Method for constructing spectrum-effect relationship of dried orange peel from different producing areas on basis of electrochemical fingerprint spectrum combined with antioxidant activity

Abstract

The invention relates to a method for constructing a spectrum-effect relationship of dried orange peel from different producing areas based on electrochemical fingerprints in combination with antioxidant activity. The method comprises the following steps: establishing electrochemical fingerprints of dried orange peel from different producing areas under the conditions that the temperature is 35 DEG C, the concentration of sulfuric acid is 1mol / L, the concentration of ceric ammonium sulfate is 0.005 mol / L, the concentration of malonic acid is 0.45 mol / L and the concentration of sodium bromate is 0.3 mol / L; an ABTS free radical scavenging rate method with high sensitivity and stability is selected for antioxidant activity evaluation. Meanwhile, a rapid and accurate spectrum-activity relationship method is established by analyzing the spectrum-activity relationship between the electrochemical fingerprints of the dried orange peel from different producing areas and the semi-inhibitory concentration of the ABTS free radical scavenging rate in combination with Pearson correlation. The method has certain scientific value and wide application value.

Description

Technical Field

[0001] The invention belongs to the technical field of Chinese medicinal material analysis, and relates to a method for constructing spectrum-effect relationship of tangerine peel from different origins based on electrochemical fingerprint combined with antioxidant activity. Background Art

[0002] Tangerine peel is the dried mature peel of the citrus plant (Citrus reticulata Blanco) and its cultivated varieties of the Rutaceae family. The cultivated varieties mainly include Chachi (Citrus reticulata'Chachi') (Guangzhou tangerine peel), Dahongpao (Citrus reticulata'Dahongpao'), Wenzhou mandarin (Citrus reticulata'Unshiu'), and Fu tangerine (Citrus reticulata'Tangerina'). Generally, the peel needs to be naturally aged in a specific environment for more than three years, the volatile oil content is reduced, the flavonoid content is increased, and it shows medicinal effects before it can be called tangerine peel. Fresh citrus peel contains a large amount of volatile oil, has a strong and irritating smell, so it cannot be used as medicine and is not called tangerine peel. my country's tangerine peel production areas are mainly distributed in Guangdong, Fujian, Zhejiang, Sichuan, Chongqing, Jiangxi, Hunan, Hubei and other places. It is rich in chemical components, including flavonoids, volatile oils, alkaloids, limonoids and trace elements. Tangerine peel is a traditional medicinal material that can be used as both medicine and food. Tangerine peel tastes bitter and is warm in nature. Its main functions are to regulate qi and strengthen the spleen, dry dampness and resolve phlegm. It has therapeutic effects on abdominal distension, poor appetite, vomiting and diarrhea, and cough with phlegm. Modern pharmacological studies have shown that tangerine peel has antioxidant, anti-inflammatory, cough and phlegm-relieving, lipid-lowering, blood pressure-lowering, digestion-promoting, anti-allergic, antibacterial, anti-tumor, anti-platelet aggregation, anti-aging, cardiotonic, anti-shock, liver protection and neuroprotective effects. Tangerine peel from different origins is affected by multiple factors such as the planting environment, soil composition, and climatic conditions, resulting in differences in the content, activity, and efficacy of tangerine peel flavonoids. Therefore, it is of great significance to establish the spectrum-effect relationship of tangerine peel from different origins.

[0003] As a common tea drink and snack food, tangerine peel has a wide market demand. However, the current identification of tangerine peel mainly adopts sensory methods, UV-visible spectrophotometry and liquid chromatography-mass spectrometry. Although the sensory method is simple and easy to use, it is easily interfered by subjective factors and it is difficult to achieve standardized inspection; UV-visible spectrophotometry has the advantages of simple operation, good stability and low analysis cost for detecting the content of flavonoids in tangerine peel, but the color displayed by this method will change with the change of color development temperature and time, so the colorimetric method has poor reproducibility; and although liquid chromatography-mass spectrometry has the advantages of accurate detection and high sensitivity, it has the disadvantages of high price, high cost and cumbersome operation. In contrast, electrochemical fingerprinting has the advantages of low cost, convenient operation, rapidity, high sensitivity and good reproducibility. Zhang Zeshuai (Zhang Zeshuai, Pei Jierong, Ye Ruiping, et al. Identification of honeysuckle, honeysuckle and their mixtures by nonlinear chemical oscillation fingerprint [J]. Chinese Patent Medicine, 2023, 45(06): 2067-2072.) established the chemical oscillation fingerprint of honeysuckle, honeysuckle and their mixtures in the KBrO3-MnSO4-H2SO4-CH3COCH3 oscillation system. The results showed that the chemical oscillation fingerprints of honeysuckle, honeysuckle and their mixtures were significantly different, especially during the oscillation period. Electrochemical fingerprints can be used to directly distinguish easily confused medicinal materials. Cheng Wangxing (Cheng Wangxing, Guan Yi, Chen Jia, et al. Study on the chemical fingerprints of red peony root from different origins in an oscillating system [J]. Journal of Analysis and Testing, 2011, 30(08):937-940.) used a BZ oscillation system to study the electrochemical fingerprints of red peony root from different origins. The red peony root from different origins (Bozhou, Anhui, Heze, Shandong, Chifeng, Inner Mongolia, Hailar) not only had significantly different chemical fingerprint shapes, but also had significant differences in the main parameters. Fang Xuanqi (Fang Xuanqi, Wang Xiali, Wang Fangbin, et al. Identification of the authenticity and origin of Gastrodia elata by nonlinear chemical fingerprint [J]. Journal of Food Safety and Quality, 2018, 9(17): 4693-4699.) established electrochemical fingerprints of Gastrodia elata and its counterfeit products such as Mirabilis jalapa root, dahlia, banana root and potato, and found that Gastrodia elata and its counterfeit products not only have significantly different oscillation curves, but also have significantly different characteristic parameters such as induction time and oscillation life. Gastrodia elata and its counterfeit products can be directly distinguished by the intuitive difference in electrochemical fingerprints. This shows that electrochemical fingerprints can be used to identify the authenticity of Chinese medicinal materials from different origins.

