A citrus composite extract rich in flavonoids and preparation method thereof

Abstract

The present invention relates to the technical field of citrus plant extracts, and in particular to a citrus composite extract rich in flavonoids and a preparation method thereof. A preparation method for a citrus composite extract rich in flavonoids, comprising: taking citrus plant peel, crushing, adding water, nano zinc oxide and rhamnolipid, and obtaining a first filtrate and a first filter residue after ball milling; the first filtrate is concentrated and dried under reduced pressure to a constant weight, an ethanol solution is added for ultrasonic extraction, and filtered to obtain a second filtrate and a second filter residue; the first filter residue and the second filter residue are combined, and ethyl acetate is added for extraction to obtain an extract; the second filtrate and the extract are combined, concentrated and dried under reduced pressure to obtain a citrus composite extract rich in flavonoids. The improved preparation method of the citrus composite extract rich in flavonoids in the present application not only improves the extraction efficiency of the bioactive components in the peel of citrus fruits, but also significantly improves the antioxidant activity and antibacterial activity of the extract.

Description

Technical Field

[0001] The invention relates to the technical field of citrus plant extracts, in particular to a flavonoid-rich citrus composite extract and a preparation method thereof. Background Art

[0002] With the improvement of people's living standards and the enhancement of health awareness, consumers have increasing requirements for the safety and functionality of products. Traditional drugs and synthetic additives have side effects or safety hazards in some aspects. Therefore, it is particularly urgent to develop natural plant extracts as substitutes. Citrus plants are common fruits, and their extracts have a variety of biologically active ingredients, such as flavonoids, vitamin C, carotenoids, etc. These ingredients show significant biological activity in anti-oxidation, anti-inflammatory, and antibacterial aspects. Therefore, they have broad application prospects in the fields of biomedicine and health products, and have become a popular ingredient in the biomedicine and even health products industry.

[0003] However, the traditional extraction method of citrus peel extract often has poor extraction effect, so the content of bioactive components in the extracted extract is low and the bioactivity is also low, which greatly reduces the skin care effect of citrus peel extract, making it difficult to meet the demand for high value-added products in the fields of biomedicine and health care products, and limiting the application of citrus peel extract in the fields of biomedicine and health care products. Therefore, it is necessary to develop a new extraction method for citrus peel to improve the retention rate of bioactive components in citrus peel. Summary of the invention

[0004] In order to improve the retention rate of bioactive components in the peel of citrus plants, the present application provides a citrus composite extract rich in flavonoids and a preparation method thereof. The improved preparation method of the citrus composite extract rich in flavonoids in the present application not only improves the extraction efficiency of bioactive components in the peel of citrus fruits, but also significantly improves the biological activity of the citrus composite extract, especially improves the antioxidant activity and antibacterial activity of the citrus composite extract.

[0005] In the first aspect, the present application provides a method for preparing a flavonoid-rich citrus composite extract using the following technical scheme:

[0006] A method for preparing a flavonoid-rich citrus composite extract comprises the following steps:

[0007] Step 1: Take the fruit of a citrus plant, remove the pulp, keep the peel, crush it, add 5 to 10 times the volume of water to soak it, then add 2% to 3% of nano zinc oxide by weight of the peel and 0.2% to 0.5% of rhamnolipid by weight of the peel, ball mill at a speed of 1400 to 1500 r / min for 3 to 4 hours, filter, and obtain a first filtrate and a first filter residue for standby use;

[0008] Step 2: The first filtrate is concentrated and dried under reduced pressure to a constant weight, and 10 to 15 times the volume of 65% to 70% ethanol solution is added for redissolution. After ultrasonic extraction, the mixture is allowed to stand for precipitation and filtered to obtain a second filtrate and a second filter residue for later use;

[0009] Step 3: Combine the first filter residue and the second filter residue, add 2 to 3 times the volume of ethyl acetate, stir evenly, extract, extract 2 to 3 times in total, and combine the extracts;

[0010] Step 4: Combine the second filtrate and the extract, concentrate under reduced pressure and dry, to obtain a citrus composite extract rich in flavonoids.

