Method for extracting anthocyanin in orange osmanthus fruits by using mixed solution of acetone and ethanol

Abstract

The invention relates to a method for extracting anthocyanin in orange osmanthus fruits by using a mixed solution of acetone and ethanol, which comprises the following steps of: extracting plant pigment in the orange osmanthus fruits by using acetone and ethanol in different proportions as extracting solutions, and further purifying to prepare the orange osmanthus anthocyanin. The anthocyanin of the orange osmanthus fruits is extracted under the normal-temperature (25 DEG C) dark condition, so that volatilization of an extraction solvent and photolysis of the anthocyanin are avoided. The extraction rate of the anthocyanin in the orange osmanthus fruits is increased along with the increase of the ratio of acetone to ethanol, and the anthocyanin content (51.92 + / -2.34 g / g powder) and the total phenol content (13.92 + / -0.98 mg gallic acid / g anthocyanin) have the highest value when the ratio of acetone to ethanol reaches 80: 20, which indicates that the anthocyanin in the orange osmanthus fruits can be effectively extracted by the method disclosed by the invention.

Description

Technical Field

[0001] The invention belongs to the technical field of anthocyanin extraction, and particularly relates to a method for extracting anthocyanin from osmanthus fragrans fruits by utilizing a mixed solution of acetone and ethanol. Background Art

[0002] Anthocyanidins are a class of water-soluble pigments found widely in plants and belong to the flavonoid class of compounds. Derived from nature, they are numerous, have a wide range of effects, and are low in toxic side effects. As an active ingredient with health benefits, they have become a hot topic in antioxidant research and development, playing an increasingly important role in the prevention and treatment of cardiovascular disease. If rationally developed and utilized, they have great economic value and represent a promising botanical medicine.

[0003] Osmanthus fragrans Lour., a member of the Oleaceae family, is an evergreen broadleaf shrub or small tree species endemic to China. Its ecological and economic value have garnered increasing attention. my country boasts numerous varieties of sweet osmanthus, characterized by high quality, abundant yield, and a rich, fragrant aroma. The fruit of sweet osmanthus fragrans, rich in anthocyanins, represents a resource with enormous potential for utilization.

[0004] Solvent extraction is the most commonly used method for extracting anthocyanins. The principle is to select a solvent with high solubility for anthocyanins and low solubility for other impurities based on the solubility properties of various plant components in the solvent. Common solvents include water, methanol, ethanol, acetone, etc. Solvent extraction is low-cost, simple, and easy to operate. The present invention uses acetone and ethanol as the extracting solution to extract anthocyanins from osmanthus fragrans fruit at room temperature (25°C). Summary of the Invention

[0005] The present invention aims to provide a method for extracting anthocyanins from osmanthus fragrans fruits by using a mixed solution of acetone and ethanol.

[0006] To achieve the above-mentioned and other related purposes, the present invention provides a technical solution: a method for extracting anthocyanins from osmanthus fragrans fruits using a mixed solution of acetone and ethanol, comprising the following steps:

[0007] Step 1: clean the osmanthus fruit, split it open and pick out the core to obtain the osmanthus peel; grind the dried osmanthus peel to obtain osmanthus peel powder;

[0008] Step 2: Acetone and ethanol are mixed in a volume ratio of 20-100:80-0 to obtain an extraction solution; the osmanthus peel powder is added to the extraction solution, and stirred at room temperature in the dark for 20-30 hours; after the extraction, the mixed solution is filtered to remove the residue; the solution after the initial filtration is further centrifuged, and the supernatant is taken, and the supernatant is filtered twice with filter paper to remove impurities in the solution to obtain an extract;

[0009] Step 3: First, swell the macroporous resin with ethanol aqueous solution for 10-15 hours, then filter to remove the solvent, then soak it with NaOH solution and HCl solution for 10-15 hours respectively, and finally rinse the treated macroporous resin with distilled water and pack it into a column to obtain a macroporous resin column;

[0010] Step 4: pouring the extract obtained in step 2 into a macroporous resin column, performing gradient elution with ethanol aqueous solution with volume fractions of 10%, 30%, 50%, 70% and 90%, respectively, and concentrating the obtained anthocyanin solution first. The obtained concentrated solution is placed in a freeze dryer and freeze-dried; finally, anthocyanin from osmanthus peel is obtained.

[0011] The preferred technical solution is: in step 2, the centrifugal process conditions are: rotation speed 15000-25000 rpm.

[0012] The preferred technical solution is: in step 3, the macroporous resin is macroporous resin Amberlite XAD-7, with a density of 1.05 g / mL, a particle size of 20-60 mesh, and a specific surface area of ​​450 m 2 / g, pore volume 1.14mL / g, average pore diameter 90nm.

[0013] The preferred technical solution is: the process conditions for true freeze drying are: vacuum gauge pressure -0.09MPa to -0.10MPa, and temperature -80 to -60°C.

