Extraction method and application of procyanidine in purple rice

Abstract

The invention discloses an extraction method and application of procyanidine in purple rice, and belongs to the technical field of raw material extraction. The method for extracting the procyanidine in the purple rice comprises the following steps: crushing the purple rice to obtain purple rice powder; and mixing the purple rice powder with an ethanol water solution with the pH value of 3, performing ultrasonic treatment, heating, centrifuging, and collecting supernate, thereby obtaining the procyanidine extracting solution. According to the method for extracting the proanthocyanidins in the purple rice, an ultrasonic-assisted ethanol / water co-extraction method is selected, the influence of ultrasonic time, ethanol volume fraction, water bath time and water bath temperature on the proanthocyanidins extraction rate is explored, and the extraction process of the proanthocyanidins in the purple rice is optimized by adopting a response surface method; and optimal extraction conditions are obtained. The extraction method is simple and convenient, high in extraction efficiency and accurate in response surface method analysis result, provides a theoretical basis for an extraction process before deep processing of the procyanidine in the purple rice, and has a very good application value.

Description

Technical Field

[0001] The invention relates to the technical field of raw material extraction, and in particular to a method for extracting proanthocyanidins from purple rice and application thereof. Background Art

[0002] Purple rice (Oryza satiua), also known as glutinous rice and purple pearl, has the effects of nourishing yin and kidney, strengthening spleen and warming liver, improving eyesight and promoting blood circulation, nourishing skin and treating neurasthenia. Purple rice contains a variety of nutrients such as vitamins, folic acid, protein and mineral elements, and is rich in active substances such as anthocyanins, flavonoids and phenolic acids. It is a rare rice variety that combines edible and medicinal uses. The main reason why purple rice has good medicinal effects is that it contains more proanthocyanidins.

[0003] Experiments have shown that proanthocyanidins can effectively remove free radicals in the human body. They are extremely effective natural antioxidants, free radical scavengers, and ultraviolet absorbers. Their antioxidant capacity is 50 times that of vitamin E and 20 times that of vitamin C. They can effectively remove excess free radicals in the human body to prevent and treat diseases caused by free radical oxidation, and can also strengthen the human body's defense system. Proanthocyanidins, with their natural and non-toxic advantages, have been widely used as important raw materials for the preparation of drugs for the prevention and treatment of a series of diseases such as cardiovascular diseases, tumors, skin damage, retinopathy, diabetes, Alzheimer's disease, edema, allergic inflammation, and functional health foods and cosmetics.

[0004] With the increasing application of proanthocyanidins, it is imperative to study a simpler and more efficient extraction process of proanthocyanidins from purple rice. The existing methods for extracting proanthocyanidins from purple rice include organic solvent and water co-extraction, ultrasonic assisted extraction, enzyme extraction, microwave assisted extraction and two-phase aqueous system extraction. However, these existing extraction methods still face problems such as low extraction efficiency and complicated extraction process, and still need to be further improved. Summary of the invention

[0005] The object of the present invention is to provide an extraction method and application of proanthocyanidins in purple rice to solve the above-mentioned problems in the background technology. The present invention designs a method for extracting proanthocyanidins in purple rice, selects an ultrasound-assisted ethanol / water co-extraction method, and explores the effects of ultrasound time, ethanol volume fraction, water bath time, and water bath temperature on the extraction rate of proanthocyanidins, and optimizes the extraction process of proanthocyanidins in purple rice using the response surface methodology to obtain the optimal extraction conditions.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] One of the technical solutions of the present invention is to provide a method for extracting proanthocyanidins from purple rice, comprising the following steps:

[0008] The purple rice is crushed to obtain purple rice powder;

[0009] The purple rice powder is mixed with an ethanol aqueous solution with a pH value of 3, ultrasonicated, heated, and then centrifuged to collect a supernatant to obtain a proanthocyanidin extract.

[0010] Preferably, the pulverizing is pulverizing until the powder passes through an 80-mesh sieve.

[0011] Preferably, the ethanol concentration in the ethanol aqueous solution at pH 3 is 32-37 vol%.

[0012] Preferably, the ratio of the purple rice powder to the ethanol aqueous solution with a pH of 3 is 1 g:8 mL.

[0013] Preferably, the ultrasonication time is 30-40 min.

[0014] Preferably, the heating temperature is 60-70°C and the heating time is 60-80 minutes.

[0015] Preferably, the centrifuge speed is 5000r / min and the time is 5min.

[0016] Preferably, the ethanol concentration in the ethanol aqueous solution at pH 3 is 33.56 vol%; the ultrasonic time is 32.53 min; the heating temperature is 62.90° C. and the heating time is 68.61 min.