[0011] In the above technical scheme, the present application optimizes step 1 and directly extracts fresh peels, thereby significantly reducing the loss of bioactive ingredients in the extraction process, thereby more effectively maintaining and retaining the native biological activity of these bioactive ingredients, and innovatively introducing nano zinc oxide and rhamnolipids, utilizing the high specific surface area and reactivity of nanomaterials, and the synergistic effect of rhamnolipids, thereby significantly improving the extraction efficiency of bioactive substances in the peel. Specifically, nano zinc oxide effectively breaks the cell walls of peel cells during the ball milling process, so that the bioactive ingredients in the cells are released more smoothly, and rhamnolipids are used to optimize the surface tension of the extract, further promoting The dissolution process of the bioactive ingredients was improved, the bioactivity of these ingredients was effectively maintained, and the high quality and bioactivity of the extracted products were ensured; by optimizing step 2, the bioactive ingredients such as flavonoids in the extract were further enriched through reduced pressure concentration and drying, ethanol re-dissolution, and ultrasonic extraction, and the non-target ingredients such as pectin in the peel were separated, thereby improving the purity and bioactivity of the extract; by optimizing step 3, the bioactive ingredients such as flavonoids were effectively separated by ethyl acetate extraction, and the extraction rate of the bioactive ingredients was further improved; finally, by optimizing the reduced pressure concentration and drying conditions of step 4, the stability and activity of the bioactive ingredients in the final extract were ensured.

[0012] Preferably, in step 1, the soaking in 5 to 10 times the volume of water is performed for 30 to 50 minutes at a temperature of 60 to 70°C, and the ball milling temperature is 50 to 70°C.

[0013] In the above technical solution, the present application further optimizes the conditions of water extraction in step 1 to avoid the destruction of bioactive components by high temperature while ensuring the extraction efficiency. In addition, the present application optimizes the temperature of ball milling to ensure the reactivity of nano zinc oxide and rhamnolipid and ensures the complete destruction of the peel cell wall during the ball milling process, thereby further increasing the content of bioactive components in the extract.

[0014] Preferably, the reduced pressure concentration and drying conditions of the first filtrate in step 2 are: 55-60°C, -0.09-0.03MPa, and the ultrasonic extraction is performed at 55-60°C for 50-60min.

[0015] In the above technical scheme, the present application further optimizes the drying and ultrasonic extraction conditions of step 2, which not only ensures the stability and activity of bioactive ingredients such as flavonoids, but also better separates non-target components, better enriches bioactive ingredients such as flavonoids, and increases the content of effective ingredients in the extract.

[0016] Preferably, the extraction conditions of step 3 are: 55-60° C., 0.3-0.5 MPa, and each extraction time is 30-50 min.

[0017] In the above technical scheme, the present application ensures the efficient extraction of bioactive ingredients such as flavonoids by further optimizing the temperature and pressure conditions in the extraction process of step 3, while reducing the interference of non-target components and further increasing the content of effective ingredients in the extract.

[0018] Preferably, the conditions for reduced pressure concentration and drying in step 4 are: 55-60° C., -0.09-0.03 MPa.

[0019] In the above technical solution, the present application further optimizes the drying conditions of step 4 to ensure the stability of the bioactive ingredients during the drying process and avoid the destruction of the ingredients by high temperature, thereby ensuring the high retention rate and high activity of the bioactive ingredients in the final product.

[0020] Preferably, the mass ratio of the peel, nano zinc oxide and rhamnolipid is 100:2.5:0.3.

[0021] In the above technical scheme, the present application further optimizes the mass ratio of peel, nano zinc oxide and rhamnolipid to ensure the synergistic effect of nano zinc oxide and rhamnolipid during the extraction process, thereby ensuring the extraction efficiency while retaining the activity of the bioactive ingredients to the maximum extent.

[0022] Preferably, the citrus plants include but are not limited to sour orange, Taiwanese lemon, lemon, Yunnan citron, orange, finger lime, etc.

[0023] Preferably, the citrus plant is Citrus aurantium.

[0024] In the above technical scheme, the present application further optimizes the composition and quality of the flavonoid-rich citrus composite extract by further selecting the species of citrus plants.

[0025] In a second aspect, the present application provides a citrus composite extract rich in flavonoids using the following technical solution:

[0026] A citrus composite extract rich in flavonoids is prepared by the preparation method of the citrus composite extract rich in flavonoids as described in the first aspect.

[0027] In the above technical scheme, the present application adopts the preparation method described in the first aspect to extract the flavonoid-rich citrus composite extract, which has a higher and more stable content of biologically active ingredients. Such extracts can be used in the fields of biomedicine, health care products, etc., to provide better antioxidant and antibacterial effects.

