An extract from the peel of the yellow peel and its application

Abstract

This invention belongs to the field of plant extraction technology, specifically relating to an extract of wampee peel and its applications. The invention employs a ternary eutectic solvent system of choline chloride-ethylene glycol-water to extract dried wampee peel powder. The resulting wampee peel extract exhibits excellent antioxidant activity, including scavenging abilities of 1,1-diphenyl-2-trinitrophenylhydrazine radicals, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radicals, and iron ion reducing antioxidant capacity. Simultaneously, it shows significant inhibitory effects on common pathogenic bacteria such as Escherichia coli and Staphylococcus aureus. It can be widely used in the preparation of antioxidant and antibacterial products, providing a new technical pathway for the high-value utilization of wampee peel.

Description

Technical Field

[0001] This invention belongs to the field of plant extraction technology. More specifically, it relates to an extract of wampee peel and its applications. Background Technology

[0002] yellow skin ( Clausena lansium *Clausena lansium* is an evergreen shrub or small tree belonging to the Rutaceae family and the *Clausena* genus. Its flesh is soft, sweet, and juicy, with a unique and refreshing flavor, making it suitable for direct consumption as well as processing into jams, wines, preserves, and other products, resulting in a high level of development and utilization. However, the peel, as a byproduct of the fruit, is often discarded due to its "low edible value and high processing costs." In fact, the peel is also rich in flavonoids, polyphenols, volatile oils, and other bioactive components. This development model, which prioritizes the flesh over the byproduct, leads to a significant waste of resources.

[0003] To explore the potential value of wampee peel, some research has been conducted. Yang Weicheng et al. used ultrasonic extraction with ethanol to extract the effective components from wampee peel and confirmed that these extracts have antioxidant activity. However, this traditional organic solvent extraction method has significant drawbacks: the composition of extracts obtained from different types of solvents varies significantly, and the biological activity (including antioxidant and antibacterial activity) of wampee peel extracts prepared by simple ethanol extraction is limited. These drawbacks greatly restrict the high-value development and industrial application of wampee peel resources.

[0004] Therefore, it is urgent to develop efficient and green extraction technologies for wampee peel, a byproduct, to obtain wampee peel extracts with high biological activity, fully explore the resource value of wampee peel byproducts, and promote the green and sustainable development of the wampee industry. Summary of the Invention

[0005] This invention addresses the problem of low utilization of wampee peel in existing research and aims to provide a wampee peel extract.

[0006] A second objective of this invention is to provide the application of the said wampee peel extract in the preparation of antioxidant products.

[0007] A third objective of this invention is to provide the application of the said wampee peel extract in the preparation of antibacterial products.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a wampee peel extract, prepared by the following steps: The dried peel powder of wampee was extracted using a eutectic solvent as the extraction medium to obtain the wampee peel extract. The eutectic solvent is composed of choline chloride, ethylene glycol and water, wherein the molar ratio of choline chloride to ethylene glycol is 1:(2~4).

[0009] This invention creatively discovers that by using a eutectic solvent system formed by compounding choline chloride, ethylene glycol, and water in a specific ratio to extract dried wampee peel powder, the resulting wampee peel extract exhibits superior antioxidant and antibacterial activity compared to wampee peel extract obtained by traditional alcohol extraction and other eutectic solvent systems. This provides a new technical pathway for the high-value utilization of wampee peel.

[0010] Preferably, the molar ratio of choline chloride to ethylene glycol is 1:(2~3).

[0011] More preferably, the molar ratio of choline chloride to ethylene glycol is 1:2.

[0012] Preferably, the ratio of the dried yellow peel powder to the eutectic solvent is 1 g: (15~35) mL.

[0013] More preferably, the ratio of the dried yellow peel powder to the eutectic solvent is 1 g: (15~25) mL, specifically 1 g: 25 mL.

[0014] Preferably, the mass of the water is 20% to 40% of the total mass of choline chloride and ethylene glycol.

[0015] More preferably, the mass of the water is 30% of the total mass of choline chloride and ethylene glycol.

[0016] Preferably, the extraction is performed using ultrasonic extraction.

[0017] Preferably, the extraction temperature of the ultrasonic extraction method is 40~60 ℃; More preferably, the extraction temperature of the ultrasonic extraction method is 50 °C.

[0018] Preferably, the extraction time of the ultrasonic extraction method is 1 to 4 hours.

[0019] More preferably, the extraction time of the ultrasonic extraction method is 2 hours.

[0020] Preferably, the ultrasonic frequency of the ultrasonic extraction method is 30~50 kHz.