[0004] Therefore, the present invention adopts electrochemical fingerprint to analyze tangerine peel from different origins, and at the same time, establishes the spectrum-effect relationship between the electrochemical fingerprint of tangerine peel and the half-inhibition concentration of ABTS free radical scavenging rate, so as to achieve a method for quickly and accurately establishing the spectrum-effect relationship between the characteristic parameters of the electrochemical fingerprint and the half-inhibition concentration of ABTS free radical scavenging rate. Summary of the invention

[0005] The object of the present invention is to provide a method for constructing a spectrum-effect relationship of dried tangerine peel from different origins based on electrochemical fingerprints combined with antioxidant activity. The sample does not need to be processed, the operation is simple, the cost is low, and the experimental results are accurate. Specifically, it includes the determination of the electrochemical fingerprint, the determination of the ABTS free radical scavenging ability, and the establishment of a spectrum-effect relationship between the characteristic parameters of the electrochemical fingerprint and the half-inhibitory concentration of the ABTS free radical scavenging rate. It includes the following steps:

[0006] (1) Add 0.1000+0.0003-0.0007g of tangerine peel powder from different origins, 12ml of 1mol / L sulfuric acid aqueous solution, 3mL of 0.005mol / L cerium ammonium sulfate aqueous solution, and 6mL of 0.45mol / L malonic acid aqueous solution to the electrolytic cell under constant temperature conditions, carefully insert the platinum electrode and saturated calomel electrode, and cover the electrolytic cell; take 3mL of 0.3mol / L sodium bromate aqueous solution, preheat for 13min, and quickly add the sodium bromate aqueous solution to the open constant temperature double-layer electrolytic cell, then click the start data collection button on the computer until the oscillation ends, and collect the electrochemical fingerprints of tangerine peel from different origins. Each sample was repeated 3 times in parallel, and the characteristic parameters were taken as the average value of the 3 groups of parallel experiments. The numerical differences in the four characteristic parameters of oscillation period, minimum potential, maximum potential and stop potential of dried tangerine peel from different origins are relatively small, but there are obvious differences in induction time, oscillation life and maximum amplitude. According to the shape and characteristic parameters of the electrochemical fingerprint, it can be seen that the chemical composition of dried tangerine peel produced in different regions may be different due to different factors such as soil moisture, fertility and precipitation in different regions.

[0007] (2) Take 88 μL of 7 mmol / L ABTS solution and add it to 5 mL of 140 mmol / L potassium persulfate. Keep it away from light for 12 hours. Take 500 μL of different concentrations of quinoa alcohol extract and ABTS and place them in a test tube. Shake well. After reacting in the dark for 6 minutes, measure the absorbance at 517 nm. Vc is used as a positive control.

[0008] (3) Take an appropriate amount of dried tangerine peel powder (passed through a No. 2 sieve) from different origins, add 80% ethanol solution at a liquid-to-solid ratio of 20:1, reflux extraction for 3 hours, filter while hot after extraction, concentrate the extract using a rotary evaporator, and then freeze-dry to obtain an alcohol extract. Accurately weigh an appropriate amount of the alcohol extract and dissolve it in 80% ethanol solution to obtain different concentrations of dried tangerine peel alcohol extracts for activity determination.

[0009] (4) Pearson correlation analysis was used to study the spectrum-effect relationship between the characteristic parameters of the electrochemical fingerprint and the half-inhibition concentration of the ABTS free radical scavenging rate. When the Pearson correlation coefficient is positive and the larger it is, the weaker the ABTS free radical scavenging activity of the sample is. Among them, the induction time and the lowest potential are negatively correlated with the half-inhibition concentration of the ABTS free radical scavenging rate, with correlation coefficients of -0.39 and -0.26, respectively; the oscillation life, maximum amplitude and highest potential are positively correlated with the half-inhibition concentration of the ABTS free radical scavenging rate, with correlation coefficients of 0.97, 0.92 and 0.88, respectively, all greater than 0.8, indicating that when the oscillation life, maximum amplitude and highest potential are larger, the value of the half-inhibition concentration of the ABTS free radical scavenging rate is larger, and the ABTS antioxidant activity of the tangerine peel of the corresponding origin is smaller; among them, the correlation coefficient between the oscillation life and the half-inhibition concentration of the ABTS free radical scavenging rate is the largest. By comparing the oscillation life and antioxidant activity of tangerine peel from different origins, it was found that as the antioxidant activity decreases, the characteristic parameter oscillation life shows an increasing trend.

