A quality evaluation method for regulating lipid metabolism efficacy of rice bran fatty alkanols based on spectrum-effect relationship

Abstract

The invention discloses a quality evaluation method for regulating lipid metabolism efficacy of rice bran fatty alkanol based on spectrum-effect relationship, and belongs to the technical field of traditional Chinese medicine, food and feed quality. The invention is based on the fingerprint spectrum of rice bran fatty alkanol with different compositions, takes lipid-regulating activity as the main research content, detects the content of rice bran fatty alkanol with different compositions, cooperates with lipid-regulating activity, combines analytic hierarchy process (AHP), grayscale correlation analysis (GRA) and partial least squares regression analysis (PLSR), constructs a spectrum-effect relationship model, screens out the common peaks related to lipid-regulating activity in the chromatographic peaks of rice bran fatty alkanol, and establishes a quality evaluation method. The evaluation method of the invention is fast, accurate and highly sensitive, provides a basis for the lipid-regulating material basis and quality evaluation of rice bran fatty alkanol, and also provides a reference for further product development of rice bran fatty alkanol.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine, food and feed quality, and in particular relates to a quality evaluation method for the efficacy of rice bran fatty alkanol in regulating lipid metabolism based on spectrum-effect relationship. Background Art

[0002] Rice bran fatty alkanols, also known as policosanols, are derived from rice bran wax and are mainly composed of higher fatty alkanols such as docosanol, tetracosanol, hexacosanol, octacosanol, and triacontanol. Octacosanol is the main characteristic component and other higher fatty alkanols form a fingerprint spectrum in a certain mass ratio. Basic research and clinical trial results show that rice bran fatty alkanols have a variety of unique physiological activities such as regulating blood lipids, resisting atherosclerosis, resisting fatigue, and enhancing immunity, and the biological activities of rice bran fatty alkanols with different fingerprints are different.

[0003] At present, the industry uses octacosanol as a marker to evaluate the quality of rice bran fatty alkanols, which cannot reflect the synergistic effect of various higher fatty alkanols in rice bran fatty alkanols, making the efficacy of the product unstable and lacking a standard and method that is clearly associated with and specifically quantified for lipid-lowering efficacy. Therefore, it has been difficult to conduct accurate and effective quality assessment of the lipid-lowering efficacy of rice bran fatty alkanols with different fingerprints. Summary of the invention

[0004] The present invention provides a quality evaluation method for regulating lipid metabolism efficacy of rice bran fatty alkanol based on spectrum-effect relationship, the method comprising the following steps:

[0005] (1) Preparation of Rice Bran Fatty Alkanols

[0006] 8-12 kg of refined rice bran wax is taken, 2-4 times the volume of anhydrous ethanol, 6-8 times the volume of No. 200 solvent oil, and 0.1-0.15 times the mass of sodium hydroxide are added, heated to boiling in a water bath, stirred and refluxed for reaction for 3-5 hours, washed with hot water to remove the lower aqueous phase, washed until the pH value is equal to 6.5-7.5, the upper clear liquid is cooled overnight, vacuum filtered to obtain a filter cake, the filter cake is desolventized and dried to obtain rice bran wax saponification crude alcohol; the prepared crude alcohol is distilled, and the fractions with a tower top temperature of 160-200° C. are collected in sections to obtain 7 kinds of rice bran fatty alkanols with different compositions;

[0007] (2) Preparation of Rice Bran Fatty Alcohol Microemulsion

[0008] 100 mg of the seven different compositions of rice bran fatty alkanols prepared in step (1) were respectively mixed with 250 mg of Tween 80, heated to 85-95° C., and then slowly added dropwise into 100 mL of boiling water while stirring to form a microemulsion with a concentration of 1.0 mg / mL;

[0009] (3) Preparation of Rice Bran Fatty Alkanol and Higher Fatty Alkanol Standard Solution

[0010] Accurately weigh docosanol (C 22 ), tetracosanol (C 24 ), hexacosanol (C 26 ), octacosanol (C 28 ), triacontanol (C 30 ) standard, and 5 mg of each of the 7 fingerprints of rice bran fatty alkanols were dissolved in n-hexane and fixed to volume with a 10 mL volumetric flask to prepare a 500 μg / mL solution;

[0011] (4) Establishment of GC fingerprints of seven different compositions of rice bran fatty alkanols