[0021] More preferably, the ultrasonic frequency of the ultrasonic extraction method is 40 kHz.

[0022] Preferably, the ultrasonic power of the ultrasonic extraction method is 200~400 W.

[0023] More preferably, the ultrasonic power of the ultrasonic extraction method is 300 W.

[0024] Preferably, the eutectic solvent is prepared by the following steps: (1) Mix choline chloride and ethylene glycol thoroughly to obtain a mixed solution; (2) Add water to the mixed solution obtained in step (1) and mix thoroughly to obtain the eutectic solvent.

[0025] More preferably, in step (1), the temperature at which the mixture is fully mixed is 75~95 ℃, more preferably 85 ℃.

[0026] More preferably, in step (1), the time for thorough mixing is 40-80 min, more preferably 60 min.

[0027] More preferably, in step (2), the temperature at which the mixture is fully mixed is 75~95 ℃, more preferably 85 ℃.

[0028] More preferably, in step (2), the time for thorough mixing is 5 to 15 minutes, more preferably 10 minutes.

[0029] Preferably, the extraction process further includes separation and purification.

[0030] More preferably, the separation and purification process includes adsorption, desorption, and drying, ultimately yielding a freeze-dried powder of wampee peel extract.

[0031] More preferably, the adsorption is performed by adding macroporous adsorption resin to the wampee peel extract, shaking thoroughly, and then filtering and collecting the macroporous adsorption resin.

[0032] More preferably, the desorption is performed by adding anhydrous methanol to the macroporous adsorption resin obtained after adsorption, shaking thoroughly, filtering and collecting the filtrate.

[0033] More preferably, the drying process involves rotary evaporation and freeze-drying of the filtrate obtained after desorption.

[0034] Optionally, the macroporous adsorption resin includes AB-8 macroporous adsorption resin.

[0035] Optionally, during the adsorption process, the mass-to-volume ratio of the macroporous adsorption resin to the extract of yellow peel is 5 g: (15~35) mL, more preferably 5 g: 25 mL.

[0036] Optionally, during the adsorption process, the temperature at which the full oscillation occurs is 20~30 ℃, more preferably 25 ℃.

[0037] Optionally, during the adsorption process, the time for sufficient oscillation is 8 to 16 hours, more preferably 12 hours.

[0038] Optionally, during the adsorption process, the rotation speed of the fully oscillating motion is 160~200 rpm, more preferably 180 rpm.

[0039] Optionally, during the desorption process, the temperature for sufficient oscillation is 20~30 ℃, more preferably 25 ℃.

[0040] Optionally, during the desorption process, the time for sufficient oscillation is 8 to 16 hours, more preferably 12 hours.

[0041] Optionally, during the desorption process, the rotational speed of the fully oscillating motion is 80~140 rpm, more preferably 120 rpm.

[0042] Optionally, during the desorption process, the volume ratio of the anhydrous methanol to the wampee peel extract is 1:(0.3~0.7), more preferably 1:0.5.

[0043] Optionally, during the drying process, the temperature of the rotary evaporation is 34~38℃, specifically 36℃.

[0044] Optionally, during the drying process, the rotary evaporation time is 1 to 3 hours, specifically 2 hours.

[0045] Optionally, during the drying process, the freeze-drying temperature is -30 to -20°C, more preferably -25°C.

[0046] Preferably, the dried wampee peel powder is obtained by pre-treating the wampee peel.

[0047] More preferably, the pretreatment includes drying, pulverizing, and sieving.

[0048] Optionally, the drying is carried out at 40~60 ℃, specifically 55 ℃.

[0049] Optionally, the sieving process involves passing the pulverized wampee peel powder through a 40-60 mesh (more preferably 50 mesh) standard inspection sieve, and taking the sieved powder, which is the wampee peel powder.

[0050] This invention also protects the use of the yellow peel extract in the preparation of antioxidant products.

[0051] Furthermore, the antioxidant product has at least one of the following antioxidant capabilities: 1,1-diphenyl-2-trinitrophenylhydrazine free radical scavenging capability, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) free radical scavenging capability, or iron ion reduction antioxidant capability.

[0052] Optionally, the product may include pharmaceuticals, health products, or daily chemical products.

[0053] Alternatively, the daily chemical products may include cosmetics.

[0054] This invention also protects the use of the yellow peel extract in the preparation of antibacterial products.

[0055] Furthermore, the antibacterial product has an inhibitory effect on Escherichia coli and / or Staphylococcus aureus.

[0056] Optionally, the product may include pharmaceuticals or daily chemical products.