[0017] The beneficial technical effects of the present invention are as follows:

[0018] The present invention designs a method for extracting proanthocyanidins from purple rice, selects an ultrasound-assisted ethanol / water co-extraction method, and explores the effects of ultrasound time, ethanol volume fraction, water bath time, and water bath temperature on the extraction rate of proanthocyanidins. The extraction process of proanthocyanidins in purple rice is optimized by using the response surface methodology, and the optimal extraction conditions are obtained.

[0019] This extraction method is simple and convenient, with high extraction efficiency and accurate analysis results by response surface methodology. It provides a theoretical basis for the extraction process of purple rice proanthocyanidins before deep processing and has good application value.

[0020] Under the action of the ultrasonic wave of the present invention, the release, diffusion and dissolution of proanthocyanidins in purple rice are accelerated, thereby significantly improving the extraction efficiency of proanthocyanidins in purple rice. Proanthocyanidins in purple rice can form intermolecular hydrogen bonds with ethanol and water in the reaction system, making it easier to dissolve in the extract, thereby increasing the extraction rate of proanthocyanidins in the extraction process of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 is the absorbance of different reference solutions as the absorption wavelength changes.

[0023] Figure 2 This is the standard curve of proanthocyanidins.

[0024] Figure 3 The effect of light on the stability of proanthocyanidins in the proanthocyanidin extract of Example 1.

[0025] Figure 4 The effect of ultrasonic time on the extraction rate of proanthocyanidins in the extract.

[0026] Figure 5 This is the effect of ethanol volume fraction on the extraction rate of proanthocyanidins in the extract.

[0027] Figure 6 The effect of water bath temperature on the extraction rate of proanthocyanidins in the extract.

[0028] Figure 7 The effect of water bath time on the stability of proanthocyanidins in the extract.

[0029] Figure 8 The interactive effect of ultrasonic time and ethanol volume fraction on the extraction rate of proanthocyanidins in purple rice. (a) is the front view and (b) is the top view.

[0030] Fig. 9 The interactive effect of ultrasonic time and water bath temperature on the extraction rate of proanthocyanidins in purple rice. (a) is the front view and (b) is the top view.

[0031] Fig.10 The interactive effect of ultrasonic time and water bath time on the extraction rate of proanthocyanidins in purple rice. (a) is the front view and (b) is the top view.

[0032] Fig.11 The interactive effect of ethanol volume fraction and water bath temperature on the extraction rate of proanthocyanidins in purple rice. (a) is the front view and (b) is the top view.

[0033] Fig.12 The interactive effect of ethanol volume fraction and water bath time on the extraction rate of proanthocyanidins in purple rice. (a) is the front view and (b) is the top view.

[0034] Fig.13 The interactive effect of water bath time and water bath temperature on the extraction rate of proanthocyanidins in purple rice. (a) is the front view and (b) is the top view. DETAILED DESCRIPTION

[0035] Now, various exemplary embodiments of the present invention are described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention.

[0036] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0037] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in the present invention are open-ended terms, meaning including but not limited to.

[0039] Oligomeric Proantho Cyanidins (OPC) are polyphenolic hydroxyl polymers, and a partial structural formula thereof is shown below.

[0040]

[0041] Since proanthocyanidins contain polyphenolic hydroxyl structures, they have certain reducing properties. Experiments have shown that proanthocyanidins can efficiently remove free radicals in the human body and are extremely effective natural antioxidants, free radical scavengers, and ultraviolet absorbers. Their antioxidant capacity is 50 times that of vitamin E and 20 times that of vitamin C. They can effectively remove excess free radicals in the human body to prevent and treat diseases caused by free radical oxidation, and can also strengthen the human body's defense system.