[0012] GC chromatographic analysis conditions are as follows: detector: FID; chromatographic column: DB-5HT, specification: 30m×320μm×0.1μm; column temperature: 260℃ for 1min, then increase to 300℃ at a rate of 5℃ / min, and maintain for 11min; vaporization chamber temperature: 300℃; detector temperature: 300℃; carrier gas N2 flow rate: 25.0mL / min; hydrogen flow rate: 30.0mL / min; auxiliary gas (Air) flow rate: 400mL / min; injection volume: 1μL; split ratio: 30:1;

[0013] (5) Evaluate the lipid-regulating activity of rice bran fatty alkanols with different compositions through in vitro cell experiments;

[0014] (6) Grey correlation analysis and PLSR were used to establish the spectrum-effect relationship between the common peaks in the fingerprint of rice bran fatty alkanols and lipid-regulating activity;

[0015] (7) Based on the spectrum-activity relationship between the different common peaks of rice bran fatty alkanols and their lipid-regulating activity, a quality standard was established to detect the quality of rice bran fatty alkanols.

[0016] Preferably, the step (4) further comprises the step of using a higher fatty alkanol standard substance for qualitative analysis, and using an area normalization method to calculate the mass percentage of each peak of seven different compositions of rice bran fatty alkanols to determine characteristic peaks and extract common peak data.

[0017] More preferably, in the step (4), the number of common peaks in the GC fingerprints of seven different compositions of rice bran fatty alkanols is 38.

[0018] More preferably, the specific operation of step (5) is as follows: L-02 cells are randomly divided into a control group, a model group and a medication group, the control group uses a basal culture medium, the basal culture medium is RPMI 1640 culture medium containing 10% fetal bovine serum; the model group adds 300 μM / LFFA on the basis of the basal culture medium, wherein the concentration ratio of oleic acid to palmitic acid is 2:1; the medication group adds 100 μg / mL of 8 different rice bran fatty alkanols on the basis of the culture medium of the model group, and after continuing to culture for 24 hours, the contents of TC and TG in the cells are detected.

[0019] More preferably, the step (6) uses the grey correlation analysis method to establish the spectrum-effect relationship between the common peaks in the rice bran fatty alkanol fingerprint and the lipid-regulating activity, and specifically comprises the following steps:

[0020] (1) Determine the reference sequence and comparison sequence in the data sample: select the lipid-regulating activity index as the reference sequence and the relative peak area of ​​the characteristic peak in the fingerprint spectrum as the comparison sequence, which are denoted by x i (t), y j (t), i=1, 2, 3,..., n; j=1, 2, 3,..., n; t=1, 2, 3,..., k;

[0021] (2) Dimensionless processing of original data;

[0022] (3) Calculation of correlation coefficient ξ ij (t) and the correlation β ij .

[0023] More preferably, for a reference sequence x i (t) There are several comparison sequences y j (t), the correlation coefficient ξ between each comparison sequence and the reference sequence at each time ij (t) can be calculated by the following formula:

[0024]

[0025] The correlation degree β ij The calculation formula is as follows:

[0026]

[0027] More preferably, the step (6) uses PLSR to establish the spectrum-effect relationship between the common peaks in the fingerprint spectrum of rice bran fatty alkanols and lipid-regulating activity, and the specific steps are as follows:

[0028] (1) Using the analytic hierarchy process, the initial weight coefficients of the efficacy indicators were calculated;

[0029] (2) Taking the relative peak area of ​​each common peak in the fingerprint as X and the total efficacy data of rice bran fatty alkanol as Y, the partial least squares regression analysis was performed using the software SIMCA 14.1 to calculate the regression coefficient and VIP value of each X corresponding to Y, where the regression coefficient represents the contribution of each X to Y. The regression coefficient was used to model the regression equation:

