvious antihypertensive effect on hypertension, so it plays an important role in the prevention and auxiliary treatment of hypertension, and has a good application prospect.

Description of drawings

Fig. 1 is the HPLC chemical fingerprint of active extract of mulberry fruit.

Detailed ways

The present invention has carried out animal experiments on the above-mentioned mulberry fruit, mulberry juice and active extract of mulberry fruit, showing that the three are non-toxic and have no side effects, and have obvious antihypertensive effect on spontaneously hypertensive rats. The specific test results are as follows:

Test Example 1: Acute Toxicity Test

The results of the acute toxicity test in mice show that the maximum concentration and maximum administration volume are given to mice by intragastric administration, and the maximum administration dose is 65.0 g/kg, which is about 120 times of the clinically recommended dose.

Test Example 2: Hypotensive Effect of Mulberry Juice on Spontaneously Hypertensive Rats

(1) Purpose of the experiment

To study the effect of single oral administration and multiple oral administration for 3 consecutive days on the blood pressure of spontaneously hypertensive rats (SHR).

(2) Test material

① Test sample

Name: Mulberry Juice

Source: Natural Medicine Center of Fudan University

Batch number: SS02011

Appearance: dark brown liquid

② Test animals

Name: Spontaneously Hypertensive Rats

Gender: Male

Weight: 220---250g

Source: Shanghai Institute of Hypertension

Certificate of Qualification: No. 30-35, Shanghai Medical Experimental Animal Approval

Breeding: Breeding in positive pressure purification and ventilation animal room, room temperature 25±2°C, free access to food and water.

③Testing equipment

CRS-III computer rat heart rate and blood pressure instrument (Shanghai Institute of Hypertension); 101-2 electric thermostat (Shanghai Experimental Instrument Factory).

④Experimental method

After the test animals were purchased, the rats were put into a manometer for training for 2 weeks, and the blood pressure was measured every other day from the second week. Before the manometry, the animals were warmed in an electric heating box at 37±1°C, and after 15 minutes, the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of the rat tail artery were measured with a CRS-III computer rat heart rate and blood pressure instrument. The spontaneously hypertensive rats were equally divided into 2 groups, 8 in each group, which were the negative control group and the test drug mulberry extract group. The administration volume was 4ml/rat. Achillea gum. Once a day, for 3 consecutive days, blood pressure was measured before administration and 4 hours after administration.

(3) Experimental results

① Statistical test

Experimental data are expressed as mean ± standard deviation. Statistical test compares the average value of the difference between the time point after administration and before administration in each group with the difference at the same time point in the negative control group, and calculates the significance of the difference with a double test distribution t test between groups.

②Test results

After oral administration of the mulberry extract for the first time, the blood pressure decreased rapidly, and there was a significant antihypertensive effect in 4 hours. The systolic blood pressure and diastolic blood pressure decreased by -29.0±10.8mmHg and -35.8±9.9mmHg respectively, and they still had -23.8±9.9mmHg by 24 hours. 16.5mmHg and -26.8±20.1mmHg, compared with the negative control group, the difference is very significant (P<0.01); after 24 hours of oral administration again, the systolic blood pressure and diastolic blood pressure decreased significantly after 4 hours of administration, which were -2.8±9.2 mmHg and -3.0±12.4mmHg; but there was still a decline at 48 hours, and the differences from before the start of the test were -34.0±7.8mmHg and -35.0±11.6mmHg; when the drug was administered at 48 hours, the situation was the same as that at the 24th hour Similar to administration, blood pressure did not drop significantly within 4 hours of administration. In the negative control group, the blood pressure was basically stable within 48 hours, and there was no significant difference from that before administration. The results are shown in Table 1.

The test results show that the mulberry juice sample has obvious antihypertensive effect, and the effect lasts for a long time, but the antihypertensive effect has a certain limit, so the acute antihypertensive effect on the second and third days of administration is smaller than that of the first administration, but there is still a small amount of antihypertensive effect. Decline.

Table 1 Effect of oral administration of mulberry juice on blood pressure in spontaneously hypertensive rats

*P<0.05, material P<0.01 compared with the negative control group

Test example 3. Effect of mulberry juice on blood pressure, hemodynamics and cardiac cycle in spontaneously hypertensive rats

(1) Purpose of the experiment:

Carotid artery real-time blood pressure recording method was used to further test the effect of mulberry juice on blood pressure, hemodynamics and cardiac cycle in spontaneously hypertensive rats (SHR).