[0028] In summary, the present application includes at least one of the following beneficial technical effects:

[0029] The present application skips the drying and crushing steps in conventional extraction methods and directly extracts fresh peels, which not only reduces the process steps but also significantly reduces the loss of bioactive ingredients during the extraction process, thereby more effectively maintaining and retaining the native biological activity of these bioactive ingredients.

[0030] This application innovatively introduces nano zinc oxide and rhamnolipids in the extraction process, utilizing the high specific surface area and reactivity of nanomaterials and the synergistic effect of rhamnolipids to significantly improve the extraction efficiency of bioactive substances in the peel and ensure the biological activity of the bioactive ingredients.

[0031] The present application significantly improves the synergistic effect of nano zinc oxide and rhamnolipid by adopting a ball milling process in combination with a water extraction method, further effectively increases the content and purity of bioactive ingredients such as flavonoids in the extract, and at the same time ensures the stability and activity of these ingredients.

[0032] The present application further improves the extraction efficiency by optimizing the extraction steps, through re-ultrasonic extraction and precipitation of the first filtrate, and through combined extraction of the first filter residue and the second filter residue, enriches bioactive ingredients such as flavonoids, and effectively separates non-target ingredients such as pectin, thereby improving the purity and biological activity of the extract. DETAILED DESCRIPTION

[0033] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Example 1

[0034] A method for preparing a flavonoid-rich citrus composite extract comprises the following steps:

[0035] Step 1: replace the fruit of sour orange, dig out the pulp, keep 10kg of peel, break it into 200-300 mesh residue, add 8 times the volume of water, control the temperature to 65°C, soak for 40 minutes, then add 0.25kg of nano zinc oxide (particle size between 50 and 100nm) and 0.03kg of rhamnolipid (purchased from Hubei Xinyuhong Biomedicine Technology Co., Ltd., with an effective ingredient content of 90% or more), control the temperature to 60°C, ball mill at a speed of 1450r / min for 3.5h, filter, and obtain the first filtrate and the first filter residue for later use.

[0036] Step 2: The first filtrate is first concentrated and dried under reduced pressure at 60°C and -0.04MPa to constant weight, then 12 times the volume of 70% ethanol solution is added for redissolution, ultrasonic extraction is performed at 60°C for 55 minutes, and then the mixture is allowed to stand for precipitation and filtered to obtain a second filtrate and a second residue for later use.

[0037] Step 3: Combine the first filter residue and the second filter residue, add 2.5 times of ethyl acetate and stir evenly, extract at 60°C and 0.4 MPa for 40 min, repeat 3 times, combine the extracts and set aside.

[0038] Step 4: Combine the second filtrate and the extract, concentrate and dry under reduced pressure at 60° C. and −0.04 MPa to obtain a citrus composite extract rich in flavonoids. Example 2

[0039] A method for preparing a flavonoid-rich citrus composite extract, which is different from Example 1, comprises the following steps:

[0040] Step 1: replace the fruit of sour orange, dig out the pulp, keep 10kg of peel, break it into 200-300 mesh residue, add 5 times volume of water, control the temperature to 70°C, soak for 30 minutes, then add 0.2kg of nano zinc oxide and 0.02kg of rhamnolipid, control the temperature to 70°C, ball mill at a speed of 1400r / min for 3-4h, filter, obtain the first filtrate and the first filter residue, and set aside.

[0041] Step 2: The first filtrate is concentrated and dried under reduced pressure at 60°C and -0.03MPa to constant weight, then 15 times the volume of 65% ethanol solution is added for redissolution, ultrasonic extraction is performed at 60°C for 50 minutes, and then the mixture is allowed to stand for precipitation and filtered to obtain a second filtrate and a second residue for later use.

[0042] Step 3: Combine the first filter residue and the second filter residue, add 3 times the volume of ethyl acetate and stir evenly, extract at 60°C and 0.3MPa for 30min, repeat 3 times in total, combine the extracts and set aside.