[0057] Further, optionally, the daily chemical products include at least one of cosmetics and disinfectants.

[0058] The present invention has the following beneficial effects: This invention employs a ternary eutectic solvent system of choline chloride-ethylene glycol-water for the extraction of active ingredients from dried wampee peel powder. Compared with wampee peel extracts obtained through traditional ethanol-water solutions and other eutectic solvent systems, the wampee peel extract obtained by this invention exhibits superior antioxidant properties, including 1,1-diphenyl-2-trinitrophenylhydrazine free radical scavenging ability, 2,2'-azido-bis(3-ethylbenzothiazoline-6-sulfonic acid) free radical scavenging ability, and iron ion reducing antioxidant capacity. Furthermore, it shows significantly enhanced inhibitory activity against common pathogenic bacteria such as Escherichia coli and Staphylococcus aureus. Therefore, it can be widely applied in the preparation of antioxidant and antibacterial products, providing a new technological pathway for the high-value utilization of wampee peel. Attached Figure Description

[0059] Figure 1 This is a comparison chart of the 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radical scavenging ability of the extracts of wampee prepared in Examples 1 to 5 and Comparative Examples 1 to 4. Different letters represent significant differences, while the same letters represent no significant differences. TE represents the equivalent of 6-hydroxy-2,5,7,8-tetramethylbenzodihydropyran-2-carboxylic acid (Trolox), and DW represents the mass of dried wampee peel powder.

[0060] Figure 2 This is a comparison chart of the iron ion reducing antioxidant capacity (FRAP) of the extracts of wampee prepared in Examples 1 to 5 and Comparative Examples 1 to 4. Different letters represent significant differences, while the same letters represent no significant differences. TE represents the equivalent of 6-hydroxy-2,5,7,8-tetramethylbenzodihydropyran-2-carboxylic acid (Trolox), and DW represents the mass of dried wampee peel powder.

[0061] Figure 3This is a comparison chart of the 2,2'-adiazono-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radical scavenging abilities of the rutin solution (positive control), Examples 1-5, and Comparative Examples 1-4. Different letters represent significant differences, while the same letters represent no significant differences. TE represents the equivalent of 6-hydroxy-2,5,7,8-tetramethylbenzodihydropyran-2-carboxylic acid (Trolox), and DW represents the mass of dried rutin peel powder.

[0062] Figure 4 The lyophilized powder of the wampee peel extract prepared in Example 1 and Comparative Example 4 is effective against Escherichia coli (E. coli). Escherichia coli ) and Staphylococcus aureus ( Staphylococcus aureus Comparison of growth inhibition effects of ( ). Detailed Implementation

[0063] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0064] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0065] The dried wampee peel powder of the present invention is obtained by pre-processing seedless wampee peel from Yunan (harvested from the World Wampee Park in Yunan, Guangdong) as raw material. The specific pre-processing includes the following steps: (1) Raw material screening: Select fresh seedless yellow peel fruits from Yunan that are uniform in size, free from mechanical damage and pests; (2) Cleaning and peeling: After thoroughly rinsing the surface of the seedless yellow peel fruit of Yunan with running water, peel off the whole peel; (3) Drying and pulverizing: Place the peel in a 55 ℃ constant temperature drying oven overnight to dry to constant weight (weigh every 3 hours, and the difference in weight of the peel between two consecutive weighings is less than ±0.2 mg), then pulverize and pass through a 50 mesh standard inspection sieve, collect the peel powder after sieving, and obtain yellow peel dry powder, which is sealed and protected from light for later use.

[0066] Example 1: Extract from the peel of the yellow peel fruit The preparation of the extract from the peel of the yellow peel includes the following steps: (1) Mix choline chloride and ethylene glycol in a molar ratio of 1:2, and stir magnetically for 60 min in a water bath at 85 °C. Add water equivalent to 30% of the mass of choline chloride and ethylene glycol, mix well, and stir magnetically for 10 min in a water bath at 85 °C. After cooling, a clear and transparent solution is obtained, which is the eutectic solvent (DES solvent).

[0067] (2) Weigh 1 g of dried yellow peel powder, add 25 mL of the above DES solvent to the dried yellow peel powder, and extract by ultrasonication (40 kHz, 300 W) at 50℃ for 2 h. After extraction, centrifuge and take the supernatant. The supernatant is the yellow peel extract.

[0068] Example 2: Extract from the peel of the yellow peel fruit The difference from Example 1 is that water equivalent to 20% of the total mass of choline chloride and ethylene glycol is added.

