Method for improving the content of polymethoxylated flavones in blood orange peel by ultrasonic treatment

Abstract

The present application belongs to the field of edible processing, and particularly relates to a method for improving the accumulation efficiency and content of polymethoxyflavones in blood orange peels by ultrasonic treatment. The method comprises the following steps: placing fresh Tarocco blood oranges after cleaning and air-drying in an ultrasonic device for water bath ultrasonic treatment, and then storing the blood oranges at a low temperature of 8±1 DEG C for 1-40 days; peeling the blood orange peels after low-temperature storage, adding a methanol solution to the peels under oscillation in the dark for extraction, and then high-speed centrifugation; and filtering the obtained supernatant to obtain peel extract containing polymethoxyflavones. The present application greatly improves the accumulation efficiency and content of polymethoxyflavones in blood orange peels. The method of the present application is simple in operation and stable in effect, and is expected to be used for the reuse of blood orange peels.

Description

Technical Field

[0001] This invention belongs to the field of food processing, specifically relating to a method for increasing polymethoxyflavonoids in blood orange peel through ultrasonic treatment. Background Technology

[0002] Tarocco blood orange (C. sinensis L. Osbeck) is a cultivated variety of sweet orange belonging to the Rutaceae family and the Citrus genus. Its peel is rich in polymethoxyflavonoids, a class of compounds formed by two or more O-methylation modifications of flavonoids. These compounds possess rich biological activities, including anti-inflammatory, anticancer, and antiviral properties, and have high medicinal and health benefits. However, studies have shown that polymethoxyflavonoid content initially increases and then decreases during fruit development, with lower levels at the harvest stage. Existing blood orange processing techniques lack effective methods to enhance this content, resulting in the peel not being effectively processed and utilized.

[0003] Existing similar methods include the following:

[0004] 1. The invention CN107011308A, "Method for separating and purifying polymethoxyflavonoids from Ougan fruit," describes the following steps:

[0005] (1) Sample extraction: freeze-dried Ougan peel powder was extracted with 95% ethanol by ultrasonic extraction at a solid-liquid ratio of 1:20 and an extraction temperature of 25℃ for 45 min each time. After vacuum filtration, the supernatants were combined and evaporated to dryness on a rotary evaporator until no ethanol phase was found. The supernatants were then dissolved in deionized water.

[0006] (2) Sugar removal and fractionation of Sep-C18 solid phase extraction column: Take the aqueous solution obtained in step (1) and load it onto the Sep-C18 solid phase extraction column. After loading, the solid phase extraction column is first eluted with deionized water for 25 column bed volumes to remove impurities with high polarity. Then, it is eluted with methanol gradient. The eluent is evaporated to dryness at 36°C on a rotary evaporator to obtain 100% eluent rich in polymethoxyflavones.

[0007] (3) HSCCC further separated and purified hesperidin, tangeretin and 5-norhesperidin.

[0008] 2. Invention CN108727324A, entitled "A Method for Separating and Purifying Citrus Polymethoxyflavonoids," discloses the following steps:

[0009] 1) Extraction

[0010] The citrus peel residue was mixed with an 85%-95% (v / v) ethanol aqueous solution and extracted with ultrasonic treatment at a temperature below 45℃ for 30 min-2 h. The supernatant was collected and concentrated to obtain a crude extract.

[0011] 2) Enrichment and Impurity Removal

[0012] The pretreated HPD300 macroporous resin was packed into a chromatography column. The crude extract was pumped into the chromatography column. A 3%-10% (v / v) ethanol aqueous solution was pumped into the chromatography column to wash and remove impurities. A 90%-95% (v / v) ethanol aqueous solution was pumped into the chromatography column to desorb. The eluent from the desorbing stage was collected, concentrated, and freeze-dried to obtain a PMF mixture.

