Extraction method of apocynum venetum flavonoid compound

Abstract

The invention discloses a novel method for extracting an apocynum venetum flavonoid compound, which comprises the following steps of: mixing apocynum venetum powder which is crushed and sieved by a 60-mesh sieve with ethanol with a certain concentration, putting into a high-temperature and high-pressure reaction kettle, and reacting at a certain temperature and pressure; after the reaction is completed, the cooling coil is communicated with cold water to cool the reaction kettle, and after the thermocouple thermometer displays that the temperature is lower than the normal-pressure boiling point of ethanol, pressure is released, and discharged materials are collected. The collected feed liquid is subjected to centrifugation and suction filtration, filtrate is collected, and flavonoid compound filtrate is obtained. And measuring the absorbance of the compound by using a NaNO2-Al (NO3)-NaOH colorimetric method and an ultraviolet spectrophotometer, so as to obtain the extraction rate of the flavonoid compound. The novel method for extracting the flavonoid compounds in the apocynum venetum through the subcritical fluid extraction method has the advantages of being high in extraction efficiency, simple in process, green in process, convenient to operate, low in equipment investment cost and the like. The active ingredients can be maintained to the greatest extent, and the method has very important practical significance on actual industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural plant chemical extraction, and particularly relates to a new method for extracting flavonoid compounds from Apocynum venetum. Background Art

[0002] With the acceleration of the modern life rhythm, health care has become a hot topic pursued by people. It is recorded in "Compendium of Materia Medica" that "Apocynum venetum tastes sweet, bitter and cool, and belongs to the liver meridian". Apocynum venetum has high medicinal value and can clear heat and reduce fire, improve sleep, treat edema, lower blood pressure and blood lipid, treat heart failure, delay aging, resist radiation, etc. Relevant pharmacological studies have shown that Apocynum venetum contains various trace elements and drug components required by the human body, such as selenium, flavonoid compounds, tannins (tannins), catechins, fatty acid alcohol esters, alcohols, coumarins, organic acids, sugars, amino acids, mineral elements, etc. Clinical research has found that the root decoction of Apocynum venetum has the effects of slowing down the heart rate, diuresis, and strengthening the heart, and can be developed into a drug for treating heart diseases, nephritis and other related diseases. After its tender leaves are steamed, fried, kneaded and brewed as tea, it not only has pharmacological activities such as lowering blood pressure, blood lipid, blood sugar, and antidepressant, but also has health care functions such as anti-allergy, anti-cancer, anti-radiation, antioxidant and anti-aging.

[0003] The main active ingredient in Apocynum venetum is flavonoid compounds. Research has proved that flavonoid compounds can play a protective role in different diseases. Generally, flavonoid compounds have a variety of pharmacological activities, including preventing and treating cardiovascular and cerebrovascular diseases. For example, they can reduce the brittleness of blood vessels, improve the permeability of blood vessels, lower blood lipid and cholesterol, prevent and treat senile hypertension, cerebral hemorrhage, coronary heart disease, angina pectoris, dilate coronary blood vessels, and increase coronary blood flow. Many flavonoid components have activities of relieving cough, reducing phlegm, relieving asthma and antibacterial, and at the same time have the effects of protecting the liver, detoxifying the liver, antifungal, treating acute and chronic hepatitis, liver cirrhosis, and anti-free radicals and antioxidant, and are widely used in the fields of medicine, health care and food, etc.

[0004] At present, the extraction of flavonoid compounds mainly adopts methods such as warm soaking method, solvent extraction method, microwave treatment method, ultrasonic treatment method, supercritical fluid extraction method, etc. Although the traditional solvent extraction method has a simple process, its extraction efficiency is not high, the separation is difficult and the extraction time is long. Although the emerging ultrasonic extraction method and microwave extraction method have higher extraction efficiency, the impurities in the extraction liquid are more, and the requirements for equipment are high. The enzymatic extraction method is green and environmentally friendly, but the enzyme consumption is large, the cost is high, and the extraction time is long. The supercritical fluid extraction method has high extraction efficiency and good separation effect, but it has high requirements for equipment and there are certain safety hazards. The subcritical fluid extraction method not only has high extraction efficiency, good separation effect, low requirements for reaction conditions and equipment, but also is green and environmentally friendly and has low cost.