[0030] Y=-11.3006+0.0108X1+0.0050X2+0.0447X3+0.0112X4+0.0374X5-0.0109X6-0.2722X7-0.0240X8+0.1766X9

[0031] More preferably, the quality standard in step (7) is: selecting a fingerprint peak with a correlation degree greater than 0.5 in the grey correlation analysis and a negative correlation with the lipid-regulating activity in the PLSR analysis as a characteristic peak, and then formulating a quality evaluation method for rice bran wax by calculating the ratio of the selected characteristic peak to the total peak area.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The invention is based on the fingerprints of rice bran fatty alkanols with different compositions, takes lipid-regulating activity as the main research content, detects the content of rice bran fatty alkanols with different compositions, cooperates with lipid-regulating activity, combines analytic hierarchy process (AHP), grayscale correlation analysis (GRA) and partial least squares regression analysis (PLSR), constructs a spectrum-effect relationship model, and screens out the common peaks related to lipid-regulating activity in the chromatographic peaks of rice bran fatty alkanols. The evaluation method of the invention is fast, accurate and highly sensitive, provides a basis for the material basis and quality evaluation of the lipid-regulating effect of rice bran fatty alkanols, and also provides a reference for further product development of rice bran fatty alkanols. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is the fingerprint overlay and common peaks of rice bran fatty alkanol in Example 1.

[0035] Figure 2-3 This is a comparison chart of the lipid-regulating ability of rice bran fatty alcohols with different fingerprints in Example 1.

[0036] Figure 4 This is an overview diagram of the PLSR model in Example 1.

[0037] Figure 5 It is the permutation test diagram of the PLSR model in Example 1.

[0038] Figure 6 It is the total peak lipid-regulating VIP value in Example 1.

[0039] Figure 7is the regression coefficient of lipid-regulating ability in Example 1.

[0040] Figure 8 This is the animal experiment verification result in Example 1. DETAILED DESCRIPTION

[0041] Example 1

[0042] This embodiment provides a quality evaluation method for regulating lipid metabolism efficacy of rice bran fatty alkanols based on spectrum-effect relationship, which comprises the following steps:

[0043] 1. Preparation of rice bran fatty alkanol: Take 10 kg of refined rice bran wax (purchased from Gaoan Qinghe Oil Co., Ltd.), add 3 times the volume of anhydrous ethanol, 7 times the volume of No. 200 solvent oil, and 0.12 times the mass of sodium hydroxide, heat to boiling in a water bath, stir and reflux for 4 hours, wash 3 times with 5 times the volume of hot water, remove the lower aqueous phase, wash until the pH value is about equal to 7, cool the upper clear liquid overnight, vacuum filter to obtain a filter cake, desolventize the filter cake, and dry it to obtain rice bran wax saponification crude alcohol; under the conditions of a working pressure of 20 Pa, a kettle temperature of 210-250°C, and a reflux ratio of 1:10, distill the prepared crude alcohol, and collect it at intervals of 2-3°C at the top temperature of the tower The fractions with a top temperature of 160-200°C (18 sections, namely fractions P101, P102, ... P118) were mixed at a mass ratio of 10:65:25 according to the results of chromatographic analysis to obtain rice bran fatty alkanol sample S6, and the fractions P102, P107 and P113, P103, P108 and P114, P104, P109 and P115, P105, P110 and P116, P106, P111 and P117, P107, P112 and P118 were mixed at a mass ratio of 10:65:25 to obtain other samples S1-S5 and S7.

[0044] 2. Preparation of rice bran fatty alkanol test sample microemulsion: In an emulsifying device, first mix 100 mg of rice bran fatty alkanol with 250 mg of Tween 80, heat to 90°C, and then slowly add dropwise into 100 mL of boiling water while stirring to form a microemulsion with a concentration of 1.0 mg / mL;

[0045] 3. Preparation of rice bran fatty alkanol and higher fatty alkanol standard solutions: Accurately weigh docosanol (C 22 ), tetracosanol (C 24 ), hexacosanol (C 26 ), octacosanol (C 28 ), triacontanol (C 30) standard, and 5 mg of each of the 8 fingerprints of rice bran fatty alkanols were dissolved in n-hexane and fixed to volume with a 10 mL volumetric flask to prepare a 500 μg / mL solution;

[0046] 4. Establishment of GC fingerprint of rice bran fatty alcohol with different compositions, determination of characteristic peaks and extraction of common peak data: GC chromatographic analysis conditions are as follows: detector: FID; chromatographic column: DB-5HT (30m×320μm×0.1μm); column temperature: 260℃ for 1 min, then raised to 300℃ at a rate of 5℃ / min, and maintained for 11min; vaporization chamber temperature: 300℃; detector temperature: 300℃; carrier gas (N2) flow rate: 25.0mL / min; hydrogen (H2) flow rate: 30.0mL / min; auxiliary gas (Air) flow rate: 400mL / min; injection volume: 1μL; split ratio: 30:1; qualitative analysis was performed with standard products of higher fatty alcohols, and the mass percentage of each peak was calculated by area normalization method. The results are as follows: Figure 1 shown.