[0013] 3) Separation and purification

[0014] The PMF mixture was dissolved in methanol or ethanol to obtain a solution to be purified. The solution was further separated and purified by mass spectrometry-guided preparative high-performance liquid chromatography. After the purified solution was loaded onto the sample, gradient elution was performed using the mobile phase. The fractions were collected according to the retention time, concentrated and lyophilized to obtain polymethoxyflavonoid monomers.

[0015] The mobile phase consists of mobile phase A and mobile phase B, wherein mobile phase A is an acetonitrile solution of formic acid with a volume fraction of 0.1%; and mobile phase B is an aqueous solution of formic acid with a volume fraction of 0.1%.

[0016] During gradient elution, the initial percentage of mobile phase A in the mobile phase is 15%-20%; within 40-50 minutes, the percentage of mobile phase A rises to a high point of 55%-70%, and during this process, the percentage of mobile phase A remains at 28%-45% for 25-35 minutes; after the percentage of mobile phase A rises to the high point, it falls back to 15%-20% within 3-7 minutes.

[0017] 3. The invention CN113209177A, "Method for Ultrasonic-Assisted Extraction of Flavonoids from Citrus Peel," discloses the following steps:

[0018] 1) Raw material pretreatment:

[0019] Fresh citrus peels are dried and pulverized to obtain citrus peel powder;

[0020] 2) Extraction of flavonoids from citrus peel using ultrasound-assisted extraction:

[0021] 2.1) Weigh 1g of citrus peel powder and add 50-55% ethanol solution at a material-to-liquid ratio of 1g / 40-45mL, and mix thoroughly.

[0022] 2.2) Ultrasonic treatment of the mixture:

[0023] After ultrasonic extraction of the mixture obtained in step 2.1), centrifuge it, and filter the supernatant obtained by centrifugation through a filter membrane to obtain the filtrate.

[0024] The ultrasonic extraction power was 325±25W, and the ultrasonic time was 17±2min.

[0025] 2.3), fixed volume:

[0026] Add an ethanol solution with a volume concentration of 58-62% to the filtrate obtained in step 2.2) and bring the volume to 100 mL to obtain a crude flavonoid extract.

[0027] 3) Purification of flavonoids in citrus peel using macroporous resin:

[0028] 3.1) Pretreatment of crude flavonoid extract from citrus peel:

[0029] First, concentrate the crude flavonoid extract, then add an ethanol solution or ethanol with a volume concentration of ≥95% to make the ethanol concentration in the resulting liquid ≥80%. Then let it stand, discard the precipitate, take the supernatant, and concentrate the supernatant under reduced pressure until there is no alcohol odor to obtain the pretreated crude flavonoid extract.

[0030] 3.2) Wet packing of macroporous resin column: The pretreated crude flavonoid extract was prepared with ultrapure water to a concentration of 100-120 μg / mL and loaded onto the column for dynamic adsorption. Then, it was eluted with ethanol solution with a volume concentration of 48-52% to obtain the eluent.

[0031] 3.3) The eluent was concentrated under reduced pressure until no alcohol odor was detected, and then freeze-dried under vacuum to obtain a citrus peel extract containing flavonoids.

[0032] 4. The invention CN1810819A, “Method for preparing citrus flavonoids from citrus peel”, discloses that: (1) crushed citrus peel and ethanol aqueous solution are placed in an extractor at a weight ratio of 1:1 to 5, and extracted under reflux for 0.5 to 1 hour. After filtration, the filtrate is concentrated to obtain citrus peel extract. The obtained citrus peel extract is then suspended in water and centrifuged and filtered to obtain a clear filtrate; (2) under 20 to 30°C, the clear filtrate obtained in step (1) is first adsorbed by polystyrene-type or cross-linked acrylonitrile adsorption resin, and then eluted with an eluent in a gradient from low to high concentration. The eluent is collected and concentrated to obtain the target substance.

