Component analysis method of dried orange peel enzyme with liver protection effect

Abstract

The invention discloses a component analysis method of a dried orange peel enzyme with a liver protection effect, and belongs to the technical field of food processing and detection. The component analysis method disclosed by the invention integrates extraction and content determination methods of four components including total flavonoids, polysaccharides, proteins and amino acids in the dried orange peel enzyme, and the component detection result can efficiently and intuitively reflect the quality of the dried orange peel enzyme. Results show that the pericarpium citri reticulatae enzyme rich in total flavonoids, polysaccharides, proteins and amino acids detected by the component analysis method provided by the invention has good health care and nutritional values, and animal experiments verify that the pericarpium citri reticulatae enzyme shows an obvious liver protection effect and can be used for preparing the medicine for preventing or treating liver injury. The component analysis method provided by the invention can be used for quality detection and identification of the dried orange peel enzyme and expectation of the medicinal function of the dried orange peel enzyme product, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of food processing and detection, and in particular to a component analysis method of tangerine peel enzyme having liver-protecting effect. Background Art

[0002] Guangdong dried orange peel is a famous traditional Chinese medicinal material and one of the eight Lingnan Chinese medicinal materials protected by legislation in Guangdong. It is the dried and mature peel of Xinhui orange (tea branch orange), which is mainly produced in Xinhui, Guangdong. It has the effects of regulating qi and strengthening the spleen, harmonizing the stomach and stopping vomiting, and drying dampness and resolving phlegm. It is worth noting that the current development and utilization of Xinhui orange is mostly limited to its peel. Because the flesh of Xinhui orange is sour and has many seeds, its flesh is mostly discarded, resulting in serious waste of resources.

[0003] According to the investigation, the pulp of Xinhui oranges also contains rich active ingredients, such as flavonoids, polysaccharides, vitamins, etc. Attempts have been made to develop related products for the pulp of Xinhui oranges on the market, such as enzymes, jams, farmyard manure, etc., especially Xinhui orange enzymes. Enzymes are mainly made from fresh fruits, vegetables, medicinal and edible ingredients, animal and plant extracts, etc., and a certain amount of carbon source (such as sugars) is added. Then, a type of fermentation product obtained by natural microbial fermentation includes nutrients such as oligosaccharides, amino acids, organic acids, and functional ingredients such as bioactive enzymes (proteases, superoxide dismutases, lipases, etc.), alkaloids, etc. A large number of studies have shown that plant enzymes have the effects of moisturizing the intestines and promoting bowel movements, sobering up and protecting the liver, enhancing immunity, anti-tumor, and degrading residual pesticides in vegetables.

[0004] At present, there is no research on the determination of the content of the four active ingredients in tangerine peel enzyme, namely total flavonoids, polysaccharides, proteins, and amino acids. There are few studies on the biological activity of tangerine peel enzyme, and there is no report on the role of tangerine peel enzyme in protecting the liver. Citrus is rich in flavonoids and polysaccharides. Studies have shown that flavonoids have anti-tumor, anti-inflammatory, antibacterial, obesity-improving and cardiovascular protective effects, polysaccharides have antioxidant, anti-cancer, and cell immunity-enhancing activities, and proteins and amino acids are necessary nutrients for humans. It has been verified that the four types of ingredients in tangerine peel enzyme, namely total flavonoids, polysaccharides, proteins, and amino acids, are rich in content, have good health and nutritional value, and are worthy of promotion, application and development. Summary of the invention

[0005] The purpose of the present invention is to provide a component analysis method of tangerine peel enzyme with liver protection effect to solve the problems existing in the above-mentioned prior art. The component analysis method provided by the present invention can efficiently and intuitively reflect the quality of tangerine peel enzyme and predict the liver protection function of tangerine peel enzyme.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a method for analyzing the components of tangerine peel enzyme having a liver-protecting effect, comprising the steps of respectively extracting total flavonoids, polysaccharides, proteins and amino acids from the tangerine peel enzyme, and then respectively determining the contents of the total flavonoids, polysaccharides, proteins and amino acids.

[0008] Preferably, the method for extracting total flavonoids is ultrasonic extraction, and the method for determining the content of total flavonoids is ultraviolet spectrophotometry.

[0009] More preferably, the ultrasonic time of the ultrasonic extraction method is 30 min, the solvent used in the ultrasonic extraction is methanol, and the solid-liquid ratio of the tangerine peel enzyme to the methanol is 1 g:100 mL.