[0014] Due to the use of the above technical solution, the present invention has the following advantages compared with the prior art:

[0015] 1. Macroporous resin is used to purify and remove impurities, pigments, polysaccharides and other components in the osmanthus peel extract. Compared with traditional purification methods, its processing cost is lower, the efficiency is higher, and the operation method is simple and easy.

[0016] 2. Vacuum freeze-drying can maximize the retention of the functional components of anthocyanins. In a low-temperature vacuum environment, heat-sensitive natural pigments are not easily destroyed. The vacuum environment isolates oxygen and reduces the oxidation reaction of anthocyanins. At the same time, the water content of anthocyanins is reduced to 1%-3% after dehydration, effectively inhibiting microbial activity. It can be stored at room temperature for 3-5 years.

[0017] 3. When the proportion of acetone solution in the extract increases, the anthocyanin extraction rate tends to increase. This is because anthocyanin molecules contain multiple benzene rings and ether bonds, making them highly oil-soluble. At the same time, a large number of hydroxyl groups are attached to the molecular skeleton, making them very soluble in water. Acetone, which is both oil-soluble and water-soluble, matches this, increasing the solubility of anthocyanins in acetone solution and thus the extraction rate. However, when the extraction solvent is 100% acetone, the anthocyanin extraction rate decreases. This is due to the reduced dissolution of some alcohol-soluble pigments in this solvent. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Relationship between different ratios of acetone and ethanol extracts and anthocyanin content.

[0019] Figure 2 Gallic acid standard curve.

[0020] Figure 3 Relationship between different ratios of acetone and ethanol extracts and total phenol content. DETAILED DESCRIPTION

[0021] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in these embodiments.

[0022] See also Figure 1-3 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention. Therefore, they have no technical substantive significance. Any modification of the structure, change in the proportional relationship or adjustment of the size. The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional consumables and biochemical reagent stores.

[0023] Example 1: A method for extracting anthocyanins from osmanthus fragrans fruits using a mixed solution of acetone and ethanol

[0024] A method for extracting anthocyanins from osmanthus fragrans fruits using a mixed solution of acetone and ethanol, comprising the following steps:

[0025] Step 1: Sample selection, sampling, cleaning, core removal, drying and grinding into powder

[0026] Select healthy and ripe Osmanthus fragrans plants, and choose fruits with intact appearance and no obvious diseases and insect pests as samples. Place the collected fruits in clean water and gently stir to remove surface dust and impurities. Change the water several times until the peel is clean and then remove the core. Spread the washed and pitted Osmanthus fragrans peel evenly on newspaper and place it in a 37°C constant temperature incubator. Turn it over every 2 hours until the fruit is fully dried (the difference in mass before and after is less than 1g). Pour the dried Osmanthus fragrans peel into a mortar and grind it thoroughly to a particle size of ≦5μm. Place it in a dry, sealed brown container and store it in a cool and dry place for later use.

[0027] Step 2: Extraction of osmanthus peel

[0028] Mix acetone and ethanol in a volume ratio of 20:80 to prepare 100mL of extraction solution. Accurately weigh 5.0g of osmanthus peel powder and add it to the extraction solution. Place it in a magnetic stirrer and stir it in the dark at room temperature for 24 hours (25°C, 800rpm). After the extraction, the mixed solution is preliminarily filtered using a 100-mesh filter cloth to remove the residue. The solution after the initial filtration is further centrifuged (20000rpm, 10min) and the supernatant is taken. It is then filtered twice with filter paper (5μm) to remove impurities in the solution. The extract is sealed intact and placed in a refrigerator (4°C) for use.

[0029] Step 3: Macroporous resin pretreatment

[0030] A macroporous resin (Amberlite XAD-7) was selected to purify anthocyanins from the fruit peel of Osmanthus fragrans. The resin requires pretreatment before use to remove any residual inert solvent from the resin pores. First, the resin was fully swollen with 95% (v / v) ethanol in water for 12 hours, after which the excess solvent was filtered through a 100-mesh filter cloth. The resin was then soaked in 5% (v / v) NaOH and 5% (v / v) HCl solutions for 12 hours each, followed by rinsing with distilled water.

[0031] Step 4: Collection of anthocyanins from osmanthus peel

[0032] The anthocyanin solution obtained in step 2 was poured into a macroporous resin column and gradient eluted with 10%, 30%, 50%, 70% and 90% ethanol aqueous solutions, respectively. The obtained anthocyanin solution was concentrated using a vacuum rotary evaporator at 40°C for 30 seconds, and the obtained concentrate was freeze-dried in a freeze dryer for 26.5 hours. Finally, the anthocyanin powder of the osmanthus peel was obtained, placed in a brown reagent bottle, and stored at 4°C.

[0033] Step 5: Extract anthocyanin content test

[0034] This experiment adopted the pH differential method and TU-1810 UV spectrophotometer to test the anthocyanin content at 25℃.