[0005] Subcritical is the state condition of the existence of substances, referring to certain substances that exist in a fluid form at a temperature higher than their boiling point but lower than the critical temperature and at a pressure lower than their critical pressure. Fluids in the subcritical state can have properties similar to supercritical fluids at lower pressures; their density is close to that of liquids, with strong dissolution ability; their viscosity is close to that of gases, with fast mass transfer speed; their diffusion coefficient is between that of gases and liquids, with strong penetration ability; the molecular polarity changes significantly, from polar to non-polar, and it can be used to extract non-polar and weakly polar substances. The subcritical fluid extraction technology uses an organic solvent fluid in the subcritical state as the extraction solvent, and utilizes the high solubility, high permeability, and weak polarity of the fluid to extract the target extract in the material in a sealed and low-pressure environment. The subcritical fluid extraction technology has low working temperature, high efficiency, environmental protection, energy saving, no residue, and almost no damage to the target extract, and has now become an effective means to obtain high-quality flavonoids; and compared with supercritical extraction, the working pressure is very low, the equipment requirements are not high, and it is easy to scale up for industrialization. Summary of the Invention

[0006] The present invention aims to provide a new method for efficiently extracting flavonoids from Apocynum venetum.

[0007] The present invention mainly uses the subcritical fluid extraction technology to extract flavonoids from Apocynum venetum, and the specific steps are as follows:

[0008] 1. Cleaning: Simply clean the Apocynum venetum to remove floating dust.

[0009] 2. Drying: Place the Apocynum venetum in an oven and dry it at 65 °C for 24 h.

[0010] 3. Crushing: Crush the Apocynum venetum with a crusher and then pass it through a 60-mesh sieve.

[0011] 4. Subcritical fluid extraction of flavonoids: Mix the Apocynum venetum powder obtained in step 3 with ethanol of a certain concentration, put it into a high-temperature and high-pressure reaction kettle, turn on the switch of the stirring motor, turn on the heating voltage, and adjust it to the required temperature. Start timing when the temperature rises to the target temperature, and pay attention to the index of the vacuum pressure gauge. After the reaction is completed, connect the cooling coil to cold water to cool down the reaction kettle. After the thermocouple thermometer shows that the temperature is lower than the normal boiling point of ethanol, release the pressure and collect the discharged material.

[0012] 5. Purification: Place the material liquid in a centrifuge and centrifuge it at 4000 r / min for 20 min, and then perform suction filtration. The obtained filtrate is the flavonoid extract.

[0013] The solid-liquid ratio of Apocynum venetum powder to ethanol is: 1 g: 50 mL.

[0014] Ethanol concentration: 60%.

[0015] Extraction temperature: 120°C.

[0016] Extraction time: 1 h.

[0017] Ethanol concentration of the eluent: 60%.

[0018] The new method for extracting flavonoids from Apocynum venetum by the subcritical fluid extraction technology of the present invention has the advantages of high extraction efficiency, simple process, green process, convenient operation, and low equipment investment cost. It can also maximize the preservation of its biological activity and active ingredients, and is more suitable for industrial production. Description of the Drawings

[0019] Figure 1 It is the ultraviolet absorption spectrum diagram of the rutin standard curve: The rutin standard curve is drawn according to the NaNO2 - Al(NO3)3 - NaOH colorimetric method. From Figure 1 it can be seen that the rutin standard has the maximum absorbance value at a wavelength of 505 nm. Therefore, the wavelength used for measuring the concentration of flavonoids in Apocynum venetum is set at 505 nm.

