An ultrasonic method for releasing bound phenolic acids

Abstract

This invention discloses an ultrasonic method for releasing bound phenolic acids. The method involves adding fruit and vegetable powder containing phenolic acids to an 80% (v / v) methanol-water solution, extracting at room temperature, centrifuging, and rotary evaporating the supernatant until alcohol-free. The pH is adjusted to 1-2 to obtain an alcohol extract. The alcohol extract is then centrifuged, and the supernatant is extracted with a 1:1 (v / v) diethyl ether-ethyl acetate mixture. The lower aqueous phase containing bound phenolic acids is ultrasonically extracted and then dried with anhydrous sodium sulfate and rotary evaporated to dryness to obtain monomeric phenolic acids. This invention utilizes only a physical method—ultrasonic technology—to release bound phenolic acids, making it environmentally friendly. The release time for free phenolic acids is short, requiring only 10-80 minutes, a reduction of more than 3 hours. Furthermore, this invention releases seven types of bound phenolic acids in 10 minutes of ultrasonic extraction.

Description

(I) Technical Field

[0001] This invention relates to an ultrasonic method for releasing bound phenolic acids. (II) Background Technology

[0002] Phenolic acids refer to a class of phenolic compounds that are abundant and naturally occurring in plant-based foods, such as fruits, vegetables, coffee, tea, wine, beer, and olive oil. They exist in two forms: free and bound. Free phenolic acids (monomers) participate in many physiological processes in plants and animals, such as lignin formation, antioxidant activity, free radical scavenging, stimulating reactions, and toxic reactions. They can delay aging, prevent cardiovascular diseases, and possess anti-inflammatory, anti-cancer, anti-tumor, and anti-ulcer physiological effects, making phenolic acids widely used in medicine and functional foods.

[0003] However, phenolic acids in plants mostly exist in bound form. For example, bound phenolic substances account for about 50% of walnut kernels, about 77% of the total phenolic acid content in wheat flour, and about 90% in citrus peel. Monomeric phenolic acids are easily absorbed across the intestinal barrier, while large molecules are difficult to absorb. This means that bound phenolic acids limit their bioavailability, making the release of bound phenolic acids a key factor restricting their effective utilization.

[0004] The most commonly reported method in the literature is the acid-base release method, which involves first removing free phenolic acids with methanol, then reacting with sodium hydroxide solution and hydrochloric acid solution for 2 to 12 hours to obtain the released monomeric phenolic acids. Zhou Tao (Zhou Tao, Luo Chunmei, Huang Zhifang, Liu Yuhong, Liu Yunhua, Chen Yan, Tang Yina, Yi Jinhai. Comparative study of free and bound phenolic acids in Salvia miltiorrhiza before and after drying [J]. Chinese Journal of Traditional Chinese Medicine, 2020, 45(5): 1090-1096.) et al. removed free phenolic acids from Salvia miltiorrhiza and released bound phenolic acids through acid hydrolysis and alkaline hydrolysis, usually using 6 mol / L hydrochloric acid and 4 mol / L sodium hydroxide. However, this method takes too long to operate and involves the use of hazardous chemicals, which is not environmentally friendly.

[0005] Therefore, it is necessary to further research and develop methods for releasing bound phenolic acids in order to increase the content and types of monomeric phenolic acids after release, thereby improving the bioavailability of phenolic acids. (III) Summary of the Invention

[0006] The purpose of this invention is to provide an ultrasonic method for releasing bound phenolic acids, which can release bound phenolic acids into monomeric phenolic acids, thereby improving their bioavailability.

[0007] The technical solution adopted in this invention is:

[0008] This invention provides an ultrasonic method for releasing bound phenolic acids, the method comprising the following steps:

[0009] (1) Add the fruit and vegetable powder containing phenolic acid to an 80% methanol aqueous solution, soak at room temperature for 10-30h (preferably 24h), then shake in a water bath at 30-60℃ for 1-5h (preferably 40℃, 2h), centrifuge, take the supernatant and rotary evaporate until no alcohol is present, adjust the pH to 1-2, and obtain the alcohol extract.

[0010] (2) Centrifuge the alcohol extract from step (1), extract the supernatant with a 1:1 volume ratio of diethyl ether-ethyl acetate mixture (preferably 5 times), discard the upper organic phase containing free phenolic acid, and take the lower aqueous phase containing bound phenolic acid;

[0011] (3) Extract the lower aqueous phase from step (2) with ultrasound at 5-75℃ and 90-1800W for 5-60 min, then extract with a 1:1 volume ratio of diethyl ether-ethyl acetate mixed solution. Dry the lower aqueous phase with anhydrous sodium sulfate and then evaporate it to dryness to obtain monomeric phenolic acid.