[0010] (5) The antioxidant activity of tangerine peel from different origins can be intuitively reflected based on the length of the oscillation lifetime in the electrochemical fingerprint combined with specific characteristic parameters.

[0011] Beneficial effects of the present invention:

[0012] 1. The samples of the present invention do not require complicated pretreatment and can be directly crushed for use. In addition, the method studies the Chinese medicinal materials as a whole and can reflect the overall characteristics of the medicinal materials, rather than the characteristics of a certain part or a single component;

[0013] 2. Pearson correlation analysis was used to study the spectral-effect relationship between the characteristic parameters of the electrochemical fingerprint, such as induction time, oscillation period, maximum amplitude, oscillation lifetime, highest potential, lowest potential and stop potential, and the half-inhibitory concentration of ABTS free radical scavenging rate. At the same time, the antioxidant activity of tangerine peel from different origins can be intuitively reflected based on the length of the oscillation lifetime in the electrochemical fingerprint combined with specific characteristic parameters.

[0014] 3. The method is simple, fast, accurate, low-cost and has universal practicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Electrochemical fingerprints of dried tangerine peel from different origins

[0016] Figure 2 Pearson relationship between electrochemical fingerprint characteristic parameters and ABTS activity DETAILED DESCRIPTION

[0017] Electrochemical instrument: Shanghai Chenhua electrochemical workstation (CHI760E)

[0018] Embodiment 1

[0019] 1. Establishment of electrochemical fingerprints of tangerine peel from different origins

[0020] At 35°C, add 0.1000+0.0003-0.0007g of dried tangerine peel medicinal powder from different origins, and measure 12ml of 1mol / L sulfuric acid aqueous solution, 3mL of 0.005mo / L ceric ammonium sulfate aqueous solution, and 6mL of 0.45mo / L malonic acid aqueous solution in the electrolytic cell. Carefully insert the platinum electrode and saturated calomel electrode and cover the electrolytic cell. Take 3mL of 0.3mol / L sodium bromate aqueous solution, preheat for 13min, and quickly add the sodium bromate aqueous solution into an open constant temperature double-layer electrolytic cell. Then click the start data collection button on the computer until the oscillation ends, and collect the electrochemical fingerprints of dried tangerine peel from different origins. Collect 3 times for each origin, and the characteristic parameters are displayed as average values. The spectrum is as follows Figure 1 The specific average characteristic parameters are shown in Table 1.

[0021] Table 1 Influence of characteristic parameter values ​​of electrochemical fingerprint of tangerine peel from different origins

[0022]

[0023]

[0024] pass Figure 1 It is obvious that the appearance of tangerine peel from 13 origins is significantly different. Table 1 shows the average values ​​of the characteristic parameters of the electrochemical fingerprint. By calculating the RSD value (RSD < 5% means the difference is small), it is found that the numerical differences in the three characteristic parameters of oscillation period, maximum potential and stop potential of tangerine peel from different origins are small; however, it can be found that there are obvious differences in the induction time, oscillation life, maximum amplitude and minimum potential of tangerine peel from different origins, which is also the cause. Figure 1 That is the main reason why the electrochemical fingerprints of tangerine peels from different origins are quite different.

[0025] Embodiment 2

[0026] 1. ABTS free radical scavenging activity

[0027] Take an appropriate amount of tangerine peel powder (passed through No. 2 sieve) from different origins, add 80% ethanol solution at a liquid-to-solid ratio of 20:1, reflux extraction time 3h, filter while hot after extraction, use a rotary evaporator to concentrate the extract, and then freeze-dry to obtain an alcohol extract. Accurately weigh an appropriate amount of alcohol extract and dissolve it in 80% ethanol solution to obtain tangerine peel alcohol extracts of different concentrations.

[0028] Take 88 μL of 7 mmol / L ABTS solution and add it to 5 mL of 140 mmol / L potassium persulfate to react in the dark for 12 hours. Dilute the solution with ethanol to an absorbance of 0.7 ± 0.02 at 734 nm. Take 500 μL of different concentrations of tangerine peel alcohol extract and ABTS in a test tube, shake well, react in the dark for 6 minutes, and measure the absorbance at 517 nm as the experimental group (A1), measure the absorbance of ABTS and ethanol solution as the blank group (A0), and measure the absorbance of different concentrations of sample solution and ethanol solution as the control group (A2). Vc is used as the positive control. Calculate the sample clearance rate according to the following formula (1).

[0029]

[0030] The experimental results are shown in Table 2.

[0031] Table 2 Half-inhibitory concentration of ABTS free radical scavenging rate

[0032]

[0033] 2. Pearson correlation analysis

[0034