[0020] Figure 2 It is the rutin standard curve graph. From Figure 2 it can be seen that the linear regression equation of the rutin standard curve is: y = 0.01101x - 0.02101, where R 2 = 0.99939. The linear relationship between the rutin concentration and the absorbance is good within 0 - 90 mg / L. Therefore, this regression equation can be used for the quantitative determination of flavonoids.

[0021] Figure 3 It is the extraction rate of flavonoids at different ethanol concentrations. From the figure, it can be seen that when the ethanol concentration is 60%, the extraction rate reaches the maximum value of 1.35%.

[0022] Figure 4 It is the extraction rate of flavonoids at different extraction times. From the figure, it can be seen that when the extraction time is 1 h, the extraction rate reaches the maximum value of 2.04%.

[0023] Figure 5 It is the extraction rate of flavonoids at different solid - liquid ratios. From the figure, it can be seen that when the solid - to - liquid ratio is 1:50 (g / mL), the extraction rate reaches the maximum value of 2.04%.

[0024] Figure 6 It is the extraction rate of flavonoids at different extraction temperatures. From the figure, it can be seen that when the extraction temperature is 120°C, the extraction rate reaches the maximum value of 2.04%.

[0025] Figure 7 Based on the results of the single - factor experiments, three levels are selected for each factor to conduct a four - factor and three - level L 9 (3 4)Orthogonal experiment results. It can be seen from the orthogonal experiment results that:

[0026] (1) Factors affecting the extraction rate of flavonoids: extraction temperature > solid-liquid ratio > ethanol concentration > extraction time. Among them, temperature is the most important influencing factor.

[0027] (2) The optimal scheme is the 3rd group: A 1 B 3 C 3 D 3 , and the extraction rate is 1.93%, which is lower than the single-factor optimal result of 2.04%.

[0028] (3) By combining single-factor experiments and orthogonal experiments, the best extraction process for Apocynum venetum is obtained as follows: ethanol concentration 60%, extraction time 1 h, extraction temperature 120 °C, solid-liquid ratio 1 g:50 mL. Under these conditions, the extraction rate of flavonoids from the roots of Apocynum venetum is 2.04%. Specific implementation mode

[0029] The present invention will be further specifically described below in conjunction with embodiments, but is not limited thereto.

[0030] Embodiment 1

[0031] The specific steps of the new method for extracting flavonoids from Apocynum venetum by the subcritical fluid extraction technology of the present invention are as follows:

[0032] Mix the Apocynum venetum powder passing through a 60-mesh sieve obtained in the previous step with ethanol of a certain concentration, put it into a high-temperature and high-pressure reaction kettle, turn on the switch of the stirring motor, turn on the heating voltage, and adjust it to the required temperature. Start timing when the temperature rises to the target temperature, and pay attention to the index of the vacuum pressure gauge. After the reaction is completed, turn on the cooling coil to connect cold water to cool down the reaction kettle. After the temperature shown by the thermocouple thermometer is lower than the normal boiling point of ethanol, release the pressure and collect the discharged material.

[0033] Centrifuge and filter the collected liquid, collect the filtrate to obtain a filtrate of flavonoids. Let it stand for 5 minutes, take 1 mL of the filtrate and place it in a 10 mL volumetric flask, add 0.5 mL of 5% sodium nitrite solution, and let it stand for 6 min. Then add 0.5 mL of 10% aluminum nitrate solution and let it stand for 6 min. Finally, add 4 mL of 4% sodium hydroxide solution. Dilute to the scale with 60% ethanol (volume fraction) and let it stand for 15 min. After standing, place the liquid in a centrifuge and centrifuge at a speed of 8000 r / min for 10 min, take the supernatant to measure the absorbance, and thus calculate the extraction rate of flavonoids.

[0034] Ethanol concentration: 0 - 100%.