[0042] Modern experimental studies have shown that proanthocyanidins have certain pharmacological activity. In in vivo experiments, Zeng Xiaoyan et al. studied the effects of proanthocyanidins in raspberries on glucose and lipid metabolism and antioxidant effects in rats with type 2 diabetes, and concluded that proanthocyanidins can affect glucose and lipid metabolism in rats with type 2 diabetes and reduce blood sugar in rats with type 2 diabetes. Its mechanism of action may be related to improving glucose and lipid metabolism, increasing INS and liver glycogen content. In in vitro experiments, Hu Naihua et al. combined proanthocyanidins with nanotechnology and found that proanthocyanidins had a high therapeutic effect on atherosclerosis in mouse models. In gene expression experiments, Yang Lu et al. used random number table method, Isolectin GS-IB4 fluorescence staining method and protein blotting method to explore the inhibitory effect of proanthocyanidins on the formation of new blood vessels in oxygen-induced retinopathy in C57 mice. The results showed that proanthocyanidins could downregulate the expression of VEGF in the retinal tissue of oxygen OIR mice to a certain extent, thereby inhibiting the formation of retinal neovascularization. In the cell experiment, Ren Jingyi et al. tested the protective effect of proanthocyanidin B2 on rat cardiac toxicity caused by acute exposure to PM2.5 by detecting the levels of total superoxide dismutase, glutathione peroxidase, malondialdehyde, interleukin 1β, interleukin 6 and interleukin 18 in the heart. The results showed that proanthocyanidin B2 had a protective effect on cardiac damage caused by PM2.5. Wang Pan et al. treated triple-negative breast cancer cells MDA-MB-231 and breast cells MCF-10A with different concentrations of proanthocyanidins for 24 h, and then detected the activity of the two cell lines with CCK-8 kit and used Western blotting to detect. The results showed that proanthocyanidins could inhibit the proliferation and glucose metabolism of triple-negative breast cancer MDA-MB-231 cells, and inhibit the activity of Akt, mTOR and STAT3 signaling pathways.

[0043] The existing methods for extracting proanthocyanidins from purple rice include organic solvent and water co-extraction, ultrasonic-assisted extraction, enzyme extraction, microwave-assisted extraction, and two-phase aqueous system extraction. Zhang Lunning et al. used Wugongshan purple red rice and brown rice as raw materials, and used ultrasonic-assisted extraction to compare different ethanol concentrations, extraction time, extraction temperature, and solid-liquid ratio. The optimal extraction process conditions were obtained as follows: extraction time 80min, extraction temperature 60℃, ethanol concentration 60%, and solid-liquid ratio 1:8 (g / v).

[0044] There are two methods for determining the content of proanthocyanidins: UV-visible spectrophotometry and high performance liquid chromatography. UV-visible spectrophotometry is a qualitative, quantitative and structural analysis method based on the absorption characteristics of material molecules to electromagnetic radiation in the range of 200-760nm. Proanthocyanidins are polyphenolic hydroxyl polymers with strong absorption at around 280nm, which can be used for qualitative and quantitative analysis. High performance liquid chromatography is based on the different distribution coefficients of the measured substances in the two phases, so they can be gradually separated into single components or different component groups on the chromatographic column. The final separation effect is a comprehensive benefit of both thermodynamics and kinetics. Qualitative and quantitative analysis is performed through the parameters displayed in the chromatogram. However, proanthocyanidins are polymer compounds and are easily degraded. When determining the content through high performance liquid chromatography, it is easy to face the problem of difficulty in determining the standard, so UV-visible spectrophotometry is used. Some researchers have compared different methods for determining the content of proanthocyanidins and concluded that different methods have different advantages. For example, when using UV-visible spectrophotometry for content determination, there are direct determination methods, hydrochloric acid-n-butanol method, and vanillin method. The hydrochloric acid-n-butanol method has strong specificity, but is easily interfered by the color development of structural monomers such as catechins and epicatechins; the vanillin method is mainly a color development method for determining flavonoid compounds through ultraviolet light, because proanthocyanidins contain polyhydroxyphenol structures, so this method can be used for determination. Proanthocyanidins are polymer compounds that are difficult to accurately titrate and will be interfered by flavonoids in purple rice during content determination, so this experiment used ultraviolet spectrophotometry to directly determine the content of proanthocyanidins.

[0045] The relevant parameters of the main instruments used in the experiment of the present invention are shown in Table 1.

[0046] Table 1 Main instruments

[0047] Instrument Name model Manufacturer UV-Vis Spectrophotometer 752Pro Shanghai Lingguang Technology Co., Ltd. Electronic analytical balance CP214 Ohaus (Changzhou) Co., Ltd. Ultrasonic cleaning machine KQ-500B Kunshan Ultrasonic Instrument Co., Ltd. Circulating water multi-purpose vacuum pump SHZ-D(III) Shanghai Lichen Bangxi Instrument Technology Co., Ltd. Constant temperature drying oven 101-2A Shanghai Hunan Electric Furnace Oven Factory Electronic constant temperature water bath HHS-4S Shanghai Yichang Instrument Screen Factory

[0048] The purple rice used in the present invention was purchased from Loudi, Hunan, Shaoyang, Hunan, Hanzhong, Shaanxi, Pu'er, Yunnan, Qiannan, Guizhou and Harbin, Heilongjiang, anhydrous ethanol was purchased from Tianjin Yongda Chemical Reagent Co., Ltd., purified water was purchased from China Resources Yibao Beverage Co., Ltd., citric acid was purchased from Tianjin Yongda Chemical Reagent Co., Ltd., and proanthocyanidin standard products were purchased from Shanghai Solebold (95% (UV)).