[0033] However, none of the above methods can effectively extract polymethoxyflavones from blood orange peel. Summary of the Invention

[0034] The technical problem to be solved by the present invention is to provide a method for increasing the content of polymethoxyflavonoids in blood orange peel through ultrasonic treatment.

[0035] To address the aforementioned technical problems, this invention provides a method for increasing the content of polymethoxyflavonoids in blood orange peel through ultrasonic treatment, comprising the following steps:

[0036] 1) Place the washed and dried fresh Tarocco blood oranges in an ultrasonic device (e.g., an ultrasonic cleaner) and bathe them in a water bath for 0.5 to 20 minutes under ultrasonic conditions (240W, 40kHz) (water bath temperature is room temperature); to obtain ultrasonically treated blood oranges.

[0037] Air dry, generally in the shade for 10-12 hours;

[0038] 2) Store the ultrasound-treated blood oranges obtained in step 1) at a low temperature of 8±1℃ for 1 to 40 days;

[0039] 3) Peel the blood orange peel after low-temperature storage in step 2), add a methanol solution with a volume concentration of 70-80% (preferably 75%) to the peel, and extract under shaking conditions in the dark for 12±1h. Then centrifuge at high speed, and filter the supernatant obtained by filtration (using a needle filter) to obtain a peel extract containing polymethoxyflavones.

[0040] The ratio of fruit peel to methanol solution is 1g / 8-12ml (preferably 1g / 10ml).

[0041] Note: After ultrasonic stimulation, the substances inside the fruit peel need to undergo a chemical reaction to generate polymethoxyflavonoids. This process takes time, so step 2) of storage is carried out. The storage conditions set by this invention can prevent fruit rot to a certain extent. If individual fruits rot accidentally during the process, they can be discarded directly.

[0042] As an improvement to the method of increasing the content of polymethoxyflavonoids in blood orange peel by ultrasonic treatment according to the present invention, it further includes the following step 4):

[0043] The content of polymethoxyflavones in the pericarp extract obtained in step 3) was determined by high performance liquid chromatography.

[0044] As a further improvement to the method of increasing the content of polymethoxyflavonoids in blood orange peel by ultrasonic treatment according to the present invention, in step 3):

[0045] The oscillation is described as oscillating at a frequency of 150±20 rpm.

[0046] The high-speed centrifugation is carried out at 4±0.5℃ and 6000±1000rpm for 15±2 minutes.

[0047] As a further improvement to the method of ultrasonic treatment to increase the content of polymethoxyflavonoids in blood orange peel according to the present invention, in step 1): the volume of water in the ultrasonic cleaner is 6L, and 9±1 Tarocco blood oranges are placed in it (each Tarocco blood orange weighs about 0.16 to 0.23kg).

[0048] As a further improvement to the method of increasing the content of polymethoxyflavonoids in blood orange peel by ultrasonic treatment according to the present invention, the fruits that have undergone ultrasonic treatment in step 1) are placed flat with the stem facing upward (single layer placement, i.e., the fruits do not overlap) and then stored at low temperature in step 2).

[0049] As a further improvement to the method of increasing the content of polymethoxyflavonoids in blood orange peel by ultrasonic treatment according to the present invention,

[0050] In step 1), the water bath time is preferably 5 to 20 minutes;

[0051] The preferred low-temperature storage time for step 2) is 10 to 30 days (more preferably 10 to 20 days).

[0052] As a further improvement to the method of increasing the content of polymethoxyflavonoids in blood orange peel by ultrasonic treatment according to the present invention, step 4):

[0053] High-performance liquid chromatography (HPLC): Column temperature 30℃, detection wavelengths 280nm and 330nm, injection volume 10μL, flow rate 0.5ml / min, mobile phase consisting of 0.1% formic acid (0.1% aqueous formic acid solution, volume concentration 0.1%) as phase A and methanol as phase B, with gradient elution. The mobile phase ratios at different time points were as follows:

[0054] 0–20 min, Phase A 67%, Phase B 33%;

[0055] 20–35 min, phase A 67%–33%, phase B 33%–67%;

[0056] 35–45 min, phase A 33%–5%, phase B 67%–95%;

[0057] 45–51 min, Phase A 5%–0%, Phase B 95%–100%;

[0058] 51–60 min, Phase A 0%–67%, Phase B 100%–33%.