[0035] (1) Preparation of pH 1.0 buffer solution: Using a pH meter as an indicator, dissolve 1.86 g of potassium chloride in 980 mL of distilled water. Adjust the pH of the potassium chloride solution to 1.0 with 1 mol / L hydrochloric acid solution, and then make up to 1000 mL.

[0036] (2) Preparation of pH 4.5 buffer solution: Using a pH meter as an indicator, dissolve 32.81 g of anhydrous sodium acetate in 980 mL of distilled water. Adjust the pH of the potassium chloride solution to 4.5 with 1 mol / L hydrochloric acid solution, and then make up to 1000 mL.

[0037] (3) Weigh 0.1 g of anthocyanin powder from osmanthus peel and dissolve it in 50 mL of distilled water to obtain a 2 mg / mL anthocyanin aqueous solution. Take 5 mL of pH 1.0 buffer solution and 5 mL of pH 4.5 buffer solution and add them to the 2 mg / mL anthocyanin aqueous solution (5 mL). Obtain two 10 mL solutions and measure the absorbance of each solution at 530 nm and 700 nm using a UV-visible spectrophotometer. Measure each sample three times and take the average value. Finally, calculate the anthocyanin content according to the following formula:

[0038]

[0039] TAC is anthocyanin content; D f is the dilution multiple; A is the absorbance difference (A 530 -A 700 )pH1.0-(A 530 -A 700 )pH4.5; M W is the molecular weight of cyanidin-3-glucoside, 449.2 g / moL; ε is the absorptivity, 26900 L / moL·cm.

[0040] The total anthocyanin content under this extraction condition was 35.25±2.17g / g anthocyanin.

[0041] Step 6: Extract total phenol content test

[0042] (1) Preparation of gallic acid standard curve

[0043] This experiment adopted the Folin-phenol method, using a TU-1810 UV spectrophotometer at 25°C and gallic acid as a standard to calibrate the total phenol content of the substance.

[0044] (1) Accurately prepare a series of gallic acid solutions with concentrations (2, 4, 6, 8, and 10 μg / mL): Dissolve 0.1 g of gallic acid in a 100 mL volumetric flask and dilute with distilled water to the above concentration gradient. Take 1 mL of each gallic acid dilution solution in a colorimetric tube, then add 1 mL of 20% (v / v) Folin phenol reagent and 3 mL of Na2CO3 solution (20%, g / v), mix for 15 seconds, and place in a constant temperature shaking incubator (110 rpm, 25°C, dark conditions) to react for 30 minutes. Finally, measure the absorbance of the solution at 765 nm using a UV spectrophotometer. The aqueous solution serves as a blank control group.

[0045] (2) Determination of total phenol content in cinnamon peel extract

[0046] 1 mL of anthocyanin solution extracted from the fruit peel of each plant under various conditions was added to 1 mL of 20% (v / v) Folin's phenol reagent and 3 mL of 20% (g / v) sodium carbonate solution. The mixture was allowed to react in the dark for 30 minutes. The absorbance of the sample solution at 765 nm was measured using a UV-visible spectrophotometer. The total phenolic content was expressed as gallic acid equivalents (mg gallic acid / g anthocyanin). Each sample was measured three times, and the average value was taken. Finally, the total phenolic content was calculated according to the following formula:

[0047] TPC=C×V / M

[0048] Where C is the gallic acid concentration (mg / mL), V (mL) is the volume of the solution, and M (mg) is the mass of anthocyanin powder.

[0049] The total anthocyanin content under this extraction condition was 11.25±0.57mg gallic acid / g anthocyanin.

[0050] Example 2: A method for extracting anthocyanins from osmanthus fragrans fruits using a mixed solution of acetone and ethanol

[0051] A method for extracting anthocyanins from osmanthus fragrans fruits using a mixed solution of acetone and ethanol, comprising the following steps:

[0052] Step 1: Extraction of osmanthus peel

[0053] Mix acetone and ethanol in a volume ratio of 40:60 to prepare 100mL of extraction solution. Accurately weigh 5.0g of osmanthus peel powder and add it to the extraction solution. Place it in a magnetic stirrer and stir it in the dark at room temperature for 24 hours (25°C, 800rpm). After the extraction, the mixed solution is preliminarily filtered using a 100-mesh filter cloth to remove the residue. The solution after the initial filtration is further centrifuged (20000rpm, 10min) and the supernatant is taken. It is filtered twice with filter paper (5μm) to remove impurities in the solution. The extract is sealed intact and placed in a refrigerator (4°C) for use.

[0054] Step 2: Macroporous resin pretreatment

[0055] A macroporous resin (Amberlite XAD-7) was selected to purify anthocyanins from the fruit peel of Osmanthus fragrans. The resin requires pretreatment before use to remove any residual inert solvent from the resin pores. First, the resin was fully swollen with 95% (v / v) ethanol in water for 12 hours, after which the excess solvent was filtered through a 100-mesh filter cloth. The resin was then soaked in 5% (v / v) NaOH and 5% (v / v) HCl solutions for 12 hours each, followed by rinsing with distilled water.