[0010] Preferably, the method for extracting polysaccharides is a water extraction and alcohol precipitation method; and the method for determining the content of polysaccharides is a phenol-sulfuric acid method.

[0011] More preferably, the water extraction and alcohol precipitation method comprises the steps of using purified water to sonicate for 60 minutes, centrifuge, filter, prepare a water extract, and mix the water extract with anhydrous ethanol for precipitation;

[0012] The solid-liquid ratio of the tangerine peel enzyme to the purified water is 4g:45mL; the volume ratio of the water extract to the anhydrous ethanol is 1:4.

[0013] Preferably, the method for extracting protein is a cell lysis method, and the method for determining the protein content is a bicinchoninic acid kit quantitative method.

[0014] More preferably, the cell lysis method comprises the steps of centrifuging the tangerine peel enzyme, taking the supernatant, mixing it with the cell lysis solution, pipetting, centrifuging, and washing;

[0015] The volume ratio of the supernatant to the cell lysate is 10:1.

[0016] Preferably, the method for extracting amino acids is ultrasonic extraction, and the method for determining the content of amino acids is ninhydrin colorimetry.

[0017] More preferably, the ultrasonic time of the ultrasonic extraction method is 60 min, the solvent used in the ultrasonic extraction is purified water, and the solid-liquid ratio of the tangerine peel enzyme to the purified water is 1 g:10 mL.

[0018] The present invention also provides the use of the tangerine peel enzyme with liver protection effect in the preparation of a drug for preventing and / or treating liver damage.

[0019] The present invention discloses the following technical effects:

[0020] The present invention establishes a method for extracting and determining the content of four components of tangerine peel enzyme, namely, total flavonoids, polysaccharides, proteins and amino acids, and comprehensively forms a component analysis method of tangerine peel enzyme, and the test results can efficiently and intuitively reflect the quality of tangerine peel enzyme. The results show that the tangerine peel enzyme rich in four components of total flavonoids, polysaccharides, proteins and amino acids detected by the component analysis method provided by the present invention has good health and nutritional value. After animal experiment verification, the tangerine peel enzyme shows obvious liver protection effect and can be used to prepare drugs for preventing or treating liver damage. The component analysis method of the present invention can be used for quality detection and identification of tangerine peel enzyme, as well as the expected medicinal function of tangerine peel enzyme products, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 The HE staining sections and the statistical results of the physical and chemical indicators of each treatment group in Example 3 of the present invention are shown in FIG. 3; wherein, (A) is the HE staining sections of mice in the normal control group (blank control), the acetaminophen group (liver injury model) and the acetaminophen + tangerine peel enzyme group (tangerine peel enzyme extract treatment); (B) is the pathological area of ​​liver injury in the normal control group, the acetaminophen group and the acetaminophen + tangerine peel enzyme group mice; (C) is the AST level in the serum of the mice in the normal control group, the acetaminophen group and the acetaminophen + tangerine peel enzyme group mice; (D) is the ALT level in the serum of the mice in the normal control group, the acetaminophen group and the acetaminophen + tangerine peel enzyme group mice; the scale length is 100 μm; Necrotic Area is the pathological area of ​​liver damage, AST is aspartate aminotransferase, ALT is alanine aminotransferase; Control is the normal control group, APAP is the acetaminophen group, and APAP+PF is the acetaminophen+tangerine peel enzyme group; * indicates P<0.05 compared with the acetaminophen group; ** indicates P<0.01 compared with the normal control group. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] The tangerine peel enzyme stock solution used in the present invention was purchased from Guangdong Suiyue Huixiang Biotechnology Co., Ltd.

[0029] Example 1

[0030] Extraction and content determination of total flavonoids, polysaccharides, proteins and amino acids in tangerine peel enzyme stock solution:

[0031] 1. Determination of total flavonoids in tangerine peel enzyme

[0032] Accurately weigh 0.5g of tangerine peel enzyme sample in a conical flask, add 50mL of methanol and ultrasonicate for 30min, make up for the loss, filter, and the filtrate is the total flavonoid test solution. Take 4.5mL of three prepared test solutions and dilute to the scale in a 10mL volumetric flask, and use ultraviolet spectrophotometry to determine the content. Use methanol as a control and measure its absorbance at 283nm. Parallel determination 3 times, take the average value. Calculate the total flavonoid content by external standard method. The results are shown in Table 1.