[0043] Step 4: Combine the second filtrate and the extract, concentrate and dry under reduced pressure at 60° C. and −0.03 MPa to obtain a citrus composite extract rich in flavonoids. Example 3

[0044] A method for preparing a flavonoid-rich citrus composite extract, which is different from Example 1, comprises the following steps:

[0045] Step 1: replace the fruit of sour orange, dig out the pulp, keep 10kg of peel, break it into 200-300 mesh residue, add 10 times the volume of water, control the temperature to 60°C, soak for 50 minutes, then add 0.3kg of nano zinc oxide and 0.05kg of rhamnolipid, control the temperature to 50°C, ball mill at a speed of 1500r / min for 3-4 hours, filter, and obtain the first filtrate and the first filter residue for later use.

[0046] Step 2: The first filtrate is concentrated and dried under reduced pressure at 55°C and -0.09MPa to constant weight, then 10 times the volume of 70% ethanol solution is added for redissolution, ultrasonic extraction is performed at 55°C for 60 minutes, and then allowed to stand for precipitation and filtered to obtain a second filtrate and a second residue for later use.

[0047] Step 3: Combine the first filter residue and the second filter residue, add 2 times of ethyl acetate and stir evenly, extract at 55°C and 0.5MPa for 50min, repeat 2 times, combine the extracts and set aside.

[0048] Step 4: Combine the second filtrate and the extract, concentrate and dry under reduced pressure at 55° C. and −0.09 MPa to obtain a citrus composite extract rich in flavonoids. Example 4

[0049] A method for preparing a flavonoid-rich citrus composite extract, which is different from Example 1 in that the citrus plant in step 1 is replaced, and the specific steps are as follows:

[0050] Step 1: Take the fruit of Taiwanese lemon, dig out the pulp, leave 10kg of peel, break it into 200-300 mesh residue, add 8 times the volume of water, control the temperature to 65°C, soak for 40 minutes, then add 0.25kg of nano zinc oxide and 0.03kg of rhamnolipid, control the temperature to 60°C, ball mill at a speed of 1450r / min for 3.5h, filter, and obtain the first filtrate and the first filter residue for later use.

[0051] Step 2: The first filtrate is first concentrated and dried under reduced pressure at 60°C and -0.04MPa to constant weight, then 12 times the volume of 70% ethanol solution is added for redissolution, ultrasonic extraction is performed at 60°C for 55 minutes, and then the mixture is allowed to stand for precipitation and filtered to obtain a second filtrate and a second residue for later use.

[0052] Step 3: Combine the first filter residue and the second filter residue, add 2.5 times of ethyl acetate and stir evenly, extract at 60°C and 0.4 MPa for 40 min, repeat 3 times, combine the extracts and set aside.

[0053] Step 4: Combine the second filtrate and the extract, concentrate and dry under reduced pressure at 60° C. and −0.04 MPa to obtain a citrus composite extract rich in flavonoids. Example 5

[0054] A method for preparing a flavonoid-rich citrus composite extract, which is different from Example 1 in that the citrus plant in step 1 is replaced, and the specific steps are as follows:

[0055] Step 1: Take lemon fruit, dig out the pulp, leave 10kg of peel, break it into 200-300 mesh residue, add 8 times volume of water, control the temperature to 65°C, soak for 40 minutes, then add 0.25kg of nano zinc oxide and 0.03kg of rhamnolipid, control the temperature to 60°C, ball mill at a speed of 1450r / min for 3.5h, filter, obtain the first filtrate and the first filter residue, and set aside.

[0056] Step 2: The first filtrate is first concentrated and dried under reduced pressure at 60°C and -0.04MPa to constant weight, then 12 times the volume of 70% ethanol solution is added for redissolution, ultrasonic extraction is performed at 60°C for 55 minutes, and then the mixture is allowed to stand for precipitation and filtered to obtain a second filtrate and a second residue for later use.

[0057] Step 3: Combine the first filter residue and the second filter residue, add 2.5 times of ethyl acetate and stir evenly, extract at 60°C and 0.4 MPa for 40 min, repeat 3 times, combine the extracts and set aside.

[0058] Step 4: Combine the second filtrate and the extract, concentrate and dry under reduced pressure at 60° C. and −0.04 MPa to obtain a citrus composite extract rich in flavonoids. Example 6

[0059] A method for preparing a flavonoid-rich citrus composite extract, which is different from Example 1 in that the citrus plant in step 1 is replaced, and the specific steps are as follows:

[0060] Step 1: Take the fruit of Yunnan citron, dig out the pulp, leave 10kg of peel, break it into 200-300 mesh residue, add 8 times the volume of water, control the temperature to 65°C, soak for 40 minutes, then add 0.25kg of nano zinc oxide and 0.03kg of rhamnolipid, control the temperature to 60°C, ball mill at a speed of 1450r / min for 3.5h, filter, obtain the first filtrate and the first filter residue, and set aside.