[0059] As a further improvement to the method of increasing the content of polymethoxyflavonoids in blood orange peel by ultrasonic treatment according to the present invention, the polymethoxyflavonoids include sweet orange flavonoids, norihesperidin, hesperidin, 4',5,6,7-tetramethoxyflavonoids, 3,5,6,7,8,3',4'-heptamethoxyflavonoids, and naringin.

[0060] As a further improvement to the method of ultrasonic treatment to increase the content of polymethoxyflavonoids in blood orange peel of the present invention: the final content of polymethoxyflavonoids in blood orange peel was determined to be 211.01-1310.20 mg / 100g.

[0061] This invention utilizes the effects of water bath ultrasound on the structure of the plant medium and its ability to alter enzyme activity, thereby promoting the accumulation of polymethoxyflavonoids in the peel and inhibiting their degradation during storage, significantly improving the accumulation efficiency and content of polymethoxyflavonoids in blood orange peel. The method of this invention is simple to operate and has stable effects, showing promise for the reuse of blood orange peel. Detailed Implementation

[0062] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0063] Example 1:

[0064] 1) Cleaning the fruit:

[0065] Mature fruits of Tarocco blood orange (Citrus sinensis (L.) Osbeck) on the No. 8 trifoliate orange rootstock were harvested from a blood orange orchard in Zizhong County, Neijiang City, Sichuan Province (29°34′~30°24′N, 104°27′~105°07′E, subtropical humid monsoon climate). Fruits with fresh green and intact stems, free from mechanical damage and pests or diseases, were selected. The surface of the oranges was gently brushed with a sponge brush and washed with clean water (20~30℃). The fruits were then laid flat on a table with the stems facing up to air dry overnight (12h) to obtain fresh Tarocco blood oranges after washing and drying.

[0066] 2) Ultrasonic water bath

[0067] Add 6L of purified water to a water bath ultrasonic cleaner, then place 9 fresh Tarocco blood oranges (approximately 1.8kg in total) that have been washed and dried into the water, ensuring the fruit is completely submerged. Ultrasonic treatment is performed at 240W 40kHz and room temperature for 0.5min, 5min, 10min, and 20min. After treatment, the fruit is removed. The control group did not undergo ultrasonic treatment and was recorded as having a treatment time of 0s. The fruit was then dried with paper towels.

[0068] Four groups of blood oranges were obtained after ultrasonic treatment, and one group of blood oranges was not ultrasonically treated. These five groups of fruits were then subjected to steps 3) to 5) and 7) respectively:

[0069] 3) Storage

[0070] The processed fruits were arranged flat with the stems facing upwards (in a single layer, i.e., the fruits did not overlap) and stored in a refrigerator at 8±1℃. A 1cm gap was left between each sample during storage; this gap ensured the fruits had sufficient space to avoid compression and deformation while allowing for normal respiration. The fruits were checked daily for rot. Samples were taken from the fruits on day 0.

[0071] illustrate:

[0072] The temperature conditions set by this invention can prevent fruit from rotting to a certain extent. If individual fruits rot unexpectedly during the process, they can be discarded directly.

[0073] The aforementioned "Day 0" refers to the day the ultrasonic water bath was completed, before it was placed in a refrigerator for storage experiments.

[0074] 4) Blood orange peel pretreatment:

[0075] After removing the fruit from the refrigerator, gently wipe the surface with a paper towel (to remove condensation). Use a knife to cut the fruit horizontally from the equator, then make three vertical cuts along the edge. Separate the peel from the pulp starting from the cuts. Tear the peel into pieces, then crush them (particle size ≤ 1mm) to obtain peel powder.

