Preparation method of hesperidin-copper complex

Abstract

The invention discloses a preparation method of a hesperidin-copper complex, which comprises the following steps: fully dissolving copper chloride dihydrate and hesperidin solid (the purity needs to reach more than 95%) in 70% methanol according to the molar ratio of 1: 2 to obtain 0.004 mol / L methanol solution of hesperidin and copper chloride, and fully mixing; the pH of the mixed solution is adjusted to 8.5, the mixed solution is placed in a constant-temperature water bath kettle at the temperature of 48 DEG C to be subjected to water bath for 1 h, and a methanol solution of the hesperidin-copper complex is obtained. Culturing the solution at room temperature for 24 hours, and evaporating and concentrating the solution to 1 / 5 of the original volume by using a rotary evaporator; and heating and drying the concentrated solution in a constant-temperature oven to obtain the hesperidin-copper complex. According to the preparation method, a hesperidin solid is used as a starting material, a 70% methanol solution is used as a solvent, a heating catalytic reaction is performed under the alkaline condition, the reaction product reacts with copper chloride dihydrate to generate the hesperidin-copper complex, the reaction condition is simple, and a hesperidin-copper complex solid can be obtained after methanol and water are evaporated.

Description

Technical Field

[0001] The invention relates to the field of extraction and preparation of natural plant components, and in particular to a method for preparing a hesperidin-copper complex. Background Art

[0002] The research of traditional Chinese medicine is from clinic to laboratory and then to clinic, with more emphasis on inheritance and efficacy. On the basis of inheritance, it continuously develops and innovates, with curing diseases and saving lives as the primary task. Modern Chinese medicine developed from traditional Chinese medicine pays more attention to the development of innovation, new prescriptions, new drugs, new processes, new substances, new ingredients, new structures, new mechanisms of action, and pays more attention to the synchronous development with international standards. The most important signs of the modernization of Chinese medicine are the digitization and repeatability of Chinese medicine. Only in this way can the quality of Chinese medicine be stable and controllable. In the specific research and practice process of the basic research on the effective substances of Chinese medicine, the traditional research theories and concepts should be continued to be inherited. In the process of discovering effective substances, it is necessary to continuously innovate and integrate the theories and methods of Chinese and Western medicine and modern multidisciplinary disciplines, and carry out in-depth research on the basic research of the effective substances of Chinese medicine, so as to create more new situations for truly revealing the basic research of the effective substances of Chinese medicine and natural medicine, and develop a batch of new drugs that can control or cure major difficult diseases in the world today.

[0003] Hesperidin is also known as tangerine peel glycoside, which is 3,5,7-trihydroxy-4'-methoxyflavanone-7-rutinoside (C 28 H 34 O 15 ), melting point 258 ~ 262 ℃ Hesperidin has antioxidant properties, enhances capillary toughness, shortens bleeding time, reduces cholesterol, has anti-inflammatory and analgesic effects, protects blood vessels, and has certain anti-cancer effects. It can be used as a natural food pigment, natural antioxidant or natural flavor enhancer in food. Hesperidin is widely found in the peel of citrus species of Rutaceae, and the use of discarded peels can not only avoid the huge waste of resources, but also prevent environmental pollution. However, the poor water solubility of hesperidin greatly affects its utilization rate in vivo. It is reported that the water solubility and biological activity of hesperidin metal complexes are significantly improved compared with hesperidin. Hesperidin-Cu (Ⅱ) complex is a new type of drug. The complex has obvious antibacterial and superoxide scavenging activities. After hesperidin is combined with Cu (Ⅱ), its anti-inflammatory and edema effects are enhanced, and it can also promote the propulsion of gastrointestinal smooth muscle in mice. In addition, taking hesperidin or its copper complex can increase the vitamin C content in the adrenal glands of guinea pigs with vitamin C deficiency, and the effect of taking its copper complex is better than that of hesperidin. Hesperidin-Cu (Ⅱ) complex has obvious antibacterial and superoxide scavenging activities, and has therapeutic effects on rheumatic fever, rheumatoid arthritis and other degenerative diseases.