[0035] Extraction temperature: 120 °C.

[0036] Extraction time: 2 h.

[0037] The solid-liquid ratio of Apocynum venetum powder to ethanol is: 1 g : 50 mL.

[0038] Example 2

[0039] The specific steps of the new method for extracting flavonoids from Apocynum venetum by the subcritical fluid extraction technology of the present invention are as follows:

[0040] Mix the Apocynum venetum powder passing through a 60-mesh sieve obtained in the previous step with ethanol of a certain concentration, put it into a high-temperature and high-pressure reaction kettle, turn on the switch of the stirring motor, turn on the heating voltage, and adjust it to the required temperature. Start timing when the temperature rises to the target temperature, and pay attention to the index of the vacuum pressure gauge. After the reaction is completed, connect the cooling coil to cold water to cool down the reaction kettle. After the thermocouple thermometer shows that the temperature is lower than the normal boiling point of ethanol, release the pressure and collect the discharged material.

[0041] Centrifuge and filter the collected liquid, collect the filtrate to obtain a filtrate of flavonoids. Let it stand for 5 minutes, take 1 mL of the filtrate and place it in a 10 mL volumetric flask, add 0.5 mL of 5% sodium nitrite solution, and let it stand for 6 min. Then add 0.5 mL of 10% aluminum nitrate solution and let it stand for 6 min. Finally, add 4 mL of 4% sodium hydroxide solution. Dilute to the scale with 60% ethanol (volume fraction) and let it stand for 15 min. After standing, place the liquid in a centrifuge and centrifuge at a speed of 8000 r / min for 10 min, take the supernatant to measure the absorbance, and thus calculate the extraction rate of flavonoids.

[0042] Ethanol concentration: 60%.

[0043] Extraction temperature: 120 °C.

[0044] Extraction time: 0.5 - 2.5 h.

[0045] The solid-liquid ratio of Apocynum venetum powder to ethanol is: 1 g : 50 mL.

[0046] Example 3

[0047] The specific steps of the new method for extracting flavonoids from Apocynum venetum by the subcritical fluid extraction technology of the present invention are as follows:

[0048] Mix the Apocynum venetum powder passing through a 60-mesh sieve obtained in the previous step with ethanol of a certain concentration, put it into a high-temperature and high-pressure reaction kettle, turn on the switch of the stirring motor, turn on the heating voltage, and adjust it to the required temperature. Start timing when the temperature rises to the target temperature, and pay attention to the index of the vacuum pressure gauge. After the reaction is completed, connect the cooling coil to cold water to cool down the reaction kettle. After the thermocouple thermometer shows that the temperature is lower than the normal boiling point of ethanol, release the pressure and collect the discharged material.

[0049] Centrifuge and filter by suction the collected liquid material, collect the filtrate to obtain a filtrate of flavonoids. Let it stand for 5 minutes, take 1 mL of the filtrate and place it in a 10 mL volumetric flask, add 0.5 mL of 5% sodium nitrite solution, and let it stand for 6 min. Then add 0.5 mL of 10% aluminum nitrate solution and let it stand for 6 min. Finally, add 4 mL of 4% sodium hydroxide solution. Make up the volume to the mark with 60% ethanol (volume fraction) and let it stand for 15 min. After standing, place the liquid in a centrifuge and centrifuge at a speed of 8000 r / min for 10 min, take the supernatant to measure the absorbance, and thus calculate the extraction rate of flavonoids.

[0050] Ethanol concentration: 60%.

[0051] Extraction temperature: 120 °C.

[0052] Extraction time: 1 h.

[0053] The solid-liquid ratio of Apocynum venetum powder to ethanol is: 1 g: 10 mL to 1 g: 60 mL.