[0056] Step 3: Collection of anthocyanins from osmanthus peel

[0057] The anthocyanin solution obtained in step 1 was poured into a macroporous resin column and gradient eluted with 10%, 30%, 50%, 70% and 90% ethanol aqueous solutions, respectively. The obtained anthocyanin solution was concentrated using a vacuum rotary evaporator at 40° C. for 30 seconds, and the obtained concentrate was freeze-dried in a freeze dryer for 26.5 hours. Finally, the anthocyanin powder of the osmanthus peel was obtained, placed in a brown reagent bottle, and stored at 4° C.

[0058] Step 4: Extract anthocyanin content test

[0059] This experiment adopted the pH differential method and TU-1810 UV spectrophotometer to test the anthocyanin content at 25℃.

[0060] (1) Preparation of pH 1.0 buffer solution: Using a pH meter as an indicator, dissolve 1.86 g of potassium chloride in 980 mL of distilled water. Adjust the pH of the potassium chloride solution to 1.0 with 1 mol / L hydrochloric acid solution, and then make up to 1000 mL.

[0061] (2) Preparation of pH 4.5 buffer solution: Using a pH meter as an indicator, dissolve 32.81 g of anhydrous sodium acetate in 980 mL of distilled water. Adjust the pH of the potassium chloride solution to 4.5 with 1 mol / L hydrochloric acid solution, and then make up to 1000 mL.

[0062] (3) Weigh 0.1 g of anthocyanin powder from osmanthus peel and dissolve it in 50 mL of distilled water to obtain a 2 mg / mL anthocyanin aqueous solution. Take 5 mL of pH 1.0 buffer solution and 5 mL of pH 4.5 buffer solution and add them to the 2 mg / mL anthocyanin aqueous solution (5 mL). Obtain two 10 mL solutions and measure the absorbance of each solution at 530 nm and 700 nm using a UV-visible spectrophotometer. Measure each sample three times and take the average value. Finally, calculate the anthocyanin content according to the following formula:

[0063]

[0064] TAC is anthocyanin content; D f is the dilution multiple; A is the absorbance difference (A 530 -A 700 )pH1.0-(A 530 -A 700 )pH4.5; M W is the molecular weight of cyanidin-3-glucoside, 449.2 g / moL; ε is the absorptivity, 26900 L / moL·cm.

[0065] The total anthocyanin content under this extraction condition was 39.16±2.26g / g anthocyanin.

[0066] Step 5: Total phenol content test of extract

[0067] (1) Preparation of gallic acid standard curve

[0068] This experiment adopted the Folin-phenol method, using a TU-1810 UV spectrophotometer at 25°C and gallic acid as a standard to calibrate the total phenol content of the substance.

[0069] (1) Accurately prepare a series of gallic acid solutions with concentrations (2, 4, 6, 8, and 10 μg / mL): Dissolve 0.1 g of gallic acid in a 100 mL volumetric flask and dilute with distilled water to the above concentration gradient. Take 1 mL of each gallic acid dilution solution in a colorimetric tube, then add 1 mL of 20% (v / v) Folin phenol reagent and 3 mL of Na2CO3 solution (20%, g / v), mix for 15 seconds, and place in a constant temperature shaking incubator (110 rpm, 25°C, dark conditions) to react for 30 minutes. Finally, measure the absorbance of the solution at 765 nm using a UV spectrophotometer. The aqueous solution serves as a blank control group.

[0070] (2) Determination of total phenol content in cinnamon peel extract

[0071] 1 mL of anthocyanin solution extracted from the fruit peel of each plant under various conditions was added to 1 mL of 20% (v / v) Folin's phenol reagent and 3 mL of 20% (g / v) sodium carbonate solution. The mixture was allowed to react in the dark for 30 minutes. The absorbance of the sample solution at 765 nm was measured using a UV-visible spectrophotometer. The total phenolic content was expressed as gallic acid equivalents (mg gallic acid / g anthocyanin). Each sample was measured three times, and the average value was taken. Finally, the total phenolic content was calculated according to the following formula:

[0072] TPC=C×V / M

[0073] Where C is the gallic acid concentration (mg / mL), V (mL) is the volume of the solution, and M (mg) is the mass of anthocyanin powder.

[0074] The total anthocyanin content under this extraction condition was 11.96±0.46mg gallic acid / g anthocyanin.