(2) Experimental principle

A male SHR with a weight of about 200-300g was taken, and after being anesthetized with pentobarbital, a cannula was directly inserted into the carotid artery, and the blood pressure curve was traced. The injection barrel connected by the cannula is connected to the multifunctional physiological recorder, and after the instrument is calibrated and started, the blood pressure, hemodynamics, cardiac cycle and other indicators of the rat are measured.

(3) Experiment content

Select 8 spontaneously hypertensive rats (SHR) aged 10-12 weeks, weighing 180-220 grams, half male and half male. Blood pressure, hemodynamics, cardiac cycle and other indicators were measured before administration. Then, every day, each rat was orally administered (gastrically) 4 ml of mulberry juice, and 4 hours after administration on the sixth day, indexes such as blood pressure, hemodynamics, and cardiac cycle were measured again. Compared with before administration.

(4) Experimental results: See Table 2.

Table 2. Effects of mulberry juice on blood pressure, hemodynamics and cardiac cycle in spontaneously hypertensive rats

The above experimental results show that after giving mulberry juice to spontaneously hypertensive rats continuously for six days, the average diastolic blood pressure can be reduced by 22.512mmHg, and the systolic blood pressure can be reduced by an average of 18.861mmHg.

(5 Conclusion

Mulberry juice has a certain antihypertensive effect on spontaneously hypertensive rats; there is no significant difference in the hemodynamic effects of spontaneously hypertensive rats; it has a shortening effect on the cardiac cycle of spontaneously hypertensive rats, but not significantly ; The heart rate acceleration of spontaneously hypertensive rats is also not obvious.

Test example 3. Effect of mulberry active extract on blood pressure in spontaneously hypertensive rats

(1) Purpose of the test

To observe and compare the therapeutic effects of the active extract of mulberry fruit (mainly flavonoids) and the impurities removed during the purification process (mainly sugars) on spontaneously hypertensive rats (SHR) after repeated oral administration.

(2) Materials and methods

① Test sample

Name: Mulberry active extract (mainly flavonoids) and impurities removed during purification (mainly sugars)

Sample preparation: 2kg of fresh mulberry fruit was extracted with 95% ethanol and concentrated in vacuo to obtain 340g of extract. Get wherein 200g carries out Diaion HP-20 resin column chromatography. Water and 20% acetone were eluted and concentrated to obtain 189.42 g of impurity parts (SS02011-t), and 70% acetone was eluted and concentrated to obtain 5.60 g of mulberry active extract (SS02011-s).

Batch number: SS02011-t, SS02011-s

Shape: light brown and dark brown solid

Preparation: Dilute samples of batch numbers SS02011-t and SS02011-s to 0.2g/ml and 0.05g/ml with distilled water.

② Test animals

Name: Spontaneously Hypertensive Rats

Gender: Male

Weight: 220---250g

Source: Shanghai Institute of Hypertension

Certificate of Qualification: No. 30-35, Shanghai Medical Experimental Animal Approval

Breeding: Breeding in positive pressure purification and ventilation animal room, room temperature 25±2°C, free access to food and water.

③Testing equipment

CRS-III computer rat heart rate and blood pressure instrument, manufactured by Shanghai Institute of Hypertension.

101-2 electric heating constant temperature box, manufactured by Shanghai Experimental Instrument Factory.

④Experimental method

After the test animals were purchased, the rats were put into a manometer for training for 2 weeks, and the blood pressure was measured every other day from the second week. Before the manometry, the animals were warmed in an electric heating box at 37±1°C, and after 15 minutes, the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of the rat tail artery were measured with a CRS-III computer rat heart rate and blood pressure instrument. The spontaneously hypertensive rats were equally divided into 2 groups, 6 rats in each group, and the administration volume was 2ml/rat. Observe the change of blood pressure before administration and the 3rd day after administration, and measure and record blood pressure before administration and 4 hours after administration.

(3) Test results

The test results are shown in Table 3-6.

The test results show that the active extract of mulberry fruit prepared according to the above method has obvious antihypertensive effect, while the impurities removed in the preparation process have no antihypertensive effect.

Table 3 Effect of mulberry impurity parts (SS02011-t) on systolic blood pressure of spontaneously hypertensive rats (n=6)

Table 4 The effect of mulberry impurity parts (SS02011-t) on the diastolic blood pressure of spontaneously hypertensive rats (n=6)

Table 5 The effect of the active fraction of mulberry fruit (SS02011-s) on the systolic blood pressure of spontaneously hypertensive rats (n=6)

Table 6 The effect of mulberry active fraction (SS02011-s) on the diastolic blood pressure of spontaneously hypertensive rats (n=6)

The preparation of embodiment 1 mulberry fruit wine

Fresh mulberry 100g, soak in 500mL of rice wine, white wine or wine according to personal preference and drink for 3 days.