[0061] Step 2: The first filtrate is first concentrated and dried under reduced pressure at 60°C and -0.04MPa to constant weight, then 12 times the volume of 70% ethanol solution is added for redissolution, ultrasonic extraction is performed at 60°C for 55 minutes, and then the mixture is allowed to stand for precipitation and filtered to obtain a second filtrate and a second residue for later use.

[0062] Step 3: Combine the first filter residue and the second filter residue, add 2.5 times of ethyl acetate and stir evenly, extract at 60°C and 0.4 MPa for 40 min, repeat 3 times, combine the extracts and set aside.

[0063] Step 4: Combine the second filtrate and the extract, concentrate and dry under reduced pressure at 60° C. and −0.04 MPa to obtain a citrus composite extract rich in flavonoids. Example 7

[0064] A method for preparing a flavonoid-rich citrus composite extract, which is different from Example 1 in that the citrus plant in step 1 is replaced, and the specific steps are as follows:

[0065] Step 1: Take orange fruit, dig out the pulp, leave 10kg of peel, break it into 200-300 mesh residue, add 8 times volume of water, control the temperature to 65°C, soak for 40 minutes, then add 0.25kg of nano zinc oxide and 0.03kg of rhamnolipid, control the temperature to 60°C, ball mill at a speed of 1450r / min for 3.5 hours, filter, obtain the first filtrate and the first filter residue, and set aside.

[0066] Step 2: The first filtrate is first concentrated and dried under reduced pressure at 60°C and -0.04MPa to constant weight, then 12 times the volume of 70% ethanol solution is added for redissolution, ultrasonic extraction is performed at 60°C for 55 minutes, and then the mixture is allowed to stand for precipitation and filtered to obtain a second filtrate and a second residue for later use.

[0067] Step 3: Combine the first filter residue and the second filter residue, add 2.5 times of ethyl acetate and stir evenly, extract at 60°C and 0.4 MPa for 40 min, repeat 3 times, combine the extracts and set aside.

[0068] Step 4: Combine the second filtrate and the extract, concentrate and dry under reduced pressure at 60° C. and −0.04 MPa to obtain a citrus composite extract rich in flavonoids. Example 8

[0069] A method for preparing a flavonoid-rich citrus composite extract, which is different from Example 1 in that the citrus plant in step 1 is replaced, and the specific steps are as follows:

[0070] Step 1: Take the fruit of finger orange, dig out the pulp, keep 10kg of peel, break it into 200-300 mesh residue, add 8 times volume of water, control the temperature to 65°C, soak for 40 minutes, then add 0.25kg of nano zinc oxide and 0.03kg of rhamnolipid, control the temperature to 60°C, ball mill at a speed of 1450r / min for 3.5h, filter, obtain the first filtrate and the first filter residue, and set aside.

[0071] Step 2: The first filtrate is first concentrated and dried under reduced pressure at 60°C and -0.04MPa to constant weight, then 12 times the volume of 70% ethanol solution is added for redissolution, ultrasonic extraction is performed at 60°C for 55 minutes, and then the mixture is allowed to stand for precipitation and filtered to obtain a second filtrate and a second residue for later use.

[0072] Step 3: Combine the first filter residue and the second filter residue, add 2.5 times of ethyl acetate and stir evenly, extract at 60°C and 0.4 MPa for 40 min, repeat 3 times, combine the extracts and set aside.

[0073] Step 4: Combine the second filtrate and the extract, concentrate and dry under reduced pressure at 60° C. and −0.04 MPa to obtain a citrus composite extract rich in flavonoids.

[0074] Comparative Example 1

[0075] A method for preparing a citrus composite extract containing flavonoids, which is different from Example 1 in that the nano zinc oxide in step 1 is replaced by nano silver oxide (50-100 nm) in equal amounts.

[0076] Comparative Example 2

[0077] A method for preparing a citrus composite extract containing flavonoids, which is different from Example 1 in that an equal amount of nano zinc oxide in step 1 is replaced by nano silicon dioxide (50-100 nm).