[0012] Preferably, the fruit and vegetable powder containing phenolic acids in step (1) includes navel orange, tangerine, lemon, pomelo, and grapefruit; the fruit and vegetable powder is obtained by crushing fresh fruits and vegetables into pulp, freeze-drying them at -80°C.

[0013] Preferably, the volume of methanol aqueous solution used in step (1) is 1-50 mL / g based on the weight of the fruit and vegetable powder, preferably 25 mL / g.

[0014] Preferably, the alcohol extract in step (1) is prepared as follows: fruit and vegetable powder containing phenolic acid is added to an 80% methanol aqueous solution, soaked at room temperature for 24 hours, shaken in a water bath at 40°C for 2 hours, centrifuged at 7000 rpm for 10 minutes, the above process is repeated twice, the supernatant is combined, and the alcohol is evaporated at 40°C until no alcohol is present. The pH is adjusted to 1-2 with 6 mol / L hydrochloric acid to obtain the alcohol extract.

[0015] Preferably, in step (2), the alcohol extract is centrifuged at 7000 rpm for 10 min, and the supernatant is extracted 5 times with an equal volume of diethyl ether-ethyl acetate mixture. The extract is discarded and the lower aqueous phase is collected.

[0016] Preferably, in step (3), the lower aqueous phase is ultrasonically treated for 10 min at 5℃, 28 Hz, and 540 W, with a 2-second interval between ultrasonic treatments. Then, it is extracted 5 times with an equal volume of diethyl ether-ethyl acetate mixed solution. The lower aqueous phases are combined, dried with anhydrous sodium sulfate, and then rotary evaporated to dryness at 45℃ and 0.090-0.098 MPa to obtain monomeric phenolic acid.

[0017] In the release of bound phenolic acids, those skilled in the art generally consider total phenolic acids and free phenolic acids. However, there is little research on bound phenolic acids present in fruits and vegetables, and no good release methods exist. Existing release methods utilize hazardous chemicals and have long release times, making them unsuitable for practical production and utilization. Therefore, the applicant conducted experiments using environmentally friendly aqueous solutions and discovered that ultrasound can release bound phenolic acids, thereby improving their bioavailability.

[0018] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0019] (1) The method of the present invention is simple and environmentally friendly: The present invention only uses physical method - ultrasonic technology to release bound phenolic acid into free phenolic acid. The operation process only uses pure water, which is green and environmentally friendly and does not cause harm to the human body and the environment, while alkaline hydrolysis uses more hazardous chemicals.

[0020] (2) The free phenolic acid release time of the present invention is short: compared with the acid-base release reaction which requires 4-6 hours, the ultrasonic release time is shorter, requiring only 10-80 minutes, which is more than 3 hours shorter.

[0021] (3) The present invention releases a variety of free phenolic acids: the present invention releases 7 kinds of bound phenolic acids in 10 minutes of ultrasound, and effectively avoids the degradation of sinapic acid and caffeic acid under acid and alkaline conditions; while only 4 kinds are released in 1-3 hours of acid-alkaline reaction, and no protocatechuic acid, p-hydroxybenzoic acid and vanillic acid are released; only 5 kinds of phenolic acids are released in 4 hours of reaction and caffeic acid is degraded. (iv) Description of the attached drawings

[0022] Figure 1 Example 1: Release of bound phenolic acids from navel orange peel at different ultrasound times.

[0023] Figure 2 Comparative Example 1: The content of bound phenolic acids released from navel orange peel at different times by alkaline hydrolysis. (V) Detailed Implementation

[0024] 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: The navel orange (scientific name: Citrus sinensis Osb.var.brasliliensis Tanaka) described in the present invention is a sweet orange plant belonging to the Rutaceae family and the Citrus genus.

[0025] Example 1: Release of bound phenolic acids from navel orange peel

[0026] 1. Extraction of monomeric phenolic acids

[0027] (1) Take a clean, fresh navel orange that is free from spoilage and deterioration, take its peel, juice it using a juicer and homogenize it, freeze-dry it at -80℃ to obtain freeze-dried navel orange peel powder, and seal it at -20℃.

[0028] (2) Accurately weigh 2 grams of freeze-dried navel orange peel powder, extract it with 50 mL of 80% methanol aqueous solution in a water bath at 40°C for 2 hours, centrifuge at 7000 rpm for 10 minutes, and obtain the supernatant and precipitate. Repeat the extraction of the precipitate twice, combine the supernatants, and concentrate them by rotary evaporation at 40°C and 0.090-0.098 MPa until alcohol-free to obtain the methanol extract;

[0029] (3) After adjusting the pH of the methanol extract in step (2) to 1.5 with 6 mol / L hydrochloric acid aqueous solution, centrifuge at 7000 rpm for 10 minutes, take the supernatant and extract it with an equal volume of diethyl ether-ethyl acetate (1:1) mixed solution, discard the upper organic phase containing free phenolic acid, and collect the lower aqueous phase containing bound phenolic acid.