[0047] Table 1 Relative retention time of the main common peaks of 7 rice bran fatty alkanol samples (min)

[0048]

[0049]

[0050] 5. Evaluation of lipid-regulating activity of rice bran fatty alkanols with different compositions through in vitro cell experiments:

[0051] Human normal liver cell line L-02 was used as the object, and RPMI 1640 medium containing 10% fetal bovine serum was used for culture. When the cells grew to 80% density, they were randomly divided into 6 groups: 1 control group and 5 treatment groups. The control group used RPMI 1640 medium containing 10% fetal bovine serum, and the treatment group used different concentrations of rice bran fatty alkane alcohol S3 (the concentration in the culture medium was 50, 80, 100, 125, and 200 μg / mL, respectively). After 24 hours of treatment, 10 μL of CCK-8 solution was added, and the cells were further cultured at 37°C and 5% CO2 for 0.5 hours. The absorbance was measured at a wavelength of 450nm using an enzyme marker, and the added concentration of rice bran fatty alkane alcohol was screened to be 100 μg / mL.

[0052] The cultured L-02 cells were randomly divided into a control group, a model group and a drug group (7 types of rice bran fatty alkanol fingerprints): the control group used a basal culture medium (RPMI 1640 culture medium containing 10% fetal bovine serum); the model group added 300 μM / LFFA (oleic acid and palmitic acid concentration ratio of 2:1) on the basis of the basal culture medium; the drug group added 100 μg / mL rice bran fatty alkanol on the basis of the high-fat group culture medium, and after continued cultivation for 24 hours, the contents of TC and TG in the cells were detected according to the kit method. The results are shown in Table 2. Figure 2-3 shown.

[0053] Table 2 Lipid-regulating activity of seven rice bran fatty alkanols

[0054]

[0055] 6. Establish the spectrum-effect relationship of rice bran fatty alkanol in lipid regulation

[0056] 6.1 Analytical Hierarchy Process

[0057] 6.1.1 Calculation of initial weight coefficients for each efficacy index

[0058] In order to make the efficacy evaluation index clearer, the two lipid-regulating efficacy indexes (TC inhibition rate and TG inhibition rate) of rice bran fatty alkanol were comprehensively evaluated, where 1 means that both are equally important, 3 means that one is slightly important, and 5 means that one is obviously important. Based on the above subjective scoring of the two efficacy indicators, the matrix in Table 3 was obtained. The initial weight coefficient was calculated according to calculation formula ①, and the results are shown in Table 4.

[0059] Table 3 Priority matrix for pairwise comparison of two efficacy indicators

[0060]

[0061]

[0062] Table 4 Initial weight coefficients of two efficacy indicators

[0063] Efficacy index A1TC A2TG result 1 1

[0064] 6.1.2 Calculation of normalized weight coefficients

[0065] The normalized weight coefficient was calculated according to formula ②, and the results are shown in Table 5. The total efficacy data of rice bran fatty alcohol for lipid regulation was obtained according to the normalized weight coefficient, and the results are shown in Table 6.

[0066]

[0067] Table 5 Normalized weight coefficients

[0068] Efficacy index A1 TC A2TG result 0.5 0.5

[0069] Table 6 Total lipid-regulating activity data of rice bran fatty alkanols

[0070] serial number Total efficacy serial number Total efficacy S1 4.4645 S5 -18.4250 S2 -2.3553 S6 -11.4555 S3 -13.4687 S7 -20.5425 S4 -17.3220

[0071] 6.2 Grey correlation analysis method

[0072] 6.2.1 Determining the reference sequence and comparison sequence in the data sample

[0073] Select the efficacy index as the reference sequence and the relative peak area of ​​the characteristic peak in the fingerprint spectrum as the comparison sequence, which are denoted by x i (t), y j (t) (i=1,2,3,...,n; j=1,2,3,...,n; t=1,2,3,...,k).