[0049] The preparation method of the 20% citric acid solution used in the following embodiments of the present invention is as follows: accurately weigh 20g of citric acid and put it into a beaker, then add 50mL of deionized water to the beaker, stir until completely dissolved, transfer to a 100mL volumetric flask, and dilute to 100mL with deionized water to obtain a 20% citric acid solution for standby use.

[0050] The preparation method of the 37% ethanol solution at pH 3 used in the following embodiments of the present invention is as follows: accurately measure 37 mL of anhydrous ethanol, pour it into a 100 mL volumetric flask, make up to volume with purified water, mix the two, and then adjust the pH to 3 with 20% citric acid solution to obtain a 37% ethanol solution at pH 3 for use.

[0051] All raw materials used in the following examples and comparative examples of the present invention are commercially available products.

[0052] Example 1

[0053] A method for extracting proanthocyanidins from purple rice:

[0054] Purple rice (purchased from Loudi, Hunan) was placed in a 30°C drying oven and dried for 2 hours. About 100g of purple rice was weighed and crushed to pass through an 80-mesh sieve to obtain purple rice powder for later use. 1.00g of purple rice powder was weighed and placed in a 10mL centrifuge tube. 8mL of a 37% ethanol solution with a pH of 3 was added. The mixture was soaked for 10 minutes, ultrasonicated for 20 minutes, and then heated in a water bath at 80°C for 60 minutes to obtain an extract. The extract was centrifuged in a centrifuge at a speed of 5000r / min for 5 minutes. 1mL of the supernatant was added to a 10mL volumetric flask and fixed to volume, and allowed to stand for 30 minutes to prepare a proanthocyanidin extract for later use.

[0055] Effect verification

[0056] 1. Preparation of reference solution

[0057] Transfer 20 mg of the proanthocyanidin standard to a 10 mL volumetric flask, dissolve it with a small amount of 37% ethanol solution at pH 3, and then dilute it to 10 mL with 37% ethanol solution at pH 3 to prepare a 2 mg / mL proanthocyanidin stock solution. Take 0, 0.1, 0.15, 0.2, 0.25, and 0.3 mL of the proanthocyanidin stock solution, dilute it to 10 mL with 37% ethanol solution at pH 3, and mark the resulting solutions as I, II, III, IV, V, and VI reference solutions, respectively.

[0058] 2. Performance Testing

[0059] 2.1 Determine the wavelength of maximum absorption

[0060] Scan the wavelength of II, III, IV, V, and VI reference solutions at 200-400 nm, draw absorption spectra, and determine the maximum absorption wavelength.

[0061] 2.2 Standard curve drawing

[0062] At the maximum absorption wavelength, taking I as the blank control, the absorbance of reference solutions I, II, III, IV, V, and VI were measured respectively, and a standard curve was drawn with absorbance A as the ordinate and proanthocyanidin solution concentration C (μg / mL) as the abscissa.

[0063] 2.3 Precision test

[0064] Take IV reference solution and measure it 6 times continuously, calculate the RSD value and examine the precision of the instrument.

[0065] 2.4 Repeatability test

[0066] The extraction method of Example 1 was repeated 6 times to obtain 6 portions of proanthocyanidin extracts, and the RSD value was calculated to examine the repeatability of the method.

[0067] 2.5 Sample recovery test

[0068] Take 2mL of the proanthocyanidin extract of Example 1 in a 20mL volumetric flask, dilute with a 37% ethanol solution at pH 3 to obtain a test solution, measure the absorbance and record it. Then add 2mL of the test solution to three test tubes respectively, and then add a reference solution and a 37% ethanol solution at pH 3 so that the ratio of the proanthocyanidin content of the solution in the three test tubes to the proanthocyanidin content in the test solution is 0.8:1, 1:1, and 1.2:1, respectively. Shake well, let stand for 30 minutes, measure and record the absorbance A, and calculate the average recovery rate. The average recovery rate is calculated according to the following formula:

[0069]

[0070] 2.6 Stability test

[0071] The extraction method of Example 1 was repeated 3 times to prepare 3 portions of proanthocyanidin extracts. After being placed under shading, light-avoiding and sunlight conditions for 0, 1, 2, 4, 6, 8 and 10 h, a 37% ethanol solution with pH 3 was used as a blank control. The absorbance was measured and the RSD values ​​at 7 time points were calculated to investigate the stability of the proanthocyanidins in the extracts.