The preparation of embodiment 2 mulberry juice

10Kg of fresh mulberries, first directly press with a press to obtain 6.8L of juice (1), the residue is dispersed with 40L of pure water, squeezed and filtered after fully stirring, and 40L of juice (2) is collected. Combine fruit juice (1) and fruit juice (2), add an appropriate amount of pure water to make a total of 50L, stir well, add 5% 101 fruit juice clarifier, continue to stir well, let stand for 6 hours, centrifugal filter, filtrate Obtain clarified mulberry juice after sterilization.

The preparation of embodiment 3 mulberry active fraction

2Kg of fresh mulberry fruit is extracted twice with 20Kg of 95% ethanol for 2 hours each time. The ethanol is recovered in vacuum until there is no alcohol smell. The filtrate was added to a D101 macroporous resin column (2Kg) for adsorption. After all the extracts passed through the resin column, eluted with 10Kg of water and 10Kg of 20% ethanol to remove impurities, and finally eluted with 15Kg of 75% ethanol. The eluate was concentrated and vacuum-dried to obtain 60.0 g of the active extract of mulberry fruit, with a yield of 3%.

Example 4 Separation and Identification of Chemical Components of Mulberry Active Parts

By the mulberry fruit active extract of the gained of embodiment 2, when extracting with chloroform, separate out light yellow precipitate 21.0g in the mother liquor. Get 2.5g precipitate and carry out silica gel column chromatography (C-1 column), elution with chloroform:methanol 9:1, obtain seven components, component 5 (fr.17-23) continue to carry out silica gel column chromatography (C-1 -1-1 column), and eluted with chloroform:methanol 9:1 to obtain compound 1 (493 mg) and compound 2 (180 mg).

The ethyl acetate and n-butanol fractions (32.86 g) were combined and separated repeatedly by Diaion HP-20, Toyopearl, MCI, ODS and silica gel column chromatography to obtain 4 compounds of compounds 3-6.

Compound 1 is a light yellow crystal with m/z[M] ⁺ 244 measured by EIMS, and determined by ¹ H NMR spectrum analysis with the molecular formula C ₁₄ H ₁₂ O ₄ . The following structural information can be obtained from the ¹ HNMR spectrum: 1) There are four phenolic hydroxyl signals in the low-field region, δ8.56 (1H, s), 8.37 (1H, s), and there are two inferred from δ8.16 (2H, s). The chemical environment of the two phenolic hydroxyl groups is the same; 2) δ6.89 (1H, d, J = 16.5Hz), 7.34 (1H, d, J = 16.5Hz), deduced from its coupling constant that there is a trans double bond, and it is located relatively Low field, there may be a conjugation effect with the benzene ring; 3) There are six hydrogen signals in the aromatic region, δ6.24 (1H, t, J=2.1Hz), 6.53 (2H, d, J=2.1Hz) three Aromatic hydrogen into an AB2 coupling system, δ6.39 (1H, dd, J = 2.4, δ.5Hz), 6.44 (1H, d, J = 2.4Hz), 7.41 (1H, d, J = 8.5Hz) three aromatic hydrogens into an ABX coupling system. It was inferred that the compound was a stilbene compound. After checking the ¹ H and ¹³ C NMR spectrum data by consulting literature ^[13], it was determined that compound 1 was a known compound oxyresveratrol.

Compound 2 is a yellow amorphous powder with m/z [M] ⁺ 302 measured by EIMS. By analyzing ¹ H NMR spectrum, the molecular formula is deduced to be C ₁₅ H ₁₀ O ₇ . ^{The 1} H NMR spectrum shows the following hydrogen-containing functional group information: 1) 5 phenolic hydroxyl groups, one of which forms an intramolecular hydrogen bond with the carbonyl group, appears at the lowest field δ12.70, and the other 4 phenolic hydroxyl groups are δ9.47 (4H, br s ); 2) Referring to compound 3, there is an ABX system on the benzene ring in the aromatic region δ7.49 (1H, d, J=8.6Hz), 6.30 (1H, br d, J=8.6Hz) and 6.38 (1H, br d, J=8.6Hz) and 6.38 (1H, br s); 3) Signals of 2 meta-coupled aryl protons δ6.17(1H, br s), 6.14(1H, brs). Compared with the literature ((Shanghai Institute of Materia Medica, 1981; Ternai and Markhan,?), it was determined that compound 2 was morin.