[0069] The preparation of the extract from the peel of the yellow peel includes the following steps: (1) Mix choline chloride and ethylene glycol in a molar ratio of 1:2, and stir magnetically for 60 min in a water bath at 85 °C. Add water equivalent to 20% of the mass of choline chloride and ethylene glycol, mix well, and stir magnetically for 10 min in a water bath at 85 °C. After cooling, a clear and transparent solution is obtained, which is DES solvent.

[0070] (2) Weigh 1 g of dried yellow peel powder, add 25 mL of the above DES solvent to the dried yellow peel powder, and extract by ultrasonication (40 kHz, 300 W) at 50℃ for 2 h. After extraction, centrifuge and take the supernatant. The supernatant is the yellow peel extract.

[0071] Example 3: Extract from the peel of the yellow peel fruit The difference from Example 1 is that water equivalent to 40% of the total mass of choline chloride and ethylene glycol is added.

[0072] The preparation of the extract from the peel of the yellow peel includes the following steps: (1) Mix choline chloride and ethylene glycol in a molar ratio of 1:2, and stir magnetically for 60 min in a water bath at 85 °C. Add water equivalent to 40% of the mass of choline chloride and ethylene glycol, mix well, and stir magnetically for 10 min in a water bath at 85 °C. After cooling, a clear and transparent solution is obtained, which is DES solvent.

[0073] (2) Weigh 1 g of dried yellow peel powder, add 25 mL of the above DES solvent to the dried yellow peel powder, and extract by ultrasonication (40 kHz, 300 W) at 50℃ for 2 h. After extraction, centrifuge and take the supernatant. The supernatant is the yellow peel extract.

[0074] Example 4: Extract from the peel of the yellow peel fruit The difference from Example 1 is that the molar ratio of choline chloride and ethylene glycol is changed from 1:2 to 1:3.

[0075] The preparation of wampee peel extract specifically includes the following steps: (1) Mix choline chloride and ethylene glycol in a molar ratio of 1:3, and stir magnetically for 60 min in a water bath at 85 °C. Add water equivalent to 30% of the mass of choline chloride and ethylene glycol, mix well, and stir magnetically for 10 min in a water bath at 85 °C. After cooling, a clear and transparent solution is obtained, which is the eutectic solvent (DES solvent).

[0076] (2) Weigh 1 g of dried yellow peel powder, add 25 mL of the above DES solvent to the dried yellow peel powder, and extract by ultrasonication (40 kHz, 300 W) at 50℃ for 2 h. After extraction, centrifuge and take the supernatant. The supernatant is the yellow peel extract.

[0077] Example 5: Extract from the peel of the yellow peel fruit The difference from Example 1 is that the molar ratio of choline chloride and ethylene glycol is changed from 1:2 to 1:4.

[0078] The preparation of wampee peel extract specifically includes the following steps: (1) Mix choline chloride and ethylene glycol in a molar ratio of 1:4, and stir magnetically for 60 min in a water bath at 85 °C. Add water equivalent to 30% of the mass of choline chloride and ethylene glycol, mix well, and stir magnetically for 10 min in a water bath at 85 °C. After cooling, a clear and transparent solution is obtained, which is the eutectic solvent (DES solvent).

[0079] (2) Weigh 1 g of dried yellow peel powder, add 25 mL of the above DES solvent to the dried yellow peel powder, and extract by ultrasonication (40 kHz, 300 W) at 50℃ for 2 h. After extraction, centrifuge and take the supernatant. The supernatant is the yellow peel extract.

[0080] Comparative Example 1: Extract of Wampee Peel The difference from Example 1 is that ethylene glycol is replaced with lactic acid.

[0081] The preparation of wampee peel extract specifically includes the following steps: (1) Mix choline chloride and lactic acid in a molar ratio of 1:2, add water equivalent to 30% of the mass of choline chloride and lactic acid, mix well, and then heat in a water bath at 85 °C with magnetic stirring for 10 min. After cooling, a clear and transparent solution is obtained, which is DES solvent.

[0082] (2) Weigh 1 g of dried yellow peel powder, add 25 mL of the above DES solvent to the dried yellow peel powder, and extract by ultrasonication (40 kHz, 300 W) at 50℃ for 2 h. After extraction, centrifuge and take the supernatant. The supernatant is the yellow peel extract.

[0083] Comparative Example 2: Extract from the peel of the yellow peel fruit The difference from Example 1 is that ethylene glycol is replaced with malic acid.