[0074] 6.2.2 Dimensionless processing of original data

[0075]

[0076]

[0077] Table 7 Relative peak area average results of the common peaks of 7 rice bran fatty alkanols

[0078]

[0079]

[0080] 6.1.3 Calculate the absolute difference Δ of the sequence ij (t), minimum and maximum difference between the two levels Δ min , Δ max , the formula is as follows:

[0081] Δ ij (t) = |x′ i (t)-y′ j (t)|

[0082]

[0083]

[0084] 6.1.4 Calculate the correlation coefficient, where ξ ij (t) is x i For i The correlation coefficient is ρ, which is usually taken as 0.5.

[0085]

[0086] 6.1.5 Calculating the degree of association

[0087]

[0088] 6.1.6 Data processing results and conclusions

[0089] The correlation of each peak under the efficacy index was obtained through GRA analysis (see Table 8 for the results). The results showed that the correlation of each peak was greater than 0.5, indicating that each common peak had a different degree of inhibitory effect on the total efficacy, and also indicating that the efficacy was the result of the synergy of each component. The order of correlation between the total efficacy and the common peak was: V6>V8>V9>V4>V2>V1>V5>V3>V7.

[0090] Table 8 Correlation between the common peaks of rice bran fatty alkanols and lipid-regulating effects

[0091] Peak Relevance Sorting Peak Relevance Sorting V1 0.7461 6 V6 0.8193 1 V2 0.7489 5 V7 0.5110 9 V3 0.7296 8 V8 0.8001 2 V4 0.7511 4 V9 0.7610 3 V5 0.7370 7

[0092] 6.2PLSR analysis of the spectrum-effect relationship of rice bran fatty alkanols in lipid regulation

[0093] The relative peak area of ​​each common peak in the fingerprint spectrum was taken as X, and the data of rice bran fatty alkanol regulating cell activity was taken as Y. The software SIMCA 14.1 was used for partial least squares regression correlation analysis to establish the PLSR model, and its fitting parameter R 2 Y=0.750、Q 2 =0.597, indicating that the model is stable and has good predictive ability. Figure 4 . The model was tested 200 times, R 2 The intercept of the regression line and the Y axis is less than 0.4, Q 2 The intercept of the regression line and the Y axis is less than 0, indicating that there is no overfitting phenomenon and the model results are reliable. Figure 5 .

[0094] The regression equation of the model is as follows:

[0095] Y=-11.3006+0.0108X1+0.0050X2+0.0447X3+0.0112X4+0.0374X5-0.0109X6-0.2722X7-0.0240X8+0.1766X9

[0096] The results of partial least squares analysis showed that the regression coefficients of the 9 independent variables X to Y were both positive and negative, that is, there were positive and negative correlations. Because the lipid-regulating efficacy data was negatively correlated with the efficacy, the peaks with negative regression coefficients were the peaks that contributed to the efficacy: V6, V7, and V8 were negatively correlated with the lipid-regulating efficacy index and were lipid-regulating efficacy component groups. That is, as the content of these compounds increased, the inhibitory effect on TC and TG was enhanced.

[0097] The variable importance projection (VIP) value is an important indicator that reflects the ability of independent variables to explain dependent variables. Variables with a VIP value greater than 1.0 contribute greatly to the model and are considered important indicators for activity evaluation. Combining the two indicators of regression coefficient and VIP value, V7 was finally determined to be a potential lipid-lowering efficacy component in the PLSR model.

[0098] Table 5 Normalization results of relative peak area median of common peaks of 7 rice bran fatty alkanol samples

[0099] Xi S1 S2 S3 S4 S5 S6 S7 1 0.1707 2.1815 0.4967 -0.6970 -0.7042 -0.6996 -0.7480 2 0.0709 0.9699 0.4352 -0.3564 -0.3486 -0.3573 -0.4139 3 0.7447 8.5240 3.4944 -3.0310 -3.1856 -2.9976 -3.5490 4 -0.0648 2.2724 1.9612 -1.0843 -0.8368 -1.0406 -1.2071 5 0.4230 4.7756 10.5698 -4.6497 -3.7530 -2.2063 -5.1592 6 -4.8841 1.7513 7.0974 -3.4846 3.4591 0.6812 -4.6204 7 -28.6930 -19.2442 -9.9915 0.4346 9.6222 19.2132 28.6587 8 -2.3009 -1.3008 -2.2252 5.0462 3.0054 -0.9879 -1.2365 9 32.6742 -2.4177 -10.5250 5.5868 -8.8810 -9.5731 -6.8639

[0100] Table 6 Standardized results of the median activity of 7 rice bran fatty alcohols