[0004] Existing methods for extracting hesperidin include: thermal extraction, alcohol extraction and acid precipitation, alkali leaching and acid precipitation, etc. Among them, the thermal extraction method has a simple process, but the product has many impurities, low purity, and low yield. The process production cycle is long, the damage to heat-sensitive components is great, the extraction rate is low, and the solvent residue is serious. The recovery rate of alkali leaching and acid precipitation is low, only about 20%. In view of the above shortcomings, an extraction method that can effectively solve the low yield, low extraction rate and poor water solubility of hesperidin is urgently needed in the industry. Summary of the invention

[0005] Since hesperidin has very poor water solubility, it leads to low bioavailability. The present invention aims to solve this problem. It has been found that hesperidin is insoluble in water and CuCl2 is insoluble in pure methanol. Therefore, hesperidin and Cu(II) are made into a complex in 70% methanol at a ratio of 2:1. The relative stability constant lgK of the complex is 8.05, which solves the solubility problem of hesperidin and improves bioavailability. In addition, H + Therefore, in the system where Cu(Ⅱ) catalyzes the decomposition of hydroperoxide to produce free radicals, hesperidin mainly prevents hydroperoxide from decomposing into free radicals by chelating Cu(Ⅱ), and the solubility of hesperidin-copper complex in water is 1800μg / mL, which is about 100 times the solubility of original hesperidin. The biological effect of hesperidin is greatly enhanced.

[0006] Since the preparation of hesperidin is difficult and costly, the present invention makes improvements thereon. The present invention uses a hesperidin-copper complex to achieve the effect of promoting the biological performance and health of livestock and poultry. The preparation process of the hesperidin-copper complex is relatively simple and low in cost, and the equipment and reaction conditions are relatively low. The preparation process of the hesperidin-copper complex: hesperidin and copper are dissolved in 70% methanol in a ratio of 2:1, placed in a 45°C water bath for 45 minutes, and the pH is adjusted to 8.5 with NaOH. Let it stand overnight, then use a rotary evaporator to concentrate the solution, and finally dry it to obtain the complex.

[0007] The present invention is achieved through the following technical solutions:

[0008] The present invention first discloses a method for preparing a hesperidin-copper complex, comprising:

[0009] (1) dissolving copper chloride dihydrate and hesperidin solid in methanol to obtain a 0.004 mol / L methanol mixed solution of hesperidin and copper chloride; copper chloride dihydrate and hesperidin solid were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. and Shanghai Yuanye Biotechnology Co., Ltd., respectively;

[0010] (2) adding sodium hydroxide dropwise to a methanol mixed solution of hesperidin and copper chloride to adjust the pH of the mixed solution to alkaline, and then placing the mixed solution in a constant temperature water bath to obtain a methanol solution of hesperidin-copper complex;

[0011] (3) incubating the methanol solution of hesperidin-copper complex at room temperature for 24 hours, and concentrating the solution by evaporation using a rotary evaporator to obtain a concentrated solution;

[0012] (4) heating and drying the concentrated solution in a constant temperature oven to obtain a solid finished product of hesperidin-copper complex.

[0013] Furthermore, in step (1), the methanol concentration is 70%; and the molar ratio of the copper chloride dihydrate to the hesperidin solid is 1:2.

[0014] Furthermore, in step (2), the pH is adjusted to 8.5; the temperature of the constant temperature water bath is 48° C., and the water bath time is 1 hour.

[0015] Furthermore, in step (3), the water bath temperature of the rotary evaporator is 55° C., and the rotation speed is 35 r / min; the evaporation is concentrated to 1 / 5 of the original volume.

[0016] Furthermore, the temperature of the constant temperature oven in step (4) does not exceed 65°C.

[0017] Furthermore, the temperature of the constant temperature oven is 50°C.

[0018] The invention also discloses a hesperidin-copper complex prepared according to any one of the above preparation methods.

[0019] The beneficial effects of the present invention are:

[0020] The method for preparing the hesperidin-copper complex of the present invention. Hesperidin is coordinated with Cu(Ⅱ) in an alkaline 70% methanol at a ratio of 2:1, which solves the problem of low solubility of hesperidin in water and insolubility of CuCl2 in pure methanol. The relative stability constant lgK of the obtained complex is 8.05. In an alkaline environment, hesperidin can destroy the -OH in the molecule to form hydrogen bonds or covalent bonds, causing the charge to disappear, reducing intermolecular repulsion, narrowing the intermolecular distance, and increasing the packing density, thereby improving the chelating ability with Cu(Ⅱ). However, if the concentration of the alkali solution is too high, it will destroy the glycosidic bond or the flavonoid nucleus, resulting in the loss of hesperidin, and will also cause Cu 2+ Precipitation, so this method uses NaOH to adjust the solution concentration to 8.5 to avoid the above problems and ensure the chelating ability of hesperidin and Cu (Ⅱ). At the same time, this method is in a water bath at 45°C for 1h, which not only accelerates the reaction rate but also does not affect the coordination ability of hesperidin. The complex solution is concentrated by rotary evaporation and oven dried to remove the toxic methanol solution to obtain hesperidin-copper complex crystals, which have high safety and are expected to become a new type of drug for the prevention and treatment of diseases.