[0076] 5) Extraction:

[0077] Weigh 0.50 g of fruit peel powder into a 10 ml centrifuge tube, add 5 ml of 75% methanol solution for extraction, cover the sample with a black plastic bag, and extract the mixture in the dark using a variable speed shaker at 150 rpm at room temperature for 12 h; then centrifuge at 4 °C and 6000 rpm for 15 minutes using a high-speed refrigerated centrifuge; finally, collect the supernatant using a 1 ml syringe, filter the supernatant through a 0.22 μL organic syringe filter to obtain the extract (about 5 ml).

[0078] 6) Use polymethoxyflavonoid standards beforehand: sweet orange flavonoid, norihesperidin, hesperidin, 4',5,6,7-tetramethoxyflavonoid, 3,5,6,7,8,3',4'-heptamethoxyflavonoid, and naringenin as external standards, and perform high performance liquid chromatography according to step 7) below to obtain a standard curve.

[0079] Table 1

[0080]

[0081] Explanation: y = 2E - 0.7x + 0.0192, which is equivalent to y = 2 × 10 -7 x+0.0192.

[0082] 7) Determination of polymethoxyflavonoid content in blood orange peel

[0083] Transfer 500 μL of the extract to a brown liquid chromatography bottle lined with an inner tube (protect the extract from light during the assay) and analyze it using high performance liquid chromatography (Waters e2695, Waters Corporation, USA).

[0084] The instrument parameters are as follows:

[0085] The column temperature was 30℃, the detection wavelengths were 280nm and 330nm, the injection volume was 10μL, the flow rate was 0.5ml / min, and the mobile phase consisted of 0.1% formic acid (i.e., a 0.1% aqueous solution of formic acid) as phase A and methanol as phase B.

[0086] The elution conditions are:

[0087] 0–20 min, Phase A 67%, Phase B 33%;

[0088] 20–35 min, phase A 67%–33%, phase B 33%–67%;

[0089] 35–45 min, phase A 33%–5%, phase B 67%–95%;

[0090] 45–51 min, Phase A 5%–0%, Phase B 95%–100%;

[0091] 51–60 min, Phase A 0%–67%, Phase B 100%–33%.

[0092] The peak area data from the high performance liquid chromatography were substituted into the standard curves corresponding to sweet orange flavonoids, noriheptacortin, hesperidin, 4',5,6,7-tetramethoxyflavonoids, 3,5,6,7,8,3',4'-heptamethoxyflavonoids, and naringin, as described in Table 1, to obtain the content of the above components in the extract.

[0093] The contents of the above components were then added together to obtain the total polymethoxyl content in the extract. Finally, the total polymethoxyl flavonoid content in the peel was calculated.

[0094] The results of the four groups of blood oranges treated with ultrasound and the one group of blood oranges not treated with ultrasound are shown in Table 2 below.

[0095] Table 2

[0096]

[0097] Note: Taking the blood orange obtained after ultrasonic treatment for 5 minutes as an example, the data directly obtained by high performance liquid chromatography were 33066, 32372, 12350, 43324, 0, and 21075, respectively. Therefore, substituting the above data into the standard curves corresponding to sweet orange flavonoids, norihesperidin, tangeretin, 4',5,6,7-tetramethoxyflavonoids, 3,5,6,7,8,3',4'-heptamethoxyflavonoids, and naringin in Table 1, the contents of the above components in the extract were obtained as 0.22258 mg / ml, 0.086210 mg / ml, 0.0732834 mg / ml, 0.010189 mg / ml, 0, and 0.020273 mg / ml, respectively. Adding the above six values ​​together, the content of polymethoxyflavonoids in the extract was found to be 0.41253 mg / ml. After conversion, it was found that the content of polymethoxyflavonoids in the peel was 412.53 mg / 100g.