[0054] Example 4

[0055] The specific steps of the new method for extracting flavonoids from Apocynum venetum by the subcritical fluid extraction technology of the present invention are as follows:

[0056] Mix the Apocynum venetum powder passing through a 60-mesh sieve obtained in the previous step with ethanol of a certain concentration, put it into a high-temperature and high-pressure reaction kettle, turn on the switch of the stirring motor, turn on the heating voltage, and adjust it to the required temperature. Start timing when the temperature rises to the target temperature, and pay attention to the index of the vacuum pressure gauge. After the reaction is completed, turn on the cooling coil to connect cold water to cool down the reaction kettle. After the thermocouple thermometer shows that the temperature is lower than the normal boiling point of ethanol, release the pressure and collect the discharged material.

[0057] Centrifuge and filter by suction the collected liquid material, collect the filtrate to obtain a filtrate of flavonoids. Let it stand for 5 minutes, take 1 mL of the filtrate and place it in a 10 mL volumetric flask, add 0.5 mL of 5% sodium nitrite solution, and let it stand for 6 min. Then add 0.5 mL of 10% aluminum nitrate solution and let it stand for 6 min. Finally, add 4 mL of 4% sodium hydroxide solution. Make up the volume to the mark with 60% ethanol (volume fraction) and let it stand for 15 min. After standing, place the liquid in a centrifuge and centrifuge at a speed of 8000 r / min for 10 min, take the supernatant to measure the absorbance, and thus calculate the extraction rate of flavonoids.

[0058] Ethanol concentration: 60%.

[0059] Extraction temperature: 100 °C - 130 °C.

[0060] Extraction time: 1 h.

[0061] The solid-liquid ratio of Apocynum venetum powder to ethanol is: 1 g : 50 mL.

[0062] The experimental data of Examples 1-4 of the present invention show that under the conditions of a material-liquid ratio of 1:50 (g / mL), an ethanol concentration of 60%, and an extraction time of 1 h, when the extraction temperature is 120 °C, the extraction rate of flavonoids reaches a maximum value of 2.04%.

[0063] The above are only preferred embodiments of the present invention. It is easy for those skilled in the art to understand that the embodiments are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. A new method for extracting ketone compounds from Apocynum edulis, comprising the following steps: The 60-mesh sieve-grinded apocynum powder is mixed with a certain concentration of ethanol in a certain ratio, placed in a high-temperature and high-pressure reactor, and reacted at a certain temperature for a certain time. First, turn on the stirring motor switch, turn on the heating voltage, and adjust it to the required temperature. When the temperature rises to the target temperature, start timing, and pay attention to the vacuum pressure gauge index. After the reaction is completed, the cooling coil is connected to cold water to cool the reactor. After the thermocouple thermometer shows that the temperature is lower than the normal pressure boiling point of ethanol, release the pressure and collect the discharge. The collected feed liquid is filtered, and the filtrate is collected to obtain the flavonoid compound filtrate. Let it stand for 5 minutes, take 1mL of the filtrate and place it in a 10ml volumetric flask, add 0.5mL of 5% sodium nitrite solution, and let it stand for 6min. Then add 0.5mL of 10% aluminum nitrate solution and let it stand for 6min. Finally, add 4mL of 4% sodium hydroxide solution. Use 60% ethanol (volume fraction) to make up to the scale and let it stand for 15min. After standing, the liquid was placed in a centrifuge and centrifuged at 8000 r / min for 10 min. The supernatant was taken to measure the absorbance, thereby calculating the extraction rate of flavonoid compounds.

2. The novel method for extracting apocynum foetida ketone compounds as claimed in claim 1, characterized in that: The ethanol concentration was 60%.

3. The novel method for extracting apocynum foetida ketone compounds as claimed in claim 1, characterized in that: The solid-liquid ratio is 1g:50mL.

4. The novel method for extracting apocynum foetida ketone compounds as claimed in claim 1, characterized in that: The extraction time was 1 h.

5. The novel method for extracting apocynum foetida ketone compounds as claimed in claim 1, characterized in that: The extraction temperature was 120°C.

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