[0021] Through research, it was found that on the one hand, copper complexes themselves have antibacterial and superoxide scavenging activities, and also have therapeutic effects on some diseases. Hesperidin combined with them can greatly improve metabolism, maintain growth performance, etc. On the other hand, H + . In the reaction system where Cu(Ⅱ) catalyzes the decomposition of hydroperoxide into free radicals, hesperidin is added to complex Cu(Ⅱ), and the purity of the hesperidin-copper complex is detected by high performance liquid chromatography (HPLC). The purity test results show that the purity reaches 83.36%. At the same time, Fourier transform infrared spectroscopy is used to perform infrared testing on hesperidin-copper. The results show that the strong bands where the carbonyl and hydroxyl groups in hesperidin are located are replaced by new wide bands in the hesperidin-copper complex. The antibacterial activity test using the hesperidin-copper complex shows that the diameter of the antibacterial zone produced by the hesperidin-copper complex on bacteria is 25.2 mm. These facts indicate that hesperidin can form a complex with copper through the carbonyl group and its adjacent hydroxyl group, which can inhibit the decomposition of hydroperoxide and enhance the antioxidant and antibacterial abilities of hesperidin. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the infrared spectrum of hesperidin-copper complex;

[0023] Figure 2 This is the infrared spectrum of hesperidin. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is described in detail below in conjunction with specific implementation methods, but does not constitute any limitation to the present invention.

[0025] Example 1

[0026] A method for preparing a hesperidin-copper complex comprises the following steps:

[0027] (1) Copper chloride dihydrate and hesperidin solid (purity must reach 95% or more) are fully dissolved in 70% methanol at a molar ratio of 1:2 to obtain a 0.004 mol / L methanol solution of hesperidin and copper chloride, and the mixture is mixed thoroughly.

[0028] (2) The pH value of the mixed solution was adjusted to 8.5 using sodium hydroxide, and the mixed solution was placed in a constant temperature water bath at 48° C. for 1 hour to obtain a methanol solution of hesperidin-copper complex.

[0029] (3) The solution was incubated at room temperature for 24 h and concentrated to 1 / 5 of its original volume using a rotary evaporator.

[0030] (4) heating and drying the concentrated solution in a constant temperature oven (temperature not exceeding 65° C.) to obtain a solid finished product of hesperidin-copper complex.

[0031] In the preparation method of the present invention, hesperidin solid is used as a starting material, 70% methanol solution is used as a solvent, and a catalytic reaction is carried out under alkaline conditions to react with copper chloride dihydrate crystals to generate a hesperidin-copper complex. The reaction conditions are simple, and the hesperidin-copper complex solid can be obtained after evaporating methanol and water.

[0032] Example 2

[0033] A method for preparing a hesperidin-copper complex comprises the following steps:

[0034] (1) Methanol and pure water were mixed in a volume ratio of 7:3 to obtain a 70% methanol solution.

[0035] (2) Weigh 0.4888 g of hesperidin solid and add it to 1 L of 70% methanol solution. Weigh 0.068 g of copper chloride dihydrate and add it to 0.5 L of 70% methanol solution and stir evenly.

[0036] (3) Slowly adding the copper chloride solution prepared in (2) into the hesperidin solution, stirring continuously, and mixing evenly to promote a full reaction between hesperidin and copper ions.

[0037] (4) Using 1 mol / L sodium hydroxide solution, the pH of the mixed solution obtained in (3) was adjusted to 8.5 to prevent hydrolysis and precipitation of copper ions.

[0038] (5) The pH-adjusted mixed solution was placed in a constant temperature water bath at 48° C. for 1 h to obtain a methanol solution of hesperidin-copper complex, which was then incubated at room temperature for 24 h.

[0039] (6) The solution was evaporated and concentrated to 1 / 5 of the original volume using a rotary evaporator (water bath temperature: 55°C, rotation speed: 35 r / min).

[0040] (7) The concentrated solution was heated and dried in a constant temperature oven at 50° C. to obtain the hesperidin-copper complex.