[0072] 2.7 Determination of proanthocyanidin content

[0073] Purple rice from Loudi, Hunan, Shaoyang, Hunan, Hanzhong, Shaanxi, Pu'er, Yunnan, Qiannan, Guizhou, and Harbin, Heilongjiang was extracted under the optimal conditions optimized by the response surface method to obtain proanthocyanidin extracts. The concentration of the sample solution was obtained according to the standard curve, and the content of proanthocyanidins in different purple rice was calculated. The calculation formula for proanthocyanidin content is as follows:

[0074]

[0075] Wherein: C is the mass concentration of proanthocyanidins in the proanthocyanidin extract, μg / mL; D is the dilution multiple, mL; W is the mass of the purple rice sample, g.

[0076] 2.8 Single factor effect test on the extraction effect of proanthocyanidins in purple rice

[0077] 2.8.1 Ultrasonic time

[0078] The only difference from Example 1 is that the ultrasonic time is changed to 10, 30, 40, and 50 minutes to obtain anthocyanidin extract, and then the extracts prepared in Example 1 and 2.8.1 are used to investigate the effects of ultrasonic times of 10, 20, 30, 40, and 50 minutes on the extraction of purple rice proanthocyanidins.

[0079] 2.8.2 Ethanol volume fraction

[0080] The only difference from Example 1 is that the concentration of the pH 3 ethanol solution is modified to 22, 27, 32, and 42% to obtain an extract of proanthocyanidins, and then the extracts prepared in Example 1 and 2.8.2 are used to investigate the effects of pH 3 ethanol solution concentrations of 22, 27, 32, 37, and 42% on the extraction of purple rice proanthocyanidins.

[0081] 2.8.3 Water bath temperature

[0082] The only difference from Example 1 is that the water bath heating temperature is changed to 50, 60, 70, and 90°C to obtain anthocyanidin extract, and then the extracts prepared in Example 1 and 2.8.3 are used to investigate the effects of water bath temperatures of 50, 60, 70, 80, and 90°C on the extraction of purple rice proanthocyanidins.

[0083] 2.8.4 Water bath time

[0084] The only difference from Example 1 is that the water bath heating time is changed to 20, 40, 80, and 100 minutes to obtain anthocyanidin extract, and then the extracts prepared in Example 1 and 2.8.4 are used to investigate the effects of water bath times of 20, 40, 60, 80, and 100 minutes on the extraction of purple rice proanthocyanidins.

[0085] 2.9 Response surface experimental design

[0086] Based on the results of the single factor impact test, a four-factor three-level Box-Behnken Design (BBD) central composite analysis experiment was designed to obtain the fitting equation to obtain the optimal extraction conditions.

[0087] Table 2. Response surface test factors and levels

[0088]

[0089] 2.10 Response surface experiment verification

[0090] The optimal extraction conditions were obtained after analysis by response surface methodology, and 5 extraction experiments were carried out under these conditions. The error analysis was performed between the extraction rate and the optimal extraction rate predicted by the response surface methodology.

[0091] 3. Test results

[0092] 3.1 Determination of maximum absorption wavelength

[0093] Figure 1 is the absorbance of different reference solutions as the absorption wavelength changes.

[0094] Figure 1 In the table, 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml and 50 μg / ml represent II, III, IV, V and VI reference solutions respectively.

[0095] Depend on Figure 1 It can be seen that different concentrations of proanthocyanidins have a series of large and irregular absorption peaks between the wavelength of 200-250nm, a single, sharp, symmetrical and smooth absorption peak between the wavelength of 250-300nm, no absorption peak at the wavelength of 300-400nm, and a maximum absorption at the wavelength of 280nm. Therefore, the wavelength of 280nm is selected as the detection wavelength.

[0096] 3.2 Standard curve drawing

[0097] Figure 2 This is the standard curve of proanthocyanidins.

[0098] like Figure 2 It can be seen that within the mass concentration range of 0-50 μg / mL, the regression equation is y=0.0146x+0.0073 (r=0.9998), and the absorbance has a good linear relationship with the mass concentration of proanthocyanidins.

[0099] 3.3 Precision test

[0100] After measuring six times in succession in method 2.3, the absorbances of the IV reference solution were 0.447, 0.453, 0.448, 0.452, 0.448, and 0.449, respectively, with an average RSD value of 0.54%, indicating that the instrument had good precision.

[0101] 3.4 Repeatability test

[0102] After repeating the extraction method of Example 1 for 6 times, the absorbances of the obtained proanthocyanidin extracts were 0.466, 0.471, 0.488, 0.471, 0.478, and 0.470, respectively, and the average RSD value was 1.64%, indicating that the extraction method had good repeatability.

[0103] 3.5 Sample recovery test

[0104] As shown in Table 3, the average recovery rate of proanthocyanidins was 100.80% and the RSD value was 2.64%, indicating that the determination method had high accuracy.