Compound 3 is a white amorphous powder with m/z [M] ⁺ 304 measured by EIMS. The molecular formula is determined to be C ₁₅ H ₁₂ O ₇ by analyzing ¹ H NMR spectrum. ¹ HNMR shows the following hydrogen-containing functional groups: 1) There is a 5-hydroxyl signal at the lowest field, which forms an intramolecular hydrogen bond with the carbonyl group, δ11.73; 2) 3 phenolic hydroxyl signal δ9.62(1H, s), 8.45(1H, s), 8.37 (1H, s); 3) ABX system with a benzene ring δ7.33 (1H, d, J = 8.3Hz), 6.44 (1H, dd, J = 1.8, 8.3Hz), 6.48 ( 1H, d, J=1.8Hz), which is the 2′, 4′ disubstituted B ring of flavonoids; 4) 2 meta-coupled aromatic protons δ5.94 (1H, d, J=1.5Hz), 5.99 (1H, d, J=1.5Hz), the 6- and 8-position hydrogens of flavonoids; 5) δ5.50 (1H, d, J=11.4Hz), 4.88 (1H, dd, J=11.4, 3.6Hz ), 4.69 (1H, br s), inferred to be the 2-position, 3-position hydrogen and 3-hydroxyl signals of dihydroflavone, judging from the coupling constants of the 2-position and 3-position hydrogen that they are 2 ortho hydrogens coupled in trans . Based on the above information, it is deduced that the compound is 2',4',3,5,7-pentahydroxydihydroflavone. After reviewing and checking literature (Shanghai Institute of Materia Medica, 1981; Wenker and Gottlieb, 1977), it was determined that compound 3 was dihydromorin.

Compound 4: light yellow amorphous powder; ¹ H NMR (acetone-d ₆ , 500MHz) δ13.04 (1H, s, OH-5), 8.17 (1H, s, H-2), 7.46 (2H, d, J=8.6Hz, H-2', 6'), 6.91 (2H, d, J=8.6Hz, H-3', 5'), 6.42 (1H, d, J=1.8Hz, H-8), 6.29 (1H, d, J=1.8Hz, H-6); EIMS m/z 270[M] ⁺ (100), 153(42), 118(14). Compound 4 was determined to be Genistein.

Compound 5: light yellow amorphous powder; ¹ H NMR (acetone-d ₆ , 500 MHz) δ12.17 (1H, s, OH-5), 9.05 (3H, br s, OH-3, 5, 7), 8.16 (2H, d, J=8.6Hz, H-2′, 6′), 7.02 (2H, d, J=8.6Hz, H-3′, 5′), 6.54 (1H, br s, H-8 ), 6.28(1H, br s, H-6); EIMS m/z 286[M] ⁺ (100), 258(12), 229(11), 213(8), 153(14), 121(38 ). Compound 5 was determined to be Kaempferol.

Compound 6: Colorless amorphous powder; ¹ H NMR (acetone-d ₆ , 500 MHz) δ8.21 (1H, d, J=10.0Hz, H-4), 7.86 (1H, d, J=2.5Hz, H-2'), 7.32 (1H, dd, J=1.0, 2.5Hz, H-3'), 7.16 (1H, d, J=1.0Hz, H-8), 6.25 (1H, d, J=10.0 Hz, H-3), 4.34 (3H, s, OCH ₃ ); ¹³ C NMR (acetone-d ₆ , 125MHz): δ160.8 (C-2), 159.3 (C-7), 153.8 (C-9 ), 150.8(C-5), 146.2(C-2'), 139.9(C-4), 113.5(C-6), 113.3(C-3), 107.0(C-10), 106.3(C-3 '), 93.9(C-8), 60.8(OCH ₃ ); EIMS m/z 216[M] ⁺ (100), 201(34), 188(11), 173(54), 145(19). Determined Compound 6 is Bergapten.

Example 5 Chemical Fingerprint Research of Mulberry Active Extract

(1) Instruments and methods:

The HPLC-MS and HPLC-DAD systems are composed of Agilent1100 series high-performance liquid chromatography (including vacuum degasser, a quaternary pump, an autosampler, and a diode array detector); ThermoFinnigan TSQ Quantum MS-MS is equipped with an ESI ion source; the control software is Xcalibur (Reversion 1.1).

The chromatographic conditions are: Agilent Zorbax SB C18 (50mm×2.1mm) column, mobile phase: acetonitrile-H ₂ O=2:98 (A), acetonitrile-H ₂ O (90:10) (B); gradient elution: 0~20min, 10%B~100%B, 20~30min, 100%B; flow: 0.2mL/min; injection volume: 5μL, total time: 30min.