[0033] Table 1 Total flavonoids content determination results

[0034]

[0035] 2. Determination of polysaccharides in tangerine peel enzyme

[0036] Accurately weigh 4g of tangerine peel enzyme sample, add 45mL of purified water and ultrasonicate for 1h, let it stand, make up the lost weight, centrifuge, filter, take 5mL of sample and precipitate with 20mL of anhydrous ethanol (final ethanol content is 80%) for 2h, remove the supernatant and retain the precipitate after centrifugation, wash the precipitate with 5mL of acetone, remove the supernatant and retain the precipitate after centrifugation, repeat the acetone washing once, wash the precipitate with 5mL of ether and centrifuge, remove the supernatant and retain the precipitate, evaporate the precipitate to dryness and dissolve it in 10mL of purified water to obtain a polysaccharide test solution, take 2mL of each test sample and make up to volume in a 10mL volumetric flask, then take 1mL of each test sample in a test tube and add 1mL of purified water, quickly add 1mL of 6% phenol and 5mL of concentrated sulfuric acid, let it stand for 5min, then place it in a 40℃ water bath for 15min, then put it in ice water to cool and then put it to room temperature, use purified water as a control, measure its absorbance at 490nm, measure 3 times in parallel, take the average value, and calculate the polysaccharide content by external standard method. The results are shown in Table 2.

[0037] Table 2 Polysaccharide content determination results

[0038]

[0039] 3. Determination of protein in tangerine peel enzyme

[0040] The tangerine peel enzyme sample was centrifuged and filtered, 3 mL of supernatant was taken, the mass was weighed, 0.3 mL of cell lysis solution was added, the sample was pipetted 18 times, centrifuged for 20 min, the supernatant was taken out, the precipitate was washed with 2 mL of purified water, centrifuged, washed twice, the supernatant and the two washing solutions were combined in a 10 mL volumetric flask and fixed to the scale to obtain the protein test solution. 2 mL of the test sample was taken in a 10 mL volumetric flask and diluted to the scale, and the protein content was determined using the BCA kit method. The same concentration of the test sample was taken in every 3 replicate wells, and the results were averaged. The results are shown in Table 3.

[0041] Table 3 Protein content determination results

[0042]

[0043]

[0044] 4. Determination of amino acids in tangerine peel enzyme

[0045] Weigh 5g of tangerine peel enzyme sample accurately, add 50mL of purified water and sonicate for 1h, let it stand, make up for the lost weight, centrifuge, filter, and take the filtrate to get the amino acid test solution. Take 1mL of the test sample in a 25mL volumetric flask and add pH 6.5 at a concentration of 0.2mol·L-1 1.0 mL of phosphate buffer solution and 1.0 mL of 2.0% ninhydrin solution were added, heated in a water bath at 100°C for 20 min, and then diluted to the mark with water after cooling to room temperature. The absorbance was measured at 568 nm, and the measurement was repeated 3 times to obtain the average value. With water as the control, the absorbance was measured at 568 nm, and the amino acid content was calculated by the external standard method. The results are shown in Table 4.

[0046] Table 4 Amino acid content determination results

[0047]

[0048] According to Tables 1, 2, 3 and 4, tangerine peel enzyme is rich in flavonoids, polysaccharides, proteins and amino acids. The order of the content of each component is protein>polysaccharides>amino acids>total flavonoids, indicating that tangerine peel enzyme may have good health functions and nutritional value.

[0049] Example 2

[0050] Preparation of citrus peel enzyme freeze-dried powder (PF):

[0051] The tangerine peel enzyme stock solution that passed the component analysis test was further concentrated under reduced pressure at 50°C to a viscous state, and then stored in a -20°C refrigerator for 24 hours. After vacuum freeze-drying, the tangerine peel enzyme freeze-dried powder was collected and stored in a -20°C refrigerator.

[0052] Example 3

[0053] To verify the protective effect of the lyophilized powder of dried orange peel enzyme prepared in Example 2 on acute liver injury, a mouse liver injury model was induced using acetaminophen (APAP).

[0054] 1. Experimental subjects: C57 mice.

[0055] 2. Experimental groups: ① normal control group (oral administration of normal saline, Control); ② acetaminophen group (oral administration of normal saline, APAP); ③ acetaminophen + tangerine peel enzyme group (oral administration dose: 500 mg / kg, APAP+PF); 3 mice in each group.

[0056] 3. Experimental Procedure

[0057] The animals were given drugs for one week according to the groups, and the acute liver injury animal model was induced by intraperitoneal injection of acetaminophen (dose of 350 mg / kg), and the normal control group was injected with an equal amount of saline; 24 hours after the injection, 4% tribromoethanol was injected intraperitoneally for anesthesia, and the eyeballs were removed to obtain whole blood. After being placed at room temperature for 2 hours, the blood was centrifuged at 3000 rpm for 10 minutes to obtain serum, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the mouse serum were detected by biochemical analyzer. The results are shown in Figure 1 In (C) and (D).