[0075] Example 3: A method for extracting anthocyanins from osmanthus fragrans fruits using a mixed solution of acetone and ethanol

[0076] A method for extracting anthocyanins from osmanthus fragrans fruits using a mixed solution of acetone and ethanol, comprising the following steps:

[0077] Step 1: Extraction of osmanthus peel

[0078] Mix acetone and ethanol in a volume ratio of 60:40 to prepare 100mL of extraction solution. Accurately weigh 5.0g of osmanthus peel powder and add it to the extraction solution. Place it in a magnetic stirrer and stir it in the dark at room temperature for 24 hours (25°C, 800rpm). After the extraction, the mixed solution is preliminarily filtered using a 100-mesh filter cloth to remove the residue. The solution after the initial filtration is further centrifuged (20000rpm, 10min) and the supernatant is taken. It is filtered twice with filter paper (5μm) to remove impurities in the solution. The extract is sealed intact and placed in a refrigerator (4°C) for use.

[0079] Step 2: Macroporous resin pretreatment

[0080] A macroporous resin (Amberlite XAD-7) was selected to purify anthocyanins from the fruit peel of Osmanthus fragrans. The resin requires pretreatment before use to remove any residual inert solvent from the resin pores. First, the resin was fully swollen with 95% (v / v) ethanol in water for 12 hours, after which the excess solvent was filtered through a 100-mesh filter cloth. The resin was then soaked in 5% (v / v) NaOH and 5% (v / v) HCl solutions for 12 hours each, followed by rinsing with distilled water.

[0081] Step 3: Collection of anthocyanins from osmanthus peel

[0082] The anthocyanin solution obtained in step 1 was poured into a macroporous resin column and gradient eluted with 10%, 30%, 50%, 70% and 90% ethanol aqueous solutions, respectively. The obtained anthocyanin solution was concentrated using a vacuum rotary evaporator at 40° C. for 30 seconds, and the obtained concentrate was freeze-dried in a freeze dryer for 26.5 hours. Finally, the anthocyanin powder of the osmanthus peel was obtained, placed in a brown reagent bottle, and stored at 4° C.

[0083] Step 4: Extract anthocyanin content test

[0084] This experiment adopted the pH differential method and TU-1810 UV spectrophotometer to test the anthocyanin content at 25℃.

[0085] (1) Preparation of pH 1.0 buffer solution: Using a pH meter as an indicator, dissolve 1.86 g of potassium chloride in 980 mL of distilled water. Adjust the pH of the potassium chloride solution to 1.0 with 1 mol / L hydrochloric acid solution, and then make up to 1000 mL.

[0086] (2) Preparation of pH 4.5 buffer solution: Using a pH meter as an indicator, dissolve 32.81 g of anhydrous sodium acetate in 980 mL of distilled water. Adjust the pH of the potassium chloride solution to 4.5 with 1 mol / L hydrochloric acid solution, and then make up to 1000 mL.

[0087] (3) Weigh 0.1 g of anthocyanin powder from osmanthus peel and dissolve it in 50 mL of distilled water to obtain a 2 mg / mL anthocyanin aqueous solution. Take 5 mL of pH 1.0 buffer solution and 5 mL of pH 4.5 buffer solution and add them to the 2 mg / mL anthocyanin aqueous solution (5 mL). Obtain two 10 mL solutions and measure the absorbance of each solution at 530 nm and 700 nm using a UV-visible spectrophotometer. Measure each sample three times and take the average value. Finally, calculate the anthocyanin content according to the following formula:

[0088]

[0089] TAC is anthocyanin content; D f is the dilution factor; A is the absorbance difference (A 530 -A 700 )pH1.0-(A 530 -A 700 )pH4.5; M W is the molecular weight of cyanidin-3-glucoside, 449.2 g / moL; ε is the absorptivity, 26900 L / moL·cm.

[0090] The total anthocyanin content under this extraction condition was 46.92±2.18g / g anthocyanin.

[0091] Step 5: Extract total phenol content test

[0092] (1) Preparation of gallic acid standard curve

[0093] This experiment adopted the Folin-phenol method, using a TU-1810 UV spectrophotometer at 25°C and gallic acid as a standard to calibrate the total phenol content of the substance.

[0094] (1) Accurately prepare a series of gallic acid solutions with concentrations (2, 4, 6, 8, and 10 μg / mL): Dissolve 0.1 g of gallic acid in a 100 mL volumetric flask and dilute with distilled water to the above concentration gradient. Take 1 mL of each gallic acid dilution solution in a colorimetric tube, then add 1 mL of 20% (v / v) Folin phenol reagent and 3 mL of Na2CO3 solution (20%, g / v), mix for 15 seconds, and place in a constant temperature shaking incubator (110 rpm, 25°C, dark conditions) to react for 30 minutes. Finally, measure the absorbance of the solution at 765 nm using a UV spectrophotometer. The aqueous solution serves as a blank control group.