[0098] Example 2: The storage time in the refrigerator at 8±1℃ in step 3) of Example 1 was changed from 0 days to 10 days; the rest was the same as in Example 1. The results of the four groups of blood oranges after ultrasonic treatment and the one group of blood oranges without ultrasonic treatment are shown in Table 3 below.

[0099] Table 3

[0100]

[0101] Example 3: The storage time in the refrigerator at 8±1℃ in step 3) of Example 1 was changed from 0 days to 20 days; the rest was the same as in Example 1. The results of the four groups of blood oranges after ultrasonic treatment and the one group of blood oranges without ultrasonic treatment are shown in Table 4 below.

[0102] Table 4

[0103]

[0104] Example 4: The storage time in the refrigerator at 8±1℃ in step 3) of Example 1 was changed from 0 days to 30 days; the rest was the same as in Example 1. The results of the four groups of blood oranges after ultrasonic treatment and the one group of blood oranges without ultrasonic treatment are shown in Table 5 below.

[0105] Table 5

[0106]

[0107] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for increasing the content of polymethoxyflavonoids in blood orange peel through ultrasonic treatment, characterized in that... Includes the following steps: 1) Place the washed and dried fresh Tarocco blood oranges in an ultrasonic device and bathe them in a water bath for 5-20 minutes under ultrasonic conditions to obtain ultrasonically treated blood oranges; 2) After ultrasonic treatment, place the blood oranges obtained in step 1) with the stem facing upwards and store them at a low temperature of 8±1℃ for 10~30 days; 3) Peel the blood orange peel after low-temperature storage in step 2), add a 70-80% methanol solution to the peel and extract it under shaking conditions in the dark for 12±1h, then centrifuge at high speed, and filter the supernatant to obtain a peel extract containing polymethoxyflavones. The ratio of fruit peel to methanol solution is 1g / 8~12ml; The oscillation is described as oscillating at a frequency of 150±20 rpm. The high-speed centrifugation was carried out at 4±0.5℃ and 6000±1000 rpm for 15±2 minutes.

2. The method for increasing the content of polymethoxyflavonoids in blood orange peel by ultrasonic treatment according to claim 1, characterized in that... It also includes the following step 4): The content of polymethoxyflavones in the pericarp extract obtained in step 3) was determined by high performance liquid chromatography.

3. The method for increasing the content of polymethoxyflavonoids in blood orange peel by ultrasonic treatment according to claim 2, characterized in that... In step 1): the volume of water in the ultrasonic cleaner is 6L, and 9±1 Tarocco blood oranges are added.

4. The method for increasing the content of polymethoxyflavonoids in blood orange peel by ultrasonic treatment according to claim 3, characterized in that... Step 4): High-performance liquid chromatography (HPLC): Gradient elution was performed under the following conditions: column temperature 30℃, detection wavelengths 280nm and 330nm, injection volume 10μL, flow rate 0.5ml / min, and mobile phases of 0.1% formic acid (phase A) and methanol (phase B). The mobile phase ratios at different time points were as follows: 0~20min, Phase A 67%, Phase B 33%; 20~35min, Phase A 67%~33%, Phase B 33%~67%; 35~45min, Phase A 33%~5%, Phase B 67%~95%; 45~51min, Phase A 5%~0%, Phase B 95%~100%; 51~60min, Phase A 0%~67%, Phase B 100%~33%.

5. The method for increasing the content of polymethoxyflavonoids in blood orange peel by ultrasonic treatment according to claim 4, characterized in that: The polymethoxyflavonoids include sweet orange flavonoids, noriheptacortin, hesperidin, 4',5,6,7-tetramethoxyflavonoids, 3,5,6,7,8,3',4'-heptamethoxyflavonoids, and naringin.

6. The method for increasing the content of polymethoxyflavonoids in blood orange peel by ultrasonic treatment according to any one of claims 1 to 5, characterized in that: The content of polymethoxyflavonoids in blood orange peel ranges from 211.01 to 1310.20 mg / 100g.

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