[0084] The preparation of wampee peel extract specifically includes the following steps: (1) Mix choline chloride and malic acid in a molar ratio of 1:2, and stir magnetically for 60 min in a water bath at 85 °C. Add water equivalent to 30% of the mass of choline chloride and malic acid, mix well, and stir magnetically for 10 min in a water bath at 85 °C. After cooling, a clear and transparent solution is obtained, which is DES solvent.

[0085] (2) Weigh 1 g of dried yellow peel powder, add 25 mL of the above DES solvent to the dried yellow peel powder, and extract by ultrasonication (40 kHz, 300 W) at 50℃ for 2 h. After extraction, centrifuge and take the supernatant. The supernatant is the yellow peel extract.

[0086] Comparative Example 3: Extract from the peel of the yellow peel fruit The difference from Example 1 is that ethylene glycol is replaced with citric acid.

[0087] The preparation of wampee peel extract specifically includes the following steps: (1) Mix choline chloride and citric acid in a molar ratio of 1:2, and stir magnetically for 60 min in a water bath at 85 °C. Add water equivalent to 30% of the mass of choline chloride and citric acid, mix well, and stir magnetically for 10 min in a water bath at 85 °C. After cooling, a clear and transparent solution is obtained, which is DES solvent.

[0088] (2) Weigh 1 g of dried yellow peel powder, add 25 mL of the above DES solvent to the dried yellow peel powder, and extract by ultrasonication (40 kHz, 300 W) at 50℃ for 2 h. After extraction, centrifuge and take the supernatant. The supernatant is the yellow peel extract.

[0089] Comparative Example 4: Extract from the peel of the yellow peel fruit Based on the previous research results of this invention, the extract of yellow peel obtained by using 40 vol% ethanol aqueous solution in the alcohol extraction method showed the best antioxidant and antibacterial activities. Therefore, 40 vol% ethanol aqueous solution was selected to replace the DES solvent in Example 1.

[0090] The preparation of wampee peel extract specifically includes the following steps: Weigh 1 g of dried wampee peel powder, add 25 mL of 40 vol% ethanol aqueous solution to the dried wampee peel powder, and extract by ultrasonication (40 kHz, 300 W) at 50 ℃ for 2 h. After extraction, centrifuge and collect the supernatant. The supernatant obtained is the wampee peel extract.

[0091] Experimental Example 1: Determination of Antioxidant Activity The antioxidant capacity of the extracts of yellow peel obtained in Examples 1 to 5 and Comparative Examples 1 to 4 was determined.

[0092] 1. Experimental Procedure (1) Determination of the free radical scavenging ability of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) (a) Preparation of DPPH solution: Dissolve 7.88 mg DPPH in 25 mL of anhydrous ethanol, then dilute 4 times before use.

[0093] (b) Determination of the standard curve: The antioxidant capacity of DPPH was determined using a standard curve analysis of 6-hydroxy-2,5,7,8-tetramethylbenzodihydropyran-2-carboxylic acid (Trolox, CAS No.: 53188-07-1). Trolox solutions at concentrations of 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL were prepared, with corresponding scavenging rates of 8.78%, 14.45%, 26.47%, 51.50%, and 96.09%, respectively. A standard curve was plotted with Trolox solution concentration on the ordinate and scavenging rate on the abscissa, and the regression equation was calculated. The linear equation was Y = 106.86X - 3.4145, where X is the scavenging rate, Y is the Trolox content (μg / mL), and R0 = 106.86X - 3.4145. 2 = 0.9994, the result is expressed as mg TE / g DW, where TE represents Trolox equivalent and DW represents the mass of dried yellow peel powder.

[0094] (b) Scavenging rate determination: Experimental group: The extract of yellow peel fruit was diluted 40 times with 40 vol% ethanol, and 200 μL was taken and mixed with 1 mL DPPH solution. The mixture was vortexed and reacted at room temperature in the dark for 30 min. The absorbance value A1 was measured at 517 nm. Blank group: 200 μL of yellow peel extract diluted 40 times with 40 vol% ethanol was mixed with 1 mL of distilled water and the absorbance value A2 was measured. Background group: The absorbance value A3 was determined by mixing 200 μL of distilled water with 1 mL of DPPH solution.