[0095] (2) Determination of total phenol content in cinnamon peel extract

[0096] 1 mL of anthocyanin solution extracted from the fruit peel of each plant under various conditions was added to 1 mL of 20% (v / v) Folin's phenol reagent and 3 mL of 20% (g / v) sodium carbonate solution. The mixture was allowed to react in the dark for 30 minutes. The absorbance of the sample solution at 765 nm was measured using a UV-visible spectrophotometer. The total phenolic content was expressed as gallic acid equivalents (mg gallic acid / g anthocyanin). Each sample was measured three times, and the average value was taken. Finally, the total phenolic content was calculated according to the following formula:

[0097] TPC=C×V / M

[0098] Where C is the gallic acid concentration (mg / mL), V (mL) is the volume of the solution, and M (mg) is the mass of anthocyanin powder.

[0099] The total anthocyanin content under this extraction condition was 12.87±0.58mg gallic acid / g anthocyanin.

[0100] Example 4: A method for extracting anthocyanins from osmanthus fragrans fruits using a mixed solution of acetone and ethanol

[0101] A method for extracting anthocyanins from osmanthus fragrans fruits using a mixed solution of acetone and ethanol, comprising the following steps:

[0102] Step 1: Extraction of osmanthus peel

[0103] Mix acetone and ethanol in a volume ratio of 80:20 to prepare 100mL of extraction solution. Accurately weigh 5.0g of osmanthus peel powder and add it to the extraction solution. Place it in a magnetic stirrer and stir it in the dark at room temperature for 24 hours (25°C, 800rpm). After the extraction, the mixed solution is preliminarily filtered using a 100-mesh filter cloth to remove the residue. The solution after the initial filtration is further centrifuged (20000rpm, 10min) and the supernatant is taken. It is then filtered twice with filter paper (5μm) to remove impurities in the solution. The extract is sealed intact and placed in a refrigerator (4°C) for use.

[0104] Step 2: Macroporous resin pretreatment

[0105] A macroporous resin (Amberlite XAD-7) was selected to purify anthocyanins from the fruit peel of Osmanthus fragrans. The resin requires pretreatment before use to remove any residual inert solvent from the resin pores. First, the resin was fully swollen with 95% (v / v) ethanol in water for 12 hours, after which the excess solvent was filtered through a 100-mesh filter cloth. The resin was then soaked in 5% (v / v) NaOH and 5% (v / v) HCl solutions for 12 hours each, followed by rinsing with distilled water.

[0106] Step 3: Collection of anthocyanins from osmanthus peel

[0107] The anthocyanin solution obtained in step 1 was poured into a macroporous resin column and gradient eluted with 10%, 30%, 50%, 70% and 90% ethanol aqueous solutions, respectively. The obtained anthocyanin solution was concentrated using a vacuum rotary evaporator at 40° C. for 30 seconds, and the obtained concentrate was freeze-dried in a freeze dryer for 26.5 hours. Finally, the anthocyanin powder of the osmanthus peel was obtained, placed in a brown reagent bottle, and stored at 4° C.

[0108] Step 4: Extract anthocyanin content test

[0109] This experiment adopted the pH differential method and TU-1810 UV spectrophotometer to test the anthocyanin content at 25℃.

[0110] (1) Preparation of pH 1.0 buffer solution: Using a pH meter as an indicator, dissolve 1.86 g of potassium chloride in 980 mL of distilled water. Adjust the pH of the potassium chloride solution to 1.0 with 1 mol / L hydrochloric acid solution, and then make up to 1000 mL.

[0111] (2) Preparation of pH 4.5 buffer solution: Using a pH meter as an indicator, dissolve 32.81 g of anhydrous sodium acetate in 980 mL of distilled water. Adjust the pH of the potassium chloride solution to 4.5 with 1 mol / L hydrochloric acid solution, and then make up to 1000 mL.

[0112] (3) Weigh 0.1 g of anthocyanin powder from osmanthus peel and dissolve it in 50 mL of distilled water to obtain a 2 mg / mL anthocyanin aqueous solution. Take 5 mL of pH 1.0 buffer solution and 5 mL of pH 4.5 buffer solution and add them to the 2 mg / mL anthocyanin aqueous solution (5 mL). Obtain two 10 mL solutions and measure the absorbance of each solution at 530 nm and 700 nm using a UV-visible spectrophotometer. Measure each sample three times and take the average value. Finally, calculate the anthocyanin content according to the following formula:

[0113]

[0114] TAC is anthocyanin content; D f is the dilution factor; A is the absorbance difference (A 530 -A 700 )pH1.0-(A 530 -A 700 )pH4.5; M W is the molecular weight of cyanidin-3-glucoside, 449.2 g / moL; ε is the absorptivity, 26900 L / moL·cm.

[0115] The total anthocyanin content under this extraction condition was 51.92±2.34g / g anthocyanin.

[0116] Step 5: Extract total phenol content test

[0117] (1) Preparation of gallic acid standard curve

[0118] This experiment adopted the Folin-phenol method, using a TU-1810 UV spectrophotometer at 25°C and gallic acid as a standard to calibrate the total phenol content of the substance.