[0030] (4) The aqueous phase collected in step (3) was placed in a non-contact ultrasonic cell disruptor (SCIENTZ08-II, Ningbo Xinzhi Biotechnology Co., Ltd.) and ultrasonically treated for 10 min at 5℃, 28Hz, 540W, and a duty cycle of 50% (ultrasonic 2s, intermittent 2s). Then, it was extracted with an equal volume of diethyl ether-ethyl acetate (1:1) mixed solution, and the extraction was repeated 5 times. The upper extracts were combined. After drying with anhydrous sodium sulfate, the extracts were concentrated to dryness by rotary evaporation at 45℃ and 0.090-0.098 MPa. The solution was then diluted to 5 ml of methanol to obtain a methanol solution of monomeric phenolic acid after the release of bound phenolic acid.

[0031] 2. Quantitative analysis of monomeric phenolic acids

[0032] The monomeric phenolic acid was quantitatively analyzed by external standard method of high performance liquid chromatography. The results are shown in Table 1 and 2. Figure 1 The specific steps are as follows: The methanol solution of monomeric phenolic acid obtained in step (4) above is filtered through a 0.22 μm organic filter membrane and then subjected to high performance liquid chromatography analysis. The peak areas of caffeic acid, p-coumaric acid, ferulic acid and sinapic acid are measured at 320 nm, and the peak areas of protocatechuic acid, p-hydroxybenzoic acid and vanillic acid are measured at 260 nm.

[0033] High-performance liquid chromatography (HPLC) conditions: A Waters 2695 HPLC system was used, with an Agilent Zorbax SB-C18 column (250 × 4.6 mm, 5 μm) and a pre-column C18 column (30 × 4.6 mm, 5 μm) from Agilent Technologies. Mobile phase A was 4% acetic acid, and mobile phase B was pure methanol. The flow rate was 1 mL / min, and the column temperature was 40 °C. Isocratic elution (A:B = 85:15) was performed, with a sample elution time of 45 min and an injection volume of 10 μL.

[0034] Example 2: Effect of ultrasound time on the extraction of monomeric phenolic acids

[0035] The ultrasound time in step 1 of Example 1 was changed to 20, 30, 40, 50, 60, 70, and 80 min, respectively, while other operations remained the same. The results are shown in Table 1 and... Figure 1 .

[0036] Example 3: Release of monomeric phenolic acids in different fruit peel varieties

[0037] In Example 1, the navel orange peel was replaced with ponkan peel, ponkan pulp, lemon peel, pomelo peel, and pomelo pulp, respectively, while other operations were the same. The results are shown in Table 1.

[0038] Comparative Example 1: Alkaline hydrolysis releases bound phenolic acids

[0039] (1) Take a clean, fresh navel orange that is free from spoilage, take its peel, juice it using a juicer and homogenize it, freeze-dry it at -80℃ to obtain freeze-dried navel orange peel powder, and seal it at -20℃.

[0040] (2) Accurately weigh 2 grams of the freeze-dried navel orange peel powder from step (1), extract it with 50 mL of 80% methanol aqueous solution at 40°C in a water bath with shaking for 2 hours, centrifuge at 7000 rpm for 10 minutes, and obtain the supernatant and precipitate; repeat the extraction of the precipitate twice, combine the supernatants and concentrate them by rotary evaporation at 40°C and 0.090-0.098 MPa until alcohol-free, to obtain the methanol extract;

[0041] (3) Adjust the pH of the methanol extract from step (2) to 1.5 with 6 mol / L hydrochloric acid aqueous solution, then centrifuge at 7000 rpm for 10 minutes. Extract the supernatant with an equal volume of diethyl ether-ethyl acetate (1:1) mixed solution, discard the upper organic phase containing free phenolic acid, and collect the lower aqueous phase containing bound phenolic acid.

[0042] (4) Add 20 mL of 4 mol / L sodium hydroxide aqueous solution to the aqueous phase from step (3) and react in the dark for 4 hours. Then adjust the pH to 1-2 with 6 mol / L hydrochloric acid aqueous solution, centrifuge at 7000 rpm for 10 minutes, and extract the supernatant with an equal volume of diethyl ether-ethyl acetate mixture. Combine the upper extracts. After drying with anhydrous sodium sulfate, concentrate to dryness by rotary evaporation at 45℃ and 0.090-0.098 MPa, and make up to 5 mL of methanol. The result is the methanol solution of phenolic acid released in the first part.