[0095] The clearance rate formula is: Clearance rate (%) = (1- )×100% (Formula 1) (2) Determination of ferric reducing antioxidant capacity (FRAP): (a) Preparation of FRAP working solution: Take 10 mL of acetate buffer (0.1 M), mix 1 mL of 2,4,6-tris(2-pyridyl)-1,3,5-triazine (TPTZ) solution (10 mM dissolved in 40 mM hydrochloric acid solution), and 1 mL of ferric chloride solution (20 mM) to prepare FRAP working solution; (b) Determination of standard curve: The antioxidant capacity of iron ion reduction was determined using a Trolox standard curve. Trolox solutions with concentrations of 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, and 250 μg / mL were prepared, with corresponding scavenging rates of 2.08%, 3.83%, 7.86%, 15.67%, 30.18%, 44.85%, 58.07%, and 71.67%. A standard curve was plotted with Trolox solution concentration on the ordinate and scavenging rate on the abscissa, and the regression equation was calculated. The linear equation was Y = 348.51X - 2.8123, where X is the scavenging rate, Y is the Trolox content (μg / mL), and R0 = 348.51X - 2.8123. 2 = 0.9994, the result is expressed as mg TE / g DW, where TE represents Trolox equivalent and DW represents the mass of dried yellow peel powder.

[0096] (c) Scavenging rate determination: Experimental group: The extract of yellow peel fruit was diluted 40 times with 40 vol% ethanol, and 60 μL was taken. 1400 μL of FRAP working solution was added to it. The mixture was reacted at room temperature in the dark for 30 min, and the absorbance value A1 was measured at 595 nm.

[0097] Blank group: 200 μL of wampee peel extract diluted 40 times with 40 vol% ethanol was mixed with 1 mL of distilled water and the absorbance value A2 was measured. Background group: The absorbance value A3 was determined by mixing 1 mL of FRAP working solution with 200 μL of distilled water.

[0098] Calculate the clearance rate using Formula 1.

[0099] (3) Determination of the free radical scavenging ability of 2,2'-adiazon-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (a) Preparation of ABTS working solution: Mix 10 mL of ABTS (2 mM) with 100 μL of potassium persulfate (70 mM) and store at room temperature in the dark for 16 h; dilute the mixture with ethanol to obtain an absorbance of 0.7 ± 0.02 at 734 nm to obtain the ABTS working solution. (b) Determination of standard curve The antioxidant capacity of ABTS was determined using a Trolox standard curve. Trolox solutions with concentrations of 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 150 μg / mL were prepared, with corresponding scavenging rates of 10.75%, 13.46%, 19.36%, 30.72%, 52.79%, and 73.75%, respectively. A standard curve was plotted with Trolox solution concentration on the ordinate and scavenging rate on the abscissa, and the regression equation was calculated. The linear equation was Y = 227.34X - 18.803, where X is the scavenging rate, Y is the Trolox content (μg / mL), and R0 is the scavenging rate. 2 = 0.9997, the result is expressed as mg TE / g DW, where TE represents Trolox equivalent and DW represents the mass of dried yellow peel powder.

[0100] (c) Scavenging rate determination: Experimental group: 50 μL of the extract of *Clausena lansium* peel was diluted 40-fold with 40 vol% ethanol, and 2 mL of ABTS working solution was added. After reacting for 15 min, the absorbance was measured at 734 nm. Blank group: 200 μL of wampee peel extract diluted 40 times with 40 vol% ethanol was mixed with 1 mL of distilled water and the absorbance value A2 was measured. Background group: The absorbance value A3 was determined by mixing 1 mL of ABTS working solution with 200 μL of distilled water.

[0101] Calculate the clearance rate using Formula 1.

[0102] 2. Experimental Results Results of DPPH free radical scavenging capacity test: (e.g.) Figure 1 As shown, the DPPH free radical scavenging ability of the wampee peel extracts obtained in Examples 1 to 5 and Comparative Examples 1 to 2 was significantly higher than that of the wampee peel extract obtained by the alcohol extraction method in Comparative Example 4; while the DPPH free radical scavenging ability of the wampee peel extract obtained in Comparative Example 3 was significantly lower than that of the wampee peel extract obtained by the alcohol extraction method in Comparative Example 4.

[0103] Results of iron ion reducing antioxidant capacity test: (e.g.) Figure 2 As shown, the antioxidant capacity of the wampee peel extracts obtained in Examples 1 to 5 for reducing iron ions was significantly higher than that of the wampee peel extracts obtained in Comparative Examples 1 to 4.

[0104] Results of ABTS free radical scavenging capacity test: (e.g.) Figure 3 As shown, the extracts of wampee peel obtained in Examples 1 to 5 have significantly higher ABTS free radical scavenging ability than the extracts of wampee peel obtained in Comparative Examples 1 to 4.