[0105] Table 3 Sample recovery rate table

[0106]

[0107]

[0108] 3.6 Effect of light factors on method stability

[0109] Figure 3 The effect of light on the stability of proanthocyanidins in the proanthocyanidin extract of Example 1.

[0110] Depend on Figure 3 It can be seen that the three lighting conditions have little effect on the extract obtained in Example 1; when shaded, proanthocyanidins are most stable, and the RSD value of the determination deviation within 10 hours is 1.72%; when exposed to direct light, proanthocyanidins gradually begin to decompose after 2 hours; when shielded from light, they are relatively stable within 10 hours. The results show that after the sample is extracted, shaded, and the content determination is completed within 1 hour.

[0111] 3.7 Single factor experimental results and analysis

[0112] 3.7.1 Effect of ultrasonic time on the extraction rate of proanthocyanidins

[0113] Figure 4 The effect of ultrasonic time on the extraction rate of proanthocyanidins in the extract.

[0114] Depend on Figure 4It can be seen that when the ultrasonic time is 10-20min, the extraction rate of proanthocyanidins increases significantly; this is mainly because, under the action of ultrasound, the solution is fully in contact with the substance, which accelerates the release, diffusion and dissolution of proanthocyanidins in purple rice, thereby significantly improving the extraction efficiency of proanthocyanidins in purple rice. When the ultrasonic time is 30-40min, the proanthocyanidin extraction rate changes relatively smoothly; this is mainly because, at this stage, the proanthocyanidins in purple rice have almost all dissolved into the extract. When the ultrasonic time is 40-50min, the proanthocyanidin extraction rate decreases significantly; this is mainly because ultrasound has a strong mechanical shearing effect, and long-term action will destroy the structure of proanthocyanidins, resulting in a decrease in the proanthocyanidin extraction rate.

[0115] 3.7.2 Effect of ethanol volume fraction on proanthocyanidin extraction rate

[0116] Figure 5 This is the effect of ethanol volume fraction on the extraction rate of proanthocyanidins in the extract.

[0117] Depend on Figure 5 It can be seen that when the volume fraction of ethanol is 22-32%, the extraction rate of proanthocyanidins increases slowly; this is mainly because proanthocyanidins themselves are water-soluble substances and are slightly soluble in ethanol. When the volume fraction of ethanol is 32-36%, the extraction rate of proanthocyanidins increases significantly; this is mainly because proanthocyanidins are polyphenolic hydroxyl compounds that can form intermolecular hydrogen bonds with ethanol and water, making them more soluble in the extract, thereby increasing the extraction rate of proanthocyanidins. However, when the volume fraction of ethanol is 36-40%, the extraction rate of proanthocyanidins decreases; this is mainly because the ethanol concentration is too high, hydrogen bonds are formed between ethanol molecules, and the extraction rate of proanthocyanidins decreases.

[0118] 3.7.3 Effect of water bath temperature on extraction of purple rice proanthocyanidins

[0119] Figure 6 The effect of water bath temperature on the extraction rate of proanthocyanidins in the extract.

[0120] Depend on Figure 6 It can be seen that, overall, when the water bath temperature rises from 65 to 90°C, the extraction rate of proanthocyanidins decreases rapidly; this is mainly because, at around 70°C, proanthocyanidins will begin to degrade into other substances, such as flavonoid compounds anthocyanidins, which reduces the extraction rate of proanthocyanidins.

[0121] 3.7.4 Effect of water bath time on extraction of purple rice proanthocyanidins

[0122] Figure 7 The effect of water bath time on the extraction rate of proanthocyanidins in the extract.

[0123] Depend on Figure 7It can be seen that when the water bath time is 20-80min, the extraction rate of proanthocyanidins gradually increases; this is mainly because the longer the water bath time, the longer the extraction time, and the proanthocyanidins in the purple rice can be fully dissolved; and when the water bath time is 80-100min, some components in the purple rice will ferment, making the extracted proanthocyanidins unstable and decomposed, so the extraction rate of proanthocyanidins shows a downward trend at this stage.