[0043] (5) The phenolic acid methanol solution obtained in step (4) was added to 10 mL of 6 mol / L hydrochloric acid aqueous solution and reacted in a water bath at 85 °C for 1 hour. An equal volume of diethyl ether-ethyl acetate mixture was then used for extraction, and the upper extracts were combined. After drying with anhydrous sodium sulfate, the extract was concentrated to dryness by rotary evaporation at 45 °C and 0.090–0.098 MPa, and then diluted to 5 mL of methanol. The resulting product was the second portion of the released phenolic acid methanol solution. The method described in Example 1 was used for detection, and the results are shown in [Figure 1]. Figure 2 Table 1. Content of bound phenolic acids in citrus fruits released at different ultrasound times.

[0044]

[0045]

[0046] Results: The results above show that in Example 1, the release of all seven phenolic acids from the peel of navel oranges increased after 10 min of sonication. After 30 min of sonication, the release of protocatechuic acid, p-hydroxybenzoic acid, caffeic acid, and p-coumaric acid reached their highest values, at 24.05 μg / g DW, 16.10 μg / g DW, 313.15 μg / g DW, and 14.52 μg / g DW, respectively. After 50 min of sonication, the release of ferulic acid and sinapic acid was the highest, at 26.19 μg / g DW and 11.77 μg / g DW, respectively. After 60 min of sonication, the release of vanillic acid was the highest, at 10.36 μg / g DW.

[0047] Compared with Comparative Example 1, Example 1 shortened the reaction time by more than 3 hours and did not use hazardous chemicals, making it environmentally friendly. Comparative Example 1 reacted for 1-3 hours, releasing fewer types of acids, and did not release protocatechuic acid, p-hydroxybenzoic acid, or vanillic acid; after 4 hours of reaction, it released 5 types of phenolic acids, but caffeic acid was degraded. It used more hazardous chemicals in the process, and the degradation was significant.

Claims

1. An ultrasonic method for releasing bound phenolic acids, characterized in that, The method includes the following steps: (1) Add the fruit and vegetable powder containing phenolic acid to an 80% methanol aqueous solution, soak at room temperature for 10-30 h, shake in a water bath at 30-60 ℃ for 1-5 h, centrifuge, take the supernatant and rotary evaporate until no alcohol is present, adjust the pH to 1-2 to obtain the alcohol extract; the fruit and vegetable powder containing phenolic acid is navel orange, ponkan, lemon, pomelo, and pomelo. (2) Centrifuge the alcohol extract from step (1), extract the supernatant with a 1:1 volume ratio of diethyl ether-ethyl acetate mixture, discard the upper organic phase containing free phenolic acid, and take the lower aqueous phase containing bound phenolic acid; (3) The lower aqueous phase of step (2) was ultrasonically extracted for 10 min at 5℃, 28 Hz and 540W, with a 2 s interval between ultrasonication and 2 s. Then it was extracted 5 times with an equal volume of diethyl ether-ethyl acetate mixed solution. The lower aqueous phases were combined, dried with anhydrous sodium sulfate, and then rotary evaporated to dryness at 45℃ and 0.090~0.098 MPa to obtain monomeric phenolic acid.

2. The ultrasonic method for releasing bound phenolic acids as described in claim 1, characterized in that, The fruit and vegetable powder mentioned in step (1) is obtained by crushing fresh fruits and vegetables into a pulp, freeze-drying them at -80℃.

3. The ultrasonic method for releasing bound phenolic acids as described in claim 1, characterized in that, Step (1) The volume of methanol aqueous solution used is 1-50 mL / g based on the weight of the fruit and vegetable powder.

4. The ultrasonic method for releasing bound phenolic acids as described in claim 1, characterized in that, Step (1) The alcohol extract is prepared as follows: Phenolic acid-containing fruit and vegetable powder is added to an 80% methanol aqueous solution and soaked at room temperature for 24 hours. Then, it is shaken in a water bath at 40 °C for 2 hours and centrifuged at 7000 rpm for 10 minutes. The above process is repeated twice. The supernatants are combined and evaporated at 40 °C until no alcohol is present. The pH is adjusted to 1-2 with 6 mol / L hydrochloric acid to obtain the alcohol extract.

5. The ultrasonic method for releasing bound phenolic acids as described in claim 1, characterized in that, Step (2) The alcohol extract was centrifuged at 7000 rpm for 10 min. The supernatant was extracted 5 times with an equal volume of diethyl ether-ethyl acetate mixture. The extract was discarded and the lower aqueous phase was collected.

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