[0058] The mice were killed by cervical dislocation and the livers were removed and fixed in 4% paraformaldehyde solution for more than 48 hours. The livers were sliced ​​with HE staining and the pathological area of ​​liver damage was calculated. Figure 1 (A) and (B).

[0059] 4. Experimental Results

[0060] Depend on Figure 1 As shown in (A) and (B), the liver surface of mice in the normal control group (blank control) was smooth and delicate, while the liver surface of mice in the acetaminophen group (liver injury model) was rough, with a whitish overall color, and a large number of granular white fat droplets and red punctate inflammatory lesions. Compared with the acetaminophen group, the liver surface of the acetaminophen + tangerine peel enzyme group (tangerine peel enzyme extract treatment) tended to be smooth, and the level of granular white fat droplets and red punctate inflammatory lesions was significantly reduced. The results of HE staining sections of mouse livers showed that the liver cell structure of mice in the acetaminophen group was loose, the liver cells were obviously necrotic, and more fibrous connective tissues were abnormally proliferated, and obvious inflammatory cell infiltration was visible in the tissues (pathological area 46.82±8.86%). In the acetaminophen + tangerine peel enzyme group, the area of ​​liver cell necrosis and inflammatory cell infiltration was greatly reduced (pathological area 18.91±1.33%), showing a significant protective effect on liver cells.

[0061] Depend on Figure 1 As shown in (C) and (D), the AST and ALT activities of mice in the acetaminophen + tangerine peel enzyme group were 51.88±21.98U / L and 56.04±26.08U / L, respectively, which were significantly lower than those in the acetaminophen group (P<0.05), indicating a protective effect on liver cells.

[0062] The above results indicate that the tangerine peel enzyme of the present invention is rich in flavonoids, polysaccharides, proteins, amino acids and other four types of components, and exhibits excellent liver protection effects in the treatment of acute liver damage caused by acetaminophen.

[0063] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A component analysis method of tangerine peel enzyme having liver protection effect, characterized in that: The method comprises the steps of respectively extracting total flavonoids, polysaccharides, proteins and amino acids from the tangerine peel enzyme, and then respectively measuring the contents of the total flavonoids, polysaccharides, proteins and amino acids.

2. The component analysis method according to claim 1, characterized in that The method for extracting total flavonoids is ultrasonic extraction, and the method for determining the content of total flavonoids is ultraviolet spectrophotometry.

3. The component analysis method according to claim 2, characterized in that The ultrasonic extraction method uses an ultrasonic time of 30 minutes, and uses methanol as the solvent; the solid-liquid ratio of the tangerine peel enzyme to the methanol is 1 g:100 mL.

4. The component analysis method according to claim 1, characterized in that The method for extracting polysaccharides is a water extraction and alcohol precipitation method; the method for determining the content of polysaccharides is a phenol-sulfuric acid method.

5. The component analysis method according to claim 4, characterized in that The water extraction and alcohol precipitation method comprises the steps of using purified water for 60 minutes of ultrasonic treatment, centrifugation, filtration, preparing a water extract, and mixing the water extract with anhydrous ethanol for precipitation; The solid-liquid ratio of the tangerine peel enzyme to the purified water is 4g:45mL; the volume ratio of the water extract to the anhydrous ethanol is 1:

4.

6. The component analysis method according to claim 1, characterized in that The method for extracting protein is a cell lysis method, and the method for determining the protein content is a bicinchoninic acid kit quantitative method.

7. The component analysis method according to claim 6, characterized in that The cell lysis method comprises the steps of centrifuging the tangerine peel enzyme, taking the supernatant, mixing it with the cell lysis solution, blowing, centrifuging, and washing; The volume ratio of the supernatant to the cell lysate is 10:

1.

8. The component analysis method according to claim 1, wherein: The method for extracting amino acids is an ultrasonic extraction method, and the method for determining the content of amino acids is a ninhydrin colorimetric method.

9. The component analysis method according to claim 8, characterized in that The ultrasonic extraction method uses an ultrasonic time of 60 min, uses purified water as the solvent, and the solid-liquid ratio of the tangerine peel enzyme to the purified water is 1 g:10 mL.

10. Use of the tangerine peel enzyme with liver protection effect as claimed in claim 1 in the preparation of a drug for preventing and / or treating liver damage.

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