[0124] 3.8 Multi-factor experimental results and analysis

[0125] 3.8.1 Analysis of variance in response surface experiments

[0126] Table 4 Response surface experiment arrangement and results

[0127]

[0128]

[0129] Table 5 Analysis of variance of response surface quadratic model

[0130]

[0131]

[0132] From Table 4 and Table 5, we can see that the model f value is 12.85, indicating that the model is significant. There is only a 0.01% probability that such a large "f-type value" will be generated due to noise. "Pr>F" is less than 0.05, indicating that the model terms are significant. At the same time, A, B, C, D, BC, A 2 , B 2 , C 2 , D 2 are important model terms, and their values ​​are all less than 0.10, indicating that the model terms are significant. However, the lack of fit term of 0.75 means that there is a lack of fit. Relative to pure insignificant errors, insignificant misfit is good, and Adj R 2 =0.8801 and Pr R 2 =0.6936 is reasonable and consistent, and the goodness of fit is R 2 =0.9400, indicating that the response surface quadratic polynomial model has a high fit. The quadratic polynomial regression model equation is:

[0133] Y=-7107.09664+75.51340×A+380.14415×B+22.93783×C+43.08883×D+1.41096×A B+0.20891×AC+0.063356×AD+2.00000×BC+0.35959×BD+0.095891×CD-2.15605×A 2 -8.58994×B2 -0.822214×C 2 -0.46110×D 2 .

[0134] Response surface software analysis revealed that the optimal extraction conditions for proanthocyanidins from purple rice (Loudi, Hunan) were: ultrasonic time 32.53 min, ethanol volume fraction 33.56%, water bath temperature 62.90 °C, water bath time 68.61 min, and the proanthocyanidin extraction rate was 2,699.52 μg / g.

[0135] 3.8.2 Response Surface Analysis Interactions and Contours

[0136] Figure 8 The interactive effect of ultrasonic time and ethanol volume fraction on the extraction rate of proanthocyanidins in purple rice. (a) is the front view and (b) is the top view.

[0137] Figure 8 It indicates the effect of the interaction between ultrasonic time and ethanol volume fraction on the extraction of proanthocyanidins from purple rice when the water bath time and water bath temperature are at the center level. When the ethanol volume fraction is low, the proanthocyanidin extraction rate does not change much with the ultrasonic time; when the ethanol volume fraction is high, the proanthocyanidin extraction increases first and then decreases with the increase of ultrasonic time; and P value>F value<0.05, indicating that the interaction between ultrasonic time and ethanol volume fraction has a significant effect on the extraction of proanthocyanidins from purple rice.

[0138] Fig. 9 The interactive effect of ultrasonic time and water bath temperature on the extraction rate of proanthocyanidins in purple rice. (a) is the front view and (b) is the top view.

[0139] Fig. 9 The results show that when the water bath time and ethanol volume fraction are at the center level, the interaction between ultrasound time and water bath temperature affects the extraction of proanthocyanidins from purple rice. When the water bath temperature remains unchanged, the extraction of proanthocyanidins increases first and then decreases with the increase of ultrasound time.

[0140] Fig.10 The interactive effect of ultrasonic time and water bath time on the extraction rate of proanthocyanidins in purple rice. (a) is the front view and (b) is the top view.

[0141] Fig.10 It shows the effect of the interaction between ultrasonic time and water bath time on the extraction of proanthocyanidins from purple rice when the water bath temperature and ethanol volume fraction are at the center level. When the water bath time is low, the extraction rate of proanthocyanidins does not change much with the ultrasonic time; when the water bath time is high, the extraction of proanthocyanidins first increases and then decreases with the increase of ultrasonic time.

[0142] Fig.11 The interactive effect of ethanol volume fraction and water bath temperature on the extraction rate of proanthocyanidins in purple rice. (a) is the front view and (b) is the top view.

[0143] Fig.11 It indicates the effect of the interaction between ethanol volume fraction and water bath temperature on the extraction of proanthocyanidins from purple rice when the ultrasonic time and water bath time are at the center level. When the water bath temperature remains unchanged, with the increase of ethanol volume fraction, the extraction of proanthocyanidins first increases rapidly and then decreases slowly; and P value>F value<0.05, indicating that the interaction between water bath temperature and ethanol volume fraction has a significant effect on the extraction of proanthocyanidins from purple rice.

[0144] Fig.12 The interactive effect of ethanol volume fraction and water bath time on the extraction rate of proanthocyanidins in purple rice. (a) is the front view and (b) is the top view.

[0145] Fig.12 It shows the effect of the interaction between ethanol volume fraction and water bath time on the extraction of proanthocyanidins from purple rice when the ultrasonic time and water bath temperature are at the center level. When the water bath time remains unchanged, the extraction of proanthocyanidins first increases and then decreases with the increase of ethanol volume fraction.

[0146] Fig.13 The interactive effect of water bath time and water bath temperature on the extraction rate of proanthocyanidins in purple rice. (a) is the front view and (b) is the top view.

[0147] Fig.13 The results show that when the ultrasonic time and ethanol volume fraction are at the central level, the interaction of water bath time and water bath temperature affects the extraction of proanthocyanidins from purple rice. When the water bath time remains unchanged, as the water bath temperature increases, the extraction of proanthocyanidins first remains unchanged and then slowly decreases.