[0101] index S1 S2 S3 S4 S5 S6 S7 Total efficacy 15.7651 8.9453 -2.1681 -6.0214 -7.1244 -0.1549 -9.2419

[0102] 6.3 Establishment of a multi-index active ingredient evaluation method for rice bran fatty alkanols based on spectrum-effect correlation

[0103] Combining the results of GRA and PLSR, the fingerprint peaks with a correlation greater than 0.5 in GRA and a negative correlation with lipid-regulating efficacy in PLSR analysis were selected as characteristic peaks (V6, V7, V8), and the sum of their relative peak areas was recorded as A. The A of various rice bran fatty alkanols is shown in Table 7. Combined with the activity data, when the A of rice bran fatty alkanol is between 46.65% and 74.57%, the lipid-regulating effect is better. This provides a reference for the industrial production of rice bran fatty alkanols with excellent and stable lipid-regulating effects.

[0104] Table 7 A situation of various rice bran fatty alkanols (%)

[0105]

[0106]

[0107] 6.4 Animal Experiment Verification

[0108] According to the results of the cell test, the preferred fingerprint S7 was selected as the object for in vivo testing. 54 SD male rats (200±20g) were selected and randomly divided into 6 groups according to body weight, namely, the normal diet control group, the high-fat diet model group, the low-dose rice bran fatty alkanol treatment group (2mg / (kg·bw) rice bran fatty alkanol + high-fat diet), the medium-dose rice bran fatty alkanol treatment group (4mg / (kg·bw) rice bran fatty alkanol + high-fat diet), the high-dose rice bran fatty alkanol treatment group (8mg / (kg·bw) rice bran fatty alkanol + high-fat diet), and the positive control group (4mg / (kg·bw) atorvastatin calcium AC + high-fat diet). Among them, the normal diet group and the high-fat diet group were gavaged with blank emulsion, and the treatment group was given rice bran fatty alkanol S7 emulsion. The pre-test period was 1 week, feeding the basic diet, and the experimental period was 10 weeks. At the end of the 10th week, the animals were killed and their serum and liver were collected to test lipid metabolism indicators such as TC and TG. The results are shown in the table. Figure 7 .