[0078] Comparative Example 3

[0079] A method for preparing a citrus composite extract containing flavonoids, which is different from Example 1 in that the rhamnolipid in step 1 is replaced by stevioside in an equal amount.

[0080] Comparative Example 4

[0081] A method for preparing a citrus composite extract containing flavonoids, which is different from Example 1 in that the rhamnolipid in step 1 is replaced by Tween 80 in equal amounts.

[0082] Comparative Example 5

[0083] A method for preparing a citrus composite extract containing flavonoids, which is different from Example 1 in that step 1 is as follows:

[0084] Replace the fruit of sour orange, dig out the pulp, keep 10 kg of peel, break it into 200-300 mesh residue, add 8 times the volume of water, control the temperature to 65 ° C, soak for 40 minutes, then add 0.25 kg of nano zinc oxide (50-100 nm) and 0.03 kg of rhamnolipid, control the temperature to 60 ° C, shake and stir at a speed of 300 r / min for 3.5 hours, filter, and obtain the first filtrate and the first residue for later use.

[0085] Capacity determination of citrus compound extracts:

[0086] 1. Antioxidant activity determination:

[0087] Sample: The citrus composite extracts of the above-mentioned embodiments and comparative examples were dissolved and diluted 10 times by adding 95% ethanol solution.

[0088] 1.1DPPH removal rate (%):

[0089] Weigh 2.02 mg DPPH into a 50 mL volumetric flask, add 95% ethanol to dissolve, make up to volume, and shake well to obtain a 0.0404 mg / mL DPPH solution.

[0090] After the DPPH solutions with different samples were reacted in the dark for 1 h, the OD value at 517 nm was measured using an enzyme reader, and the DPPH clearance rate (%) was determined according to the following formula.

[0091] DPPH clearance rate (%) = [1- (A1-A2 / A0)] * 100%

[0092] Among them, A0 refers to the absorbance value of 100 μL DPPH solution + 100 μL 95% ethanol, A1 refers to 100 μL DPPH solution + 100 μL sample, and A2 refers to the absorbance value of 100 μL sample + 100 μL 95% ethanol.

[0093] 1.2ABTS clearance rate (%):

[0094] Mix 6.94mmol / L ABTS aqueous solution and 2.6mmol / L K2S2O8 aqueous solution and react fully, then dilute with 95% ethanol until the absorbance value at 734nm is between 0.70±0.02 to obtain ABTS+ solution.

[0095] After the ABTS+ solution with different samples was reacted in the dark for 40 min, the OD value at 750 nm was measured using an enzyme reader, and the ABTS clearance rate (%) was determined according to the following formula.

[0096] ABTS clearance rate (%) = [1-(B1-B2 / B0)]*100%

[0097] Among them, B0 refers to the absorbance value of 175μL ABTS + solution + 25μL 95% ethanol, B1 refers to 175μL ABTS + solution + 25μL sample, and B2 refers to the absorbance value of 25μL sample + 175μL 95% ethanol.

[0098] The above test results are shown in Table 1.

[0099] 2. Determination of antibacterial activity:

[0100] Sample: The citrus composite extracts of the above embodiments and comparative examples were dissolved and diluted 5 times by adding 70% ethanol solution.

[0101] Soak the sterilized and dried drug-sensitive tablet in the sample and absorb 100 μL of the calibrated bacterial solution (1-2×10 7 CFU / mL) on the agar plate and spread it evenly. After inoculating the plate, stick the drug-sensitive sheet on it so that there is no gap between it and the culture medium. Incubate it upside down at 30°C for 16-18h, and measure the diameter of the inhibition zone. The bacterial liquids are Escherichia coli and Staphylococcus aureus, respectively.

[0102] The above test results are shown in Table 1.