[0105] The results of the above three antioxidant activity assays show that: (1) The antioxidant activity of the extracts of wampee peel obtained in Examples 1 to 5 was significantly higher than that of the extracts of wampee peel obtained in Comparative Examples 1 to 4; (2) Not all eutectic solvent systems used to extract wampee peel produce wampee peel extracts with superior antioxidant capacity compared to those obtained by traditional alcohol extraction. In summary, only the ternary eutectic solvent system of choline chloride-ethylene glycol-water provided by this invention can effectively solve the technical defect of insufficient antioxidant activity in extracts with the best comprehensive antioxidant activity from the peel of wampee fruit.

[0106] Experimental Example 2: Determination of Antibacterial Activity The extracts of wampee peel obtained in Examples 1-5 exhibited similar antibacterial activity. Therefore, Example 1 was used as a representative example to determine the antibacterial activity of the wampee peel extracts obtained in Examples 1 and Comparative Example 4 against Escherichia coli and Staphylococcus aureus. Rutin, as a classic natural flavonoid antibacterial agent, has been extensively studied and proven to have antibacterial ability; therefore, rutin solution was used as a positive control. 1. Experimental Procedure (1) Sample preparation The extract of *Clausena lansium* peel was purified using macroporous adsorption resin separation and purification technology to obtain a lyophilized powder of the extract, thus eliminating the interference of solvent on the antibacterial activity assay. (a) Adsorption: 5 g of AB-8 macroporous adsorption resin was mixed with 25 mL of yellow peel extract and shaken at 25 ℃ and 180 rpm for 12 h to reach adsorption equilibrium. The macroporous adsorption resin was then separated by filtration. (b) Desorption: Add 50 mL of anhydrous methanol to the macroporous adsorption resin, shake and desorb for 12 h at 25 ℃ and 120 rpm, and collect the filtrate by filtration; (c) Drying: The filtrate was rotary evaporated at 36 °C for 2 h to remove methanol, and then freeze-dried (-25 °C) to obtain freeze-dried powder of wampee peel extract, which was used for subsequent antibacterial activity determination.

[0107] Preparation of lyophilized powder solutions of wampee peel extract: The lyophilized powder of wampee peel extract was prepared into 40 mg / mL stock solutions using 4 vol% dimethyl sulfoxide (DMSO) aqueous solution (this solvent has no antibacterial effect and can eliminate the interference of the solvent on subsequent antibacterial experiments). The stock solutions were then serially diluted with 4 vol% DMSO aqueous solution to obtain lyophilized powder solutions of wampee peel extract with concentrations of 40 mg / mL, 20 mg / mL, 10 mg / mL and 5 mg / mL respectively.

[0108] Preparation of rutin solution: A 40 mg / mL rutin solution was prepared using 4 vol% DMSO aqueous solution as a positive control.

[0109] (2) Determination of antibacterial activity by inhibition zone method After cleaning the Oxford cups, place them in petri dishes and wrap them with aluminum foil. Autoclave at 121 °C for 15 minutes, then dry them for later use. Pick the Staphylococcus aureus or Escherichia coli strains to be activated and inoculate them into Lysogeny Broth (LB) agar, respectively, and incubate at 37 °C for 24 h. Pick a single colony and inoculate it into 40 mL of LB liquid medium. Incubate at 37 °C and 150 r / min for 24 h, then adjust the bacterial concentration to 1.0 × 10⁻⁶. 7 Turbidity was quantified by measuring the absorbance (OD value) of the bacterial suspension at a wavelength of 600 nm using CFU / mL. 600 When ≈0.1, the bacterial concentration is approximately 1×10⁻⁶. 8 CFU / mL, for later use. Take 100 μL of the standardized bacterial suspension (bacterial concentration of 1.0 × 10⁻⁶). 7Add CFU / mL of bacterial solution to LB agar plates and spread it evenly across the entire surface using a disposable spreader, rotating the plate 60° each time to ensure even distribution. Use sterile forceps to pick up sterilized Oxford cups, briefly flaming the tips and cups to remove any potential contaminants, then allow to cool. Gently and steadily place the Oxford cups vertically and stably onto the spread LB agar plate, pressing lightly to ensure tight contact without gaps. Place three Oxford cups on each LB agar plate and label their positions. Add 40 mg / mL 100 μL of lyophilized wampee peel extract or 40 mg / mL rutin solution to each Oxford cup. Incubate the LB agar plate at 37 °C for 16 h. Observe colony growth and measure the diameter of the inhibition zone to compare the antibacterial effect.