The mass spectrometry parameters are as follows:

ScanTime(s) Parameter Q1 PeakWidth SprayVoltage(v) Sheathgas Aux gas CapillaryTemp(℃) Tube lens(V) SourceCID 0.5 0.7 3900 30 5 350 79 10

(2) Result analysis and judgment:

The HPLC chromatogram is shown in Figure 1. The confirmation of the corresponding compounds of each chromatographic peak in the chromatogram is mainly based on the retention time and its corresponding mass spectrum and ultraviolet spectrum characteristics, and compared with the literature (Paola Dugo et al., 2001). The analysis results are shown in Table 7.

Table 7 Chemical fingerprint characteristics of mulberry active extract

Note: pt, Petunidin; pg, Pelargonidin; pn, Peonidin; dp, Delphilidin; cy, Cyanidin; glc, glucoside; sop, sophoroside; ara, arabinoside; rut, rutinoside.

The preparation of embodiment 6 mulberry juice beverage

Prescription: mulberry juice 50L

  Honey 10L

Purified water 40L

Preparation method: Mix the 3 materials in the prescription evenly, add 100g of sodium benzoate, fully dissolve, filter and clarify, divide the clarified liquid into 250mL, and sterilize under high pressure to obtain the product. Specifications: Each liter is equivalent to 100g fresh mulberry, and each bottle (250mL) is equivalent to 25g fresh mulberry.

The preparation of embodiment 7 compound mulberry beverage

Prescription: mulberry juice 50L

Hawthorn juice 50L (pressed from 10Kg fresh hawthorn with water)

  Honey 20L

Purified water 80L

Preparation method: Mix the 3 materials in the prescription evenly, add 200g of sodium benzoate, filter and clarify after fully dissolving, divide the clarified solution into 250mL, and sterilize under high pressure to obtain the product. Specifications: Each liter is equivalent to 50g fresh mulberry and 50g fresh hawthorn, and each bottle (250mL) is equivalent to 25g fresh mulberry and 25g fresh hawthorn.

The preparation of embodiment 8 mulberry capsules

Prescription: Mulberry Active Extract 500g

Preparation method: Grind the above materials into fine powder, put into No. 0 capsules, a total of 1000 capsules, to obtain. Each capsule contains 0.5g of mulberry active extract.

Claims (11)

1. A mulberry active extract with hypotensive effect, characterized in that the extract contains oxyresveratrol, morin, dihydromorin, genistein, kaempferol, bergamot lactone and anthocyanin flavonoids Element. 2. The preparation method of the active extract according to claim 1, characterized in that the mature mulberries are extracted with 2-20 times the weight of ethanol, methanol or acetone, the extracts are combined, centrifuged, filtered and purified. 3. The preparation method of the active extract according to claim 2, characterized in that the mature mulberries are extracted with 20-100 wt% ethanol, methanol or acetone for 1-4 times, each time for 1.5-3.0 hours. 4. The preparation method according to claim 2, characterized in that the filtrate is purified through a low-polarity or non-polar macroporous adsorption resin, and the specific steps are: after all the extracts pass through the resin column, first use pure Rinse with water, and collect the eluate with ethanol. 5. The preparation method according to claim 4, characterized in that the eluate obtained from the macroporous resin is vacuum-recovered to obtain ethanol to obtain a concentrated solution. 6. The preparation method according to claim 5, characterized in that the obtained concentrated solution is directly formulated into a liquid preparation. 7. The preparation method according to claim 5, characterized in that the obtained concentrate is prepared into powder by spraying, vacuum or freeze-drying. 8. The preparation method of the active extract according to claim 1, characterized in that the mulberries are squeezed with a press to collect the fruit juice, and the residue is dispersed with 1-4 times the weight of pure water or alcohol-containing solution and fully stirred and then squeezed and filtered , collect the fruit juice, combine the two fruit juices to obtain mulberry juice. 9. The preparation method of mulberry juice according to claim 8, characterized in that, after the squeezed mulberry juice is fully stirred evenly, a clarifying agent is added, continued to fully stir evenly, and left standing for 2-4 hours to make it fully clarified. 10. The method for preparing mulberry juice according to claim 8 or 9, characterized in that the clarified liquid is filtered by pressing, suction filtration or centrifugal filtration, and the filtrate is sterilized to obtain clarified raw mulberry juice. 11. The use of the active extract of mulberry fruit according to claim 1, 2 or 9 as a drug for treating hypertension.