[0109] 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 method for detecting the efficacy of rice bran fatty alkanols in regulating lipid metabolism based on spectrum-effect relationship, It is characterized in that The method comprises the following steps: (1) Preparation of rice bran fatty alkanol 8-12 kg of refined rice bran wax is taken, 2-4 times the volume of anhydrous ethanol, 6-8 times the volume of No. 200 solvent oil, and 0.1-0.15 times the mass of sodium hydroxide are added, heated to boiling in a water bath, stirred and refluxed for reaction for 3-5 hours, washed with hot water to remove the lower aqueous phase, washed until the pH value is equal to 6.5-7.5, the upper clear liquid is cooled overnight, vacuum filtered to obtain a filter cake, the filter cake is desolventized and dried to obtain the rice bran wax saponified crude alcohol; the prepared crude alcohol is distilled, specifically: the fractions with a tower top temperature of 160-200° C. are collected at a tower top temperature interval of 2-3° C., a total of 18 sections, which are fractions P, P, P and P respectively. The fractions P101, P106 and P112 were mixed at a mass ratio of 10:65:25 to obtain rice bran fatty alkanol sample S6, and the fractions P102, P107 and P113, P103, P108 and P114, P104, P109 and P115, P105, P110 and P116, P106, P111 and P117, P107, P112 and P118 were mixed at a mass ratio of 10:65:25 to obtain other samples S1-S5 and S7, and finally a total of 7 rice bran fatty alkanols with different compositions were obtained; (2) Preparation of Rice Bran Fatty Alcohol Test Sample Microemulsion 100 mg of the seven different compositions of rice bran fatty alkanols prepared in step (1) were respectively mixed with 250 mg of Tween 80, heated to 85-95° C., and then slowly added dropwise into 100 mL of boiling water while stirring to form a microemulsion with a concentration of 1.0 mg / mL; (3) Preparation of Rice Bran Fatty Alkanol and Higher Fatty Alkanol Standard Solution Accurately weigh docosanol (C 22 ), tetracosanol (C 24 ), hexacosanol (C 26 ), octacosanol (C 28 )、triacontanol(C 30 ) standard, and 5 mg of each of the 7 fingerprints of rice bran fatty alkanols were dissolved in n-hexane and diluted to volume with a 10 mL volumetric flask to prepare a 500 μg / mL solution; (4) Establishment of GC fingerprints of seven different compositions of rice bran fatty alkanols The GC chromatographic analysis conditions were as follows: detector: FID; chromatographic column: DB-5HT, specification: 30 m×320 μm×0.1 μm; column temperature: 260°C for 1 min, then increased to 300°C at a rate of 5°C / min, and maintained for 11 min; vaporization chamber temperature: 300°C; detector temperature: 300°C; carrier gas: N 2 Flow rate: 25.0 mL / min; Hydrogen flow rate: 30.0 mL / min; Oxygen flow rate: 400 mL / min; Injection volume: 1 μL; Split ratio: 30:1; (5) Evaluate the lipid-regulating activity of rice bran fatty alkanols with different compositions through in vitro cell experiments; (6) Grey correlation analysis and PLSR were used to establish the spectrum-effect relationship between the common peaks in the fingerprint of rice bran fatty alkanols and lipid-regulating activity; (7) Establish quality standards for detecting rice bran fatty alkanols based on the spectrum-activity relationship between different common peaks of rice bran fatty alkanols and lipid-regulating activity; The step (6) of using the grey correlation analysis method to establish the spectrum-effect relationship between the common peaks in the fingerprint spectrum of rice bran fatty alkanols and lipid-regulating activity specifically comprises the following steps: (1) Determine the reference sequence and comparison sequence in the data sample: select the lipid-regulating activity index as the reference sequence and the relative peak area of ​​the characteristic peak in the fingerprint spectrum as the comparison sequence, which are recorded as , , i = 1, 2, 3, ..., n; j =1, 2, 3, ..., n; t = 1, 2, 3, ..., k; (2) Dimensionless processing of original data; (3) Calculation of correlation coefficient and relevance ; The step (6) uses PLSR to establish the spectrum-effect relationship between the common peaks in the fingerprint spectrum of rice bran fatty alkanols and lipid-regulating activity, and the specific steps are as follows: (1) Using the analytic hierarchy process, calculate the initial weight coefficient of the efficacy index; (2) Taking the relative peak area of ​​each common peak in the fingerprint as X and the total efficacy data of rice bran fatty alcohol as Y, partial least squares regression analysis was performed using the software SIMCA 14.1 to calculate the regression coefficient and VIP value of each X corresponding to Y. The regression coefficient represents the contribution of each X to Y, and the model is built based on this regression coefficient.

2. The detection method according to claim 1, It is characterized in that The step (4) also includes qualitatively analyzing the 7 different compositions of rice bran fatty alkanols using a standard substance, and calculating the mass percentage of each peak using an area normalization method to determine characteristic peaks and extract common peak data.

3. The detection method according to claim 2, It is characterized in that In the step (4), the number of common peaks in the GC fingerprint spectra of 7 different compositions of rice bran fatty alkanols is 9.

4. The detection method according to claim 3, It is characterized in that The specific operation of step (5) is as follows: L-02 cells are randomly divided into a control group, a model group and a medication group. The control group uses a basal culture medium, which is an RPMI 1640 culture medium containing 10% fetal bovine serum; the model group is supplemented with 300 μM / L FFA on the basis of the basal culture medium, wherein the concentration ratio of oleic acid to palmitic acid is 2:1; the medication group is supplemented with 100 μg / mL of 7 different rice bran fatty alcohols on the basis of the culture medium of the model group, and after continuing to culture for 24 hours, the contents of TC and TG in the cells are detected.

5. The detection method according to claim 4, It is characterized in that For a reference sequence There are several comparison sequences , the correlation coefficient between each comparison sequence and the reference sequence at each time It can be calculated by the following formula: ; In the formula, for right The correlation coefficient is ρ, which is 0.

5. , , , the absolute difference of the series , minimum and maximum difference between the two levels , The degree of association The calculation formula is as follows: .

6. The detection method according to claim 5, It is characterized in that The quality standard in step (7) is: select the fingerprint peak with a correlation degree greater than 0.5 in the grey correlation analysis and a negative correlation with the lipid-regulating activity in the PLSR analysis as the characteristic peak, and then formulate the quality evaluation method of rice bran wax by calculating the ratio of the selected characteristic peak to the total peak area.

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