[0119] (1) Accurately prepare a series of gallic acid solutions with concentrations (2, 4, 6, 8, and 10 μg / mL): Dissolve 0.1 g of gallic acid in a 100 mL volumetric flask and dilute with distilled water to the above concentration gradient. Take 1 mL of each gallic acid dilution solution in a colorimetric tube, then add 1 mL of 20% (v / v) Folin phenol reagent and 3 mL of Na2CO3 solution (20%, g / v), mix for 15 seconds, and place in a constant temperature shaking incubator (110 rpm, 25°C, dark conditions) to react for 30 minutes. Finally, measure the absorbance of the solution at 765 nm using a UV spectrophotometer. The aqueous solution serves as a blank control group.

[0120] (2) Determination of total phenol content in cinnamon peel extract

[0121] 1 mL of anthocyanin solution extracted from the fruit peel of each plant under various conditions was added to 1 mL of 20% (v / v) Folin's phenol reagent and 3 mL of 20% (g / v) sodium carbonate solution. The mixture was allowed to react in the dark for 30 minutes. The absorbance of the sample solution at 765 nm was measured using a UV-visible spectrophotometer. The total phenolic content was expressed as gallic acid equivalents (mg gallic acid / g anthocyanin). Each sample was measured three times, and the average value was taken. Finally, the total phenolic content was calculated according to the following formula:

[0122] TPC=C×V / M

[0123] Where C is the gallic acid concentration (mg / mL), V (mL) is the volume of the solution, and M (mg) is the mass of anthocyanin powder.

[0124] The total anthocyanin content under this extraction condition was 13.92±0.98mg gallic acid / g anthocyanin.

[0125] Example 5: A method for extracting anthocyanins from osmanthus fragrans fruits using a mixed solution of acetone and ethanol

[0126] A method for extracting anthocyanins from osmanthus fragrans fruits using a mixed solution of acetone and ethanol, comprising the following steps:

[0127] Step 1: Extraction of osmanthus peel

[0128] Mix acetone and ethanol in a volume ratio of 100:0 to prepare 100mL of extraction solution. Accurately weigh 5.0g of osmanthus peel powder and add it to the extraction solution. Place it in a magnetic stirrer and stir it in the dark at room temperature for 24 hours (25°C, 800rpm). After the extraction, the mixed solution is preliminarily filtered using a 100-mesh filter cloth to remove the residue. The solution after the initial filtration is further centrifuged (20,000rpm, 10min) and the supernatant is taken. It is then filtered twice with filter paper (5μm) to remove impurities in the solution. The extract is sealed intact and placed in a refrigerator (4°C) for use.

[0129] Step 2: Macroporous resin pretreatment

[0130] A macroporous resin (Amberlite XAD-7) was selected to purify anthocyanins from the fruit peel of Osmanthus fragrans. The resin requires pretreatment before use to remove any residual inert solvent from the resin pores. First, the resin was fully swollen with 95% (v / v) ethanol in water for 12 hours, after which the excess solvent was filtered through a 100-mesh filter cloth. The resin was then soaked in 5% (v / v) NaOH and 5% (v / v) HCl solutions for 12 hours each, followed by rinsing with distilled water.

[0131] Step 3: Collection of anthocyanins from osmanthus peel

[0132] The anthocyanin solution obtained in step 1 was poured into a macroporous resin column and gradient eluted with 10%, 30%, 50%, 70% and 90% ethanol aqueous solutions, respectively. The obtained anthocyanin solution was concentrated using a vacuum rotary evaporator at 40° C. for 30 seconds, and the obtained concentrate was freeze-dried in a freeze dryer for 26.5 hours. Finally, the anthocyanin powder of the osmanthus peel was obtained, placed in a brown reagent bottle, and stored at 4° C.

[0133] Step 4: Extract anthocyanin content test

[0134] This experiment adopted the pH differential method and TU-1810 UV spectrophotometer to test the anthocyanin content at 25℃.

[0135] (1) Preparation of pH 1.0 buffer solution: Using a pH meter as an indicator, dissolve 1.86 g of potassium chloride in 980 mL of distilled water. Adjust the pH of the potassium chloride solution to 1.0 with 1 mol / L hydrochloric acid solution, and then make up to 1000 mL.

[0136] (2) Preparation of pH 4.5 buffer solution: Using a pH meter as an indicator, dissolve 32.81 g of anhydrous sodium acetate in 980 mL of distilled water. Adjust the pH of the potassium chloride solution to 4.5 with 1 mol / L hydrochloric acid solution, and then make up to 1000 mL.

[0137] (3) Weigh 0.1 g of anthocyanin powder from osmanthus peel and dissolve it in 50 mL of distilled water to obtain a 2 mg / mL anthocyanin aqueous solution. Take 5 mL of pH 1.0 buffer solution and 5 mL of pH 4.5 buffer solution and add them to the 2 mg / mL anthocyanin aqueous solution (5 mL). Obtain two 10 mL solutions and measure the absorbance of each solution at 530 nm and 700 nm using a UV-visible spectrophotometer. Measure each sample three times and take the average value. Finally, calculate the anthocyanin content according to the following formula:

[0138]

[0139] TAC is anthocyanin content; D f is the dilution factor; A is the absorbance difference (A 530 -A 700 )pH1.0-(A 530 -A 700 )pH4.5; M W is the molecular weight of cyanidin-3-glucoside, 449.2 g / moL; ε is the absorptivity, 26900 L / moL·cm.