[0110] (3) Observation of viable bacterial growth by plate coating method (a) Take 0.9 mL of lyophilized powder solutions of 5 mg / mL, 10 mg / mL, and 20 mg / mL of *Clausena lansium* peel extract and 0.1 mL of 1.0 × 10⁻⁶ mollusc extract. 7 A mixture of CFU / mL E. coli was prepared to obtain a mixed culture medium.

[0111] (b) Take 0.9 mL of lyophilized powder solutions of 5 mg / mL and 10 mg / mL of wampee peel extract, and 0.1 mL of 1.0 × 10⁻⁶ lyophilized powder solutions. 7 The CFU / mL Staphylococcus aureus suspension was mixed to obtain a mixed culture medium.

[0112] The two types of mixed culture solutions were diluted 10,000 times with 4 vol% DMSO aqueous solution. 100 μL of the bacterial solution was spread on LB agar medium and incubated at 37 ℃ for 12 h. The colony growth was then observed.

[0113] 2. Experimental Results (1) Results of antibacterial activity determination by inhibition zone method Table 1. Size of the inhibition zone

[0114] The results are shown in Table 1. The inhibition zone diameter of the extract of *Clausena lansium* peel obtained in Example 1 against *Escherichia coli* was 15.4 ± 0.6 mm. Different letters indicate significant differences, while the same letter indicates no significant differences.

[0115] Compared with the rutin solution, the freeze-dried powder of wampee peel extract obtained in Example 1 showed significantly stronger activity against Escherichia coli.

[0116] Compared with the freeze-dried powder of wampee peel extract obtained in Comparative Example 4, the freeze-dried powder of wampee peel extract obtained in Example 1 showed better antibacterial activity against both tested bacteria, with a particularly significant improvement in antibacterial effect against Staphylococcus aureus.

[0117] (2) Results of observation of viable bacterial growth by plate coating method The results are as follows Figure 4 As shown, the freeze-dried powder of wampee peel extract obtained in Example 1 exhibited a more significant inhibitory effect on the growth of Escherichia coli at low concentrations (5~10 mg / mL); the freeze-dried powder of wampee peel extract obtained in Example 1 also exhibited a more significant inhibitory effect on the growth of Staphylococcus aureus at low concentrations (5~20 mg / mL); the above experimental results indicate that the freeze-dried powder of wampee peel extract obtained in Example 1 has better antibacterial properties even at lower concentrations.

[0118] The above results indicate that the present invention uses a ternary eutectic solvent system of choline chloride-ethylene glycol-water to extract wampee peel extract from dried wampee peel powder, which exhibits better antibacterial effect compared to wampee peel extract obtained by traditional alcohol extraction.

[0119] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A wampee peel extract, characterized in that, The preparation process includes the following steps: The dried peel powder of wampee was extracted using a eutectic solvent as the extraction medium to obtain the wampee peel extract. The eutectic solvent is composed of choline chloride, ethylene glycol and water, wherein the molar ratio of choline chloride to ethylene glycol is 1:(2~4). The mass of the water is 20% to 40% of the total mass of choline chloride and ethylene glycol.

2. The wampee peel extract as described in claim 1, characterized in that, The ratio of the dried yellow peel powder to the eutectic solvent is 1 g: (15~35) mL.

3. The wampee peel extract as described in claim 1, characterized in that, The extraction method is ultrasonic extraction.

4. The wampee peel extract as described in claim 3, characterized in that, The parameters of the ultrasonic extraction method include at least one of the following: (1) The extraction temperature of the ultrasonic extraction method is 40~60 ℃; (2) The extraction time of the ultrasonic extraction method is 1~4 h; (3) The ultrasonic frequency of the ultrasonic extraction method is 30~50 kHz; (4) The ultrasonic power of the ultrasonic extraction method is 200~400 W.

5. The wampee peel extract as described in claim 1, characterized in that, The extraction process also includes separation and purification.

6. The wampee peel extract as described in claim 5, characterized in that, The separation and purification process includes adsorption, desorption, and drying.

7. The use of the extract of yellow peel as described in any one of claims 1 to 6 in the preparation of antioxidant products.

8. The application as described in claim 7, characterized in that, The antioxidant product has at least one of the following antioxidant capabilities: 1,1-diphenyl-2-trinitrophenylhydrazine free radical scavenging capability, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) free radical scavenging capability, or iron ion reduction antioxidant capability.

9. The use of the extract of wampee peel according to any one of claims 1 to 6 in the preparation of antibacterial products.

10. The application as described in claim 9, characterized in that, The antibacterial product has an inhibitory effect on Escherichia coli and / or Staphylococcus aureus.

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