[0148] The above response surface and contour map can intuitively reflect the influence of interaction on the response value. The steeper the surface and the denser the contour lines, the more significant the influence. Therefore, the significance is: BC>AB>BD>AC>CD>AD.

[0149] 3.8.3 Response surface experiment verification

[0150] In the validation experiment, the extraction conditions of proanthocyanidins in purple rice were: ultrasonic time 33min, ethanol volume fraction 34%, water bath temperature 63℃, water bath time 69min. The average yield of 5 experiments was 2,701.92μg / g, which was within the predicted value (2,699.52±142.41)μg / g, and the relative error was 0.09%, indicating that the model was accurate and the method had good precision.

[0151] Table 6 Response surface validation experiment

[0152]

[0153]

[0154] 3.9 Determination of proanthocyanidin content in purple rice from different origins under the optimal conditions optimized by response surface methodology

[0155] The ultrasonic time in Example 1 was modified to 33 min, the ethanol volume fraction was modified to 34%, the water bath temperature was modified to 63° C., and the water bath time was modified to 69 min. Then, the following extraction experiments were carried out on purple rice from different origins using the method of Example 1. The experimental results are shown in Table 7.

[0156] Table 7 Determination of proanthocyanidins content in purple rice from different origins

[0157]

[0158] As shown in Table 7, Loudi purple rice and Heilongjiang purple rice have the highest relative content of proanthocyanidins, with extraction rates of 2,670.50 μg / g and 2,340.52 μg / g, respectively; Shaanxi purple rice and Guizhou purple rice have medium relative content of proanthocyanidins, with extraction rates of 1,953.91 μg / g and 1,911.90 μg / g, respectively; Yunnan purple rice and Shaoyang purple rice have low relative content of proanthocyanidins, with extraction rates of 1,435.80 μg / g and 1,697.60 μg / g, respectively.

[0159] The present invention designs a method for extracting proanthocyanidins from purple rice, selects ultrasound-assisted ethanol / water co-extraction method, explores the effects of ultrasound time, ethanol volume fraction, water bath time, and water bath temperature on the extraction rate of proanthocyanidins, and uses response surface method to optimize the extraction process of proanthocyanidins from purple rice. Through experimental results and analysis, it is known that the optimal extraction conditions for purple rice proanthocyanidins are ultrasound time of 32.53min, ethanol volume fraction of 33.56%, water bath temperature of 62.90℃, and water bath time of 68.61min, and the yield is 2,699.52μg / g. At the same time, five parallel experiments were carried out under this condition, and the average yield was 2,701.92μg / g, within the predicted value (2,699.52±142.41)μg / g range, and the relative error was 0.09%, indicating that the established regression equation model can more truly and accurately reflect the influence of the factors on the extraction results of proanthocyanidins in purple rice. The results of measuring the content of purple rice from Loudi, Hunan, Shaoyang, Hunan, Hanzhong, Shaanxi, Pu'er, Yunnan, Qiannan, Guizhou and Harbin, Heilongjiang show that the content of proanthocyanidins in purple rice from different origins is different, and the content of proanthocyanidins in purple rice from Loudi, Hunan is the highest. The present invention provides a theoretical basis for the extraction process of purple rice proanthocyanidins before deep processing.

[0160] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for extracting proanthocyanidins from purple rice, characterized in that: The following steps are involved: The purple rice is crushed to obtain purple rice powder; The purple rice powder is mixed with an ethanol aqueous solution with a pH value of 3, ultrasonicated, heated, and then centrifuged to collect a supernatant to obtain a proanthocyanidin extract.

2. The extraction method according to claim 1, characterized in that The pulverizing step is pulverizing until the powder passes through a 80-mesh sieve.

3. The extraction method according to claim 1, characterized in that The ethanol concentration in the ethanol aqueous solution at pH 3 is 32-37 vol%.

4. The extraction method according to claim 1, characterized in that The ratio of the purple rice powder to the ethanol aqueous solution with a pH of 3 is 1 g:8 mL.

5. The extraction method according to claim 1, characterized in that The ultrasonic time is 30-40 minutes.

6. The extraction method according to claim 1, characterized in that The heating temperature is 60-70°C and the heating time is 60-80 minutes.

7. The extraction method according to claim 1, characterized in that The centrifuge speed is 5000r / min and the time is 5min.

8. The extraction method according to claim 1, characterized in that The ethanol concentration in the ethanol aqueous solution at pH 3 is 33.56 vol%; the ultrasonic time is 32.53 min; the heating temperature is 62.90° C. and the heating time is 68.61 min.

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