[0140] The total anthocyanin content under this extraction condition was 47.24±2.98g / g anthocyanin.

[0141] Step 5: Extract total phenol content test

[0142] (1) Preparation of gallic acid standard curve

[0143] This experiment adopted the Folin-phenol method, using a TU-1810 UV spectrophotometer at 25°C and gallic acid as a standard to calibrate the total phenol content of the substance.

[0144] (1) Accurately prepare a series of gallic acid solutions with concentrations (2, 4, 6, 8, and 10 μg / mL): Dissolve 0.1 g of gallic acid in a 100 mL volumetric flask and dilute with distilled water to the above concentration gradient. Take 1 mL of each gallic acid dilution solution in a colorimetric tube, then add 1 mL of 20% (v / v) Folin phenol reagent and 3 mL of Na2CO3 solution (20%, g / v), mix for 15 seconds, and place in a constant temperature shaking incubator (110 rpm, 25°C, dark conditions) to react for 30 minutes. Finally, measure the absorbance of the solution at 765 nm using a UV spectrophotometer. The aqueous solution serves as a blank control group.

[0145] (2) Determination of total phenol content in cinnamon peel extract

[0146] 1 mL of anthocyanin solution extracted from the fruit peel of each plant under various conditions was added to 1 mL of 20% (v / v) Folin's phenol reagent and 3 mL of 20% (g / v) sodium carbonate solution. The mixture was allowed to react in the dark for 30 minutes. The absorbance of the sample solution at 765 nm was measured using a UV-visible spectrophotometer. The total phenolic content was expressed as gallic acid equivalents (mg gallic acid / g anthocyanin). Each sample was measured three times, and the average value was taken. Finally, the total phenolic content was calculated according to the following formula:

[0147] TPC=C×V / M

[0148] Where C is the gallic acid concentration (mg / mL), V (mL) is the volume of the solution, and M (mg) is the mass of anthocyanin powder.

[0149] The total anthocyanin content under this extraction condition was 12.63±0.57mg gallic acid / g anthocyanin.

[0150] Table 1 Anthocyanin content and total phenol content of Osmanthus fragrans peel extract under different extraction conditions.

[0151]

[0152] The above description is only used to explain the preferred embodiments of the present invention and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. A method for extracting anthocyanins from osmanthus fragrans fruits using a mixed solution of acetone and ethanol, characterized in that: The following steps are involved: Step 1: clean the osmanthus fruit, split it open and pick out the core to obtain the osmanthus peel; grind the dried osmanthus peel to obtain osmanthus peel powder; Step 2: Acetone and ethanol are mixed in a volume ratio of 20-100:80-0 to obtain an extraction solution; the osmanthus peel powder is added to the extraction solution, and stirred at room temperature in the dark for 20-30 hours; after the extraction, the mixed solution is filtered to remove the residue; the solution after the initial filtration is further centrifuged, and the supernatant is taken, and the supernatant is filtered twice with filter paper to remove impurities in the solution to obtain an extract; Step 3: First, swell the macroporous resin with ethanol aqueous solution for 10-15 hours, then filter to remove the solvent, then soak it with NaOH solution and HCl solution for 10-15 hours respectively, and finally rinse the treated macroporous resin with distilled water and pack it into a column to obtain a macroporous resin column; Step 4: pouring the extract obtained in step 2 into a macroporous resin column, performing gradient elution with ethanol aqueous solution with volume fractions of 10%, 30%, 50%, 70% and 90%, respectively, and concentrating the obtained anthocyanin solution first. The obtained concentrated solution is placed in a freeze dryer and freeze-dried; finally, anthocyanin from osmanthus peel is obtained.

2. The method for extracting anthocyanins from osmanthus fragrans fruits using a mixed solution of acetone and ethanol according to claim 1, wherein: In step 2, the centrifugal process conditions are: rotation speed 15000-25000 rpm.

3. The method for extracting anthocyanins from osmanthus fragrans fruits using a mixed solution of acetone and ethanol according to claim 1, wherein: In step 3, the macroporous resin is Amberlite XAD-7, with a density of 1.05 g / mL, a particle size of 20-60 mesh, and a specific surface area of ​​450 m 2 / g, pore volume 1.14mL / g, average pore diameter 90nm.

4. The method for extracting anthocyanins from osmanthus fragrans fruits using a mixed solution of acetone and ethanol according to claim 1, wherein: The process conditions for true freeze drying are: vacuum gauge pressure -0.09MPa to -0.10MPa, temperature -80 to -60℃.

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