[0041] Example 3

[0042] The method for preparing hesperidin-copper complex by ion exchange chromatography column method comprises the following steps:

[0043] (1) Adsorption of hesperidin

[0044] D296 resin was pretreated (soaked in 4% to 5% NaCl solution overnight, then washed with water until there was no NaCl - ) for later use, take the alkaline solution of hesperidin with a concentration of 2.43 g / l and the resin in a 500 ml conical flask, seal it, and shake it in a water bath at 60°C and 40 r / min until adsorption equilibrium;

[0045] (2) Elution and coordination reaction

[0046] Use 70% methanol as solvent and prepare a concentration of 4×10 -2 mol / L CuCl2 eluent, and then the D296 resin that has adsorbed 95% (purity) hesperidin to saturation is loaded into a Φ1.6cm×44cm exchange column and statically eluted at 55°C at a speed of 3mL / min;

[0047] (3) Collection and purification

[0048] The mixture was concentrated to 30 mL at 65-70° C., refrigerated for sedimentation, filtered, and the precipitate was washed and dried to obtain a light green solid sample of hesperidin-Cu (II) complex.

[0049] Test Example 1

[0050] Identification of a Hesperidin-Copper Complex by Infrared Spectroscopy

[0051] (1) The sample of hesperidin-copper complex is pressed into tablets to meet the measurement requirements of infrared spectrometer.

[0052] (2) Before measurement, the spectrometer is background corrected to eliminate signal interference caused by the instrument or external environmental factors.

[0053] (3) Use an infrared spectrometer to scan the sample and record the absorption of infrared light of different wavelengths.

[0054] (4) Analyze the obtained spectrum.

[0055] Test Example 2

[0056] Identification of a Hesperidin-Copper Complex

[0057] (1) Weigh 2 mg of dry solid hesperidin-copper complex, grind it thoroughly in a mortar, then add 150 mg of dry KBr and grind until it is completely mixed, with a particle size of about 2 μm.

[0058] (2) Take out about 100 mg of the mixture and put it into a clean film pressing device, place it on a tablet press, and press it at a pressure of 29.4 μPa for 1 min to make a transparent thin sheet.

[0059] (3) Place the sample slice on the sample holder and insert it into the light path of the infrared spectrometer. Use a pure KBr slice as a reference and roughly measure whether the transmittance exceeds 40%. If it reaches 40%, scan the spectrum from 400 cm-1 to 650 cm-1 according to the instrument's operating instructions. If the transmittance does not reach 40%, re-press the slice.

[0060] (4) Determine the functional groups in the molecule by identifying characteristic absorption peaks; observe the changes in the absorption peaks of specific functional groups in the hesperidin molecule after coordination with copper ions; analyze the absorption peaks in the low wavenumber region (usually 400-1500 cm-1).

[0061] The IR of hesperidin and the isolated complexes are recorded in the form of wave numbers, which occupy 4000~600cm-1 (such as Figure 1 and Figure 2 There are two major differences in IR: the strong band of carbonyl group at 1640cm-1 in hesperidin is replaced by a new wide band at 1637cm-1 in the complex, and the stretching vibration of hydroxyl group (v0-H) at 3426cm-1 in hesperidin is replaced by a new wide band at 3460cm-1 in the complex. These facts indicate that hesperidin can form a complex with copper through the carbonyl group and its adjacent hydroxyl group.

[0062] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a hesperidin-copper complex, comprising: (1) dissolving copper chloride dihydrate and hesperidin solid in methanol to obtain a 0.004 mol / L methanol mixed solution of hesperidin and copper chloride; (2) adding sodium hydroxide dropwise to a methanol mixed solution of hesperidin and copper chloride to adjust the pH of the mixed solution to alkaline, and then placing the mixed solution in a constant temperature water bath to obtain a methanol solution of hesperidin-copper complex; (3) incubating the methanol solution of hesperidin-copper complex at room temperature for 24 hours, and concentrating the solution by evaporation using a rotary evaporator to obtain a concentrated solution; (4) heating and drying the concentrated solution in a constant temperature oven to obtain a solid finished product of hesperidin-copper complex.

2. The preparation method according to claim 1, wherein: The methanol concentration in step (1) is 70%; The molar ratio of the copper chloride dihydrate to the hesperidin solid is 1:

2.

3. The preparation method according to claim 1, wherein: Step (2) wherein the pH is adjusted to 8.5; The temperature of the constant temperature water bath is 48° C., and the water bath time is 1 h.

4. The preparation method according to claim 1, wherein: The water bath temperature of the rotary evaporator in step (3) is 55° C. and the rotation speed is 35 r / min; The evaporation concentration was to 1 / 5 of the original volume.

5. The preparation method according to claim 1, wherein: The temperature of the constant temperature oven in step (4) does not exceed 65°C.

6. The preparation method according to claim 5, wherein: The constant temperature oven temperature is 50°C.

7. A hesperidin-copper complex prepared according to the preparation method according to any one of claims 1 to 6.

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