[0103] Table 1:

[0104]

[0105] From the test results in Table 1, it can be seen that the citrus composite extracts of Examples 1 to 8 have good DPPH scavenging effects, ABTS scavenging effects, and good inhibitory effects on Escherichia coli and Staphylococcus aureus. In summary, the citrus composite extracts rich in flavonoids of the present application have better antioxidant activity and antibacterial activity. Analysis and comparison of Examples 1 and 4 to 8, using the peels of 6 varieties of citrus fruits, respectively, the peels of the 6 varieties of citrus fruits all have good antioxidant activity and antibacterial activity, which also proves that the preparation method of the present application has wide applicability in extracting citrus composite extracts rich in flavonoids. The applicant further analyzed the test results of comparative example 1 and comparative examples 1 to 4, and it can be clearly seen that when nano zinc oxide is replaced by nano silver oxide or nano silicon dioxide, and when rhamnolipid is replaced by stevioside or Tween 80, the antibacterial activity and antioxidant activity are affected to a certain extent, which shows that nano zinc oxide and rhamnolipid play a key role in the composite extract. The applicant analyzed and believed that this is because the synergistic effect of the bioactive ingredients such as flavonoids in the citrus composite extract with nano zinc oxide and rhamnolipid significantly improves the extraction efficiency of the bioactive substances in the peel, and further improves the biological activity of the composite extract, enhances the stability of the complex or provides an additional antibacterial mechanism. In addition, the applicant further analyzed comparative example 1 and comparative example 5. Comparative example 5 did not use the ball milling process, but used a conventional water extraction process such as oscillating stirring extraction. The results showed that the ball milling process had a good effect in improving the extraction efficiency and biological activity. The applicant analyzed that the ball milling process can cooperate with nano zinc oxide to rupture the cell wall and increase the contact area between the extraction solvent and the cell contents, thereby improving the extraction efficiency. At the same time, under the action of the mechanical energy of the ball milling process, the synergistic effect of nano zinc oxide and rhamnolipid can also be improved, promoting the transformation of certain biologically active ingredients, and further enhancing the biological activity of the composite extract.

[0106] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for preparing a citrus composite extract rich in flavonoids, characterized in that: The following steps are involved: Step 1: Take the fruit of a citrus plant, remove the pulp, keep the peel, crush it, add 5 to 10 times the volume of water to soak it, then add 2% to 3% of nano zinc oxide by weight of the peel and 0.2% to 0.5% of rhamnolipid by weight of the peel, ball mill at a speed of 1400 to 1500 r / min for 3 to 4 hours, filter, and obtain a first filtrate and a first filter residue for standby use; Step 2: The first filtrate is concentrated and dried under reduced pressure to a constant weight, and 10 to 15 times the volume of 65% to 70% ethanol solution is added for redissolution. After ultrasonic extraction, the mixture is allowed to stand for precipitation and filtered to obtain a second filtrate and a second filter residue for later use; Step 3: Combine the first filter residue and the second filter residue, add 2 to 3 times the volume of ethyl acetate, stir evenly, extract, extract 2 to 3 times in total, and combine the extracts; Step 4: Combine the second filtrate and the extract, concentrate under reduced pressure and dry, to obtain a citrus composite extract rich in flavonoids.

2. The method for preparing a flavonoid-rich citrus composite extract according to claim 1, characterized in that: In step 1, the conditions for water immersion are: time 30-50 min, temperature 60-70° C., and the temperature of ball milling is 50-70° C.

3. The method for preparing a citrus composite extract rich in flavonoids according to claim 1, characterized in that: The reduced pressure concentration and drying conditions of the first filtrate in step 2 are: 55-60° C., -0.09-0.03 MPa, and the ultrasonic extraction is performed at 55-60° C. for 50-60 min.

4. The method for preparing a flavonoid-rich citrus composite extract according to claim 1, characterized in that: The extraction conditions of step 3 are: 55-60° C., 0.3-0.5 MPa, and each extraction time is 30-50 min.

5. The method for preparing a flavonoid-rich citrus composite extract according to claim 1, characterized in that: The conditions for reduced pressure concentration and drying in step 4 are: 55-60° C., -0.09-0.03 MPa.

6. The method for preparing a flavonoid-rich citrus composite extract according to claim 1, characterized in that: The mass ratio of the peel, nano zinc oxide and rhamnolipid is 100:2.5:0.

3.

7. The method for preparing a flavonoid-rich citrus composite extract according to claim 1, characterized in that: The citrus plants include but are not limited to the sour orange, Taiwan lemon, lemon, Yunnan citron, orange, and finger lime.

8. The method for preparing a flavonoid-rich citrus composite extract according to claim 7, characterized in that: The citrus plant is Citrus aurantium.

9. A citrus composite extract rich in flavonoids, characterized in that: The flavonoid-rich citrus composite extract is prepared by the preparation method of any one of claims 1 to 8.

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