Method for improving antioxidant activity of pithecellobium clypearia branches and leaves and application

Abstract

The invention discloses a method for improving antioxidant activity of pithecellobium clypearia branches and leaves and application. The pithecellobium clypearia branches and leaves are fermented through microorganisms, the antioxidant activity of the pithecellobium clypearia branches and leaves is improved, and the method has the advantages of being low in cost, safe, reliable, easy to use and the like and can be applied and popularized in the field of traditional Chinese medicinal material processing.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicinal material processing, and particularly relates to a method for improving the antioxidant activity of the branches and leaves of Iodes cruciata and application thereof. BACKGROUND

[0002] As an anti-inflammatory and antibacterial traditional Chinese medicinal material, Iodes cruciata has potential value as an antibiotic adjuvant. Flavonoids and phenolic acids in the branches and leaves of Iodes cruciata are effective components for exerting pharmacological effects and are closely related to antioxidant capacity. In vitro evaluation of the antioxidant activity of traditional Chinese medicinal materials generally uses three indexes of DPPH, ABTS free radical scavenging and FRAP iron reduction capacity, and the larger the numerical value is, the stronger the antioxidant capacity is. If the antioxidant capacity of the branches and leaves of Iodes cruciata can be significantly improved, the pharmacological efficacy thereof will be directly enhanced, and the application value thereof will be greatly expanded: firstly, in the field of medicine, more effective anti-inflammatory and anti-infective drugs or health products can be developed; secondly, in the field of functional food, the branches and leaves of Iodes cruciata can be used as high-efficiency natural antioxidants for preservation or function enhancement of products; and thirdly, in the field of cosmetics, the branches and leaves of Iodes cruciata can provide high-quality raw materials for development of high-end anti-aging and repair products. However, there is currently a lack of technology for improving the antioxidant activity of Iodes cruciata. SUMMARY

[0003] The primary purpose of the present application is to overcome the shortcomings and deficiencies of the prior art and provide a method for improving the antioxidant activity of the branches and leaves of Iodes cruciata.

[0004] Another purpose of the present application is to provide application of the above-mentioned method for improving the antioxidant activity of the branches and leaves of Iodes cruciata.

[0005] The purposes of the present application are achieved by the following technical solution: a method for improving the antioxidant activity of the branches and leaves of Iodes cruciata, comprising the following steps:

[0006] (1) cutting the branches and leaves of Iodes cruciata to obtain cut branches and leaves of Iodes cruciata;

[0007] (2) uniformly spraying a bacterial agent on the surface of the cut branches and leaves of Iodes cruciata, stirring to obtain treated branches and leaves of Iodes cruciata;

[0008] (3) storing the treated branches and leaves of Iodes cruciata obtained in step (2) after degassing and sealing.

[0009] In step (1), the cutting size is preferably less than 4 cm, more preferably less than 3 cm, and most preferably 1-2 cm.

[0010] In step (2), the bacterial species in the bacterial agent are preferably Bacillus altitudinis GST-24, or a complex of Bacillus altitudinis GST-24 and Lactobacillus plantarum CCZZ1.

[0011] The high-terrestris Bacillus GST-24 and the Lactobacillus plantarum CCZZ1 in the complex are in a ratio of 1:0.9-1.1 in cell number, and more preferably in a ratio of 1:1 in cell number.

[0012] The bacterial agent in step (2) is in a solid form or a liquid form, and preferably in a liquid form, so that it is more beneficial to be uniformly dispersed.

[0013] The bacterial agent in step (2) is preferably in an amount of 10 7 ~ 10 9 cfu of the bacterial agent per kilogram of the leaves of the Iodes cirrhosum, and more preferably in an amount of 10 8 cfu of the bacterial agent per kilogram of the leaves of the Iodes cirrhosum.

[0014] The storage mode in step (3) is preferably light-proof storage.

[0015] The storage time in step (3) is preferably more than 30 days, more preferably 50-80 days, and most preferably 60 days.

[0016] The method for improving the antioxidant activity of the leaves of the Iodes cirrhosum has an application in the field of processing of traditional Chinese medicinal materials.

[0017] The present application has the following advantages and effects relative to the prior art:

[0018] The present application provides a method for improving the antioxidant activity of the leaves of the Iodes cirrhosum, which improves the antioxidant capacity of the leaves of the Iodes cirrhosum through fermentation by microorganisms, and has the advantages of low cost, safety, reliability, and easy utilization. BRIEF DESCRIPTION OF DRAWINGS

[0019] Fig. 1 is a result graph of the influence of the additive on the DPPH free radical scavenging capacity of the leaves of the Iodes cirrhosum after silage.

[0020] Fig. 2 is a result graph of the influence of the additive on the ABTS free radical scavenging capacity of the leaves of the Iodes cirrhosum after silage.

[0021] Fig. 3 is a result graph of the influence of the additive on the FRAP iron-reducing antioxidant capacity of the leaves of the Iodes cirrhosum after silage.

[0022] In the graph, CK is a blank control group, which is the leaves of the Iodes cirrhosum without silage; S is a negative control group subjected to silage fermentation by replacing the bacterial liquid with an equal amount of water; CCZZ1 is an experimental group subjected to silage fermentation by adding the CCZZ1 bacterial liquid; GST-24 is an experimental group subjected to silage fermentation by adding the GST-24 bacterial liquid; DM is the dry matter; TE represents the Trolox equivalent; and different lowercase letters represent significant differences (P<0.05). DETAILED DESCRIPTION

[0023] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0024] Example 1: Silage fermentation of monkey earrings with added bacterial solution

[0025] 1. Preparation of microbial agents

[0026] Lactobacillus plantarum CCZZ1 has been disclosed in Chinese patent CN 102851233B, accession number [missing information].

[0027] CGMCC NO.6078. MRS culture was performed at 37℃ in shake flasks, followed by centrifugation at 10000 rpm and resuspending in deionized water to obtain a concentration of 1.0 × 10⁻⁶. 8 CFU CCZZ1 bacterial suspension.

[0028] Bacillus hygroscopicus GST-24 has been disclosed in Chinese patent CN 114921375 B, accession number GDMCCNo:62124. It was cultured in shake flasks at 37°C using LB culture base, centrifuged at 10000 rpm, and resuspended in deionized water to obtain a concentration of 1.0 × 10⁻⁶. 8 CFU GST-24 bacterial suspension.

[0029] Preparation of compound microbial agent: CCZZ1 bacterial suspension and GST-24 bacterial suspension were mixed at a volume ratio of 1:1 to obtain compound microbial agent.

[0030] 2. Experimental Procedure

[0031] A silage experiment was conducted using branches and leaves of *Hylocereus undatus* (sourced from a *Hylocereus undatus* planting base in Qianfeng Village, Liangjing Town, Huizhou City). The conventional nutrient components and microbial counts of fresh *Hylocereus undatus* branches and leaves are shown in Table 1. The results of the antioxidant activity tests of fresh *Hylocereus undatus* branches and leaves are shown in Table 3. Figs. 1-3 As shown. The materials were cut to less than 3cm, mixed thoroughly, and divided into the following groups: CCZZ1 treatment group (abbreviated as CCZZ1 in the table and figure), GST-24 treatment group (abbreviated as GST-24 in the table and figure), compound microbial agent treatment group (abbreviated as CCZZ1+GST-24 in the table and figure), blank control group (i.e., fresh monkey ear fungus branches and leaves, abbreviated as CK in the table and figure), and negative control group (i.e., using an equal volume of sterile water instead of the bacterial solution, abbreviated as S in the table and figure). Approximately 1.0 × 10⁻⁶ bacteria were added per kg of fresh monkey ear fungus branches and leaves. 8cfu or equal volume of water. About 200 g per bag, 3 replicates for each treatment, 4 treatments in total, 12 bags, vacuum packaging machine after air sealing, placed in the dark, room temperature fermentation for 60 d, then open and analyze the fermentation quality, microbial quantity. The leaves of A. paniculata were extracted with methanol, the ratio of material to liquid was 1:50 (g / mL), and the antioxidant activity was analyzed. The fermentation quality of A. paniculata leaves is shown in Table 2; the results of antioxidant activity are shown in Table 3. Figs. 1-3 and Table 3.

[0032] Table 1 Nutritional components and microbial quantity of fresh A. paniculata leaves

[0033]

[0034] Note: FM, fresh matter; DM, dry matter.

[0035] Table 2 Fermentation quality of A. paniculata leaves

[0036] Note: DM, dry matter; TN, total nitrogen; ND, not detected; SEM, standard error of mean; the same lowercase letters in the same column represent significant difference (P<0.05); **, P<0.01.

[0037] Table 3 Test results of antioxidant activity of A. paniculata leaves

[0038] Note: The DPPH scavenging rate of each treatment was measured at a mass concentration of A. paniculata of 0.12 mg / ml; the ABTS scavenging rate of each treatment was measured at a mass concentration of A. paniculata of 0.20 mg / ml; SEM, standard error of mean; the same lowercase letters in the same column represent significant difference (P<0.05).

[0039] The results of DPPH free radical scavenging capacity are shown in Table 3 and Fig. 1 It can be seen that the DPPH free radical scavenging capacity of the CK group is the lowest (238.97 mg·TE g -1 DM), the DPPH free radical scavenging capacity of the S group is significantly improved (P<0.05), reaching 422.15 mg·TE g -1 DM, about 1.77 times that of the CK group; the DPPH free radical scavenging capacity of the CCZZ1 group (437.29 mg·TE g -1 DM) is slightly higher than that of the S group; the DPPH free radical scavenging capacity of the GST-24 group (494.71 mg·TE g -1 DM) is 2.07 times that of the CK group; the DPPH free radical scavenging capacity of the CCZZ1+GST-24 group (479.27 mg·TE g -1The DPPH free radical scavenging rate of the monkey ear ring branch leaves with a mass concentration of 0.12 mg / mL increased from 34.21% of the CK to 68.12%.

[0040] The results of the ABTS free radical scavenging capacity are shown in Table 3 and Fig. 2 Fig. 2, and it can be seen that the ABTS free radical scavenging capacity of the CK group was 602.89 mg·TE g -1 DM. The ABTS free radical scavenging capacity of the CCZZ1 group (653.89 mg·TE g -1 DM) was higher than that of the CK group, but had no significant difference with that of the S group (670.43 mg·TE g -1 DM); the ABTS free radical scavenging capacity of the GST-24 group (691.56 mg·TE g -1 DM) was significantly higher than that of the S group (P<0.05); the ABTS free radical scavenging capacity of the CCZZ1+GST-24 group (792.77 mg·TE g -1 DM) was 1.32 times and 1.18 times that of the CK group and the S group, respectively, and was significantly higher than that of the CCZZ1 group and the GST-24 group (P<0.05). The ABTS free radical scavenging rate of the monkey ear ring branch leaves with a mass concentration of 0.20 mg / mL increased from 46.48% of the CK to 66.69%.

[0041] The results of the FRAP iron-reducing antioxidant capacity are shown in Table 4 and Fig. 3 Fig. 3, and it can be seen that the FRAP iron-reducing antioxidant capacity of each treatment group after ensiling (the S group, the CCZH1 group and the GST-24 group were 393.36, 402.48 and 415.83 mg·TE g -1 DM, respectively) was significantly higher than that of the CK group (P<0.05). The FRAP iron-reducing antioxidant capacity of the CCZZ1+GST-24 group reached the highest value among all the treatments (418.31 mg·TE g -1 DM), which was about 1.27 times that of the CK group (328.55 mg·TE g -1 DM).

[0042] After calculation, the antioxidant comprehensive index of the CK group was 67.63, that of the S group was 87.98, that of the CCZH1 group was 89.03, that of the GST-24 group was 95.55, and that of the CCZZ1+GST-24 group was 98.96. In terms of the antioxidant activity comprehensive index, the CCZZ1+GST-24 group was the best, followed by the GST-24 group.

[0043] In summary, the above results show that the CCZZ1 and the GST-24 complex used for fermenting the monkey ear ring branch leaves has a synergistic effect.

[0044] Conventional nutrient component analysis method

[0045] The dry matter content was determined after oven drying at 70°C for 48 h, and the dried sample was ground through a 1 mm sieve for the determination of nutrients. The buffering capacity was determined by titration with hydrochloric acid and sodium hydroxide. The soluble carbohydrate content was determined by the anthrone sulfuric acid method. The pH value was determined by a pH meter.

[0046] Microbial quantity analysis method

[0047] Accurately weigh 10 g of the sample into a sterile self-sealing bag, add 90 mL of sterile water, and shake on a 200 rpm shaker for 30 min. Dilute to different concentrations (10 -1 ~ 10 -5 ), and determine the microbial quantity by the plate coating method. Lactic acid bacteria are counted using MRS solid medium, aerobic bacteria are counted using nutrient agar medium, and yeast and mold are counted using Bengal red (tiger red) agar medium. MRS solid medium, nutrient agar medium, and Bengal red (tiger red) agar medium are used for counting, respectively. Lactic acid bacteria are counted under anaerobic conditions at 30°C for 2-3 days, aerobic bacteria are counted under aerobic conditions at 30°C for 1-2 days, and yeast and mold are counted under aerobic conditions at 30°C for 2-3 days.

[0048] Analysis method for silage fermentation quality

[0049] Accurately weigh 20 g of the sample into a self-sealing bag, add 80 mL of distilled water to completely immerse, and place in a 4°C refrigerator for 16-18 h. After filtering with qualitative filter paper, the filtrate is collected to determine the pH value, NH3-N, and organic acids. The pH value is determined by a pH meter, and the NH3-N content is determined by the phenol-sodium hypochlorite colorimetric method. The lactic acid content is determined by the p-hydroxy biphenyl colorimetric method, and the acetic acid, propionic acid, and butyric acid contents are determined by a gas chromatograph. Filtrate pretreatment: take 1 mL of the filtrate into a centrifuge tube, add 0.2 mL of a 25% metaphosphoric acid solution containing 2 g·L -1 2-ethyl butyric acid, and place at 4°C for 30 min or more. Centrifuge at 10,000 rpm for 10 min, and take the supernatant through a 0.45 μm microporous filter for use. Vaporization chamber parameters: carrier gas N2, split ratio 40:1, sample injection amount 0.4 μL, temperature 220°C; chromatographic column parameters: HP-INNOWax capillary chromatographic column in constant flow mode, flow rate 2.0 mL·min -1 , average linear velocity 38 cm·sec -1 ; column oven parameters: programmed temperature, 120°C for 3 min, increased to 180°C at a rate of 10°C·min -1 , and maintained for 1 min, FID temperature 250°C.

[0050] Analysis method for antioxidant activity

[0051] Extract preparation: accurately weigh 0.2 g of the raw material, dry, grind the sample through a 1 mm sieve into a 15 mL centrifuge tube, add 10 mL of methanol, place in a shaker at 200 rpm in the dark for 24 h, centrifuge at 3000 rpm for 10 min, and extract the supernatant, store at 4°C until testing. Weigh 1 g of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) into 1 L of methanol to prepare a 1 g·L -1 trolox solution for use.

[0052] DPPH radical scavenging capacity determination: prepare a 0.1 mmol·L -1 2,2-diphenyl-1-trinitrohydrazine (DPPH) solution, take 0.5 mL of the trolox standard solution (0, 10, 20, 30, 40, 50, 60, 70, 80 μg·mL -1 ) and add to 4 mL of 0.1 mmol·L -1 DPPH solution to make a standard curve, take 0.5 mL of the appropriately diluted extract and add to 4 mL of 0.1 mmol·L -1 DPPH solution, mix the above solutions well, and let stand in the dark for 30 min, read the absorbance of the solution (A sample) at 517 nm, calculate the radical scavenging activity from the standard curve, and express the scavenging activity as trolox equivalent (TE). At the same time, measure the absorbance of methanol instead of DPPH solution (A blank) and the absorbance of purified water instead of extract (A control). Radical scavenging rate = [1-(A sample-A blank) / (A control-A blank)] x 100%.

[0053] ABTS radical scavenging capacity determination: mix 7 mmol·L -1 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS) solution and 2.45 mmol·L -1 potassium persulfate solution in a volume ratio of 1:1 to prepare an ABTS stock solution, transfer to a brown glass bottle, let stand at room temperature for 16 h, dilute the stock solution 20-fold to detect the absorbance at 734 nm, adjust to 0.70 ± 0.02, and reserve. Take 0.1 mL of the trolox standard solution (0, 100, 200, 300, 400, 500, 600 μg·mL -1) 0.1 mL of the extract solution was added to 2 mL of the ABTS solution to prepare a standard curve, 0.1 mL of the extract solution was added to 2 mL of the ABTS solution to prepare a standard curve, and the absorbance of the solution was read at 734 nm (A sample) after the mixture was uniformly mixed and kept in the dark for 6 min. The free radical scavenging activity was calculated from the standard curve, and the scavenging activity was expressed as TE. The absorbance of methanol (A blank) and the absorbance of purified water instead of the extract solution (A control) were also determined. The free radical scavenging rate = [1-(A sample-A blank) / (A control-A blank)] x 100%.

[0054] FRAP iron-reducing antioxidant capacity determination: 10 mmol·L -1 2,4,6-tripyridyl triazine (TPTZ) solution, 20 mmol·L -1 ferric chloride hexahydrate solution, 300 mmol·L -1 The acetate buffer (the acetate buffer is composed of 20.4 g of sodium acetate + 80 mL of glacial acetic acid, and the volume is made up to 1 L with distilled water) was mixed at a volume ratio of 1:1:10 to prepare the FRAP solution, which was heated in a 37℃ water bath for 30 min in the dark and then used. 0.5 mL of the trolox standard solution (0, 10, 20, 30, 40, 50, 60, 70, 80 μg·mL -1 ) was added to 4 mL of the FRAP solution to prepare a standard curve, 0.5 mL of the extract solution was added to 4 mL of the FRAP solution for determination, and the absorbance of the solution was read at 593 nm after the mixture was uniformly mixed and kept in the dark at 37℃ for 30 min. The reducing power was expressed as TE.

[0055] The DPPH free radical scavenging capacity, the ABTS free radical scavenging capacity, and the FRAP iron-reducing antioxidant capacity were comprehensively evaluated and ranked by the antioxidant capacity comprehensive index method. The antioxidant capacity comprehensive index score was calculated according to the following formula: (sample score / optimal score) x 100, and the average value of the scores of the three indicators was taken as the final comprehensive evaluation result.

[0056] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A method of improving antioxidant activity of Ginkgo biloba leaves, characterized by It comprises the following steps: (1) cutting the leaves of Monochasma, to obtain cut leaves of Monochasma; (2) spraying the bacterial agent on the surface of the cut leaves of Monochasma, and stirring to obtain treated leaves of Monochasma; (3) storing the treated leaves of Monochasma in a sealed container.

2. The method of claim 1, wherein the cut leaves of Monochasma are cut to a length of less than 4 cm.

3. The method of claim 1, wherein the bacterial agent comprises Bacillus altitudinis GST-24, or a mixture of Bacillus altitudinis GST-24 and Lactobacillus plantarum CCZZ1.

4. The method of claim 3, wherein the mixture of Bacillus altitudinis GST-24 and Lactobacillus plantarum CCZZ1 is in a ratio of 1:0.9-1.1 by cell number.

5. The method of claim 1, wherein the bacterial agent is applied to the leaves of Monochasma in an amount of 0.1-0.5 mL per 100 g of leaves.

6. The method of claim 1, wherein the treated leaves of Monochasma are stored in the dark.

7. The method of claim 1, wherein the treated leaves of Monochasma are stored for more than 30 days.

8. Use of the method of any one of claims 1-7 for processing of traditional Chinese medicinal materials. The amount of the bacterial agent used in step (2) is 10 7 ~10 9 cfu of the bacterial agent per kilogram of the leaves of the monkey ear ring. ​ ​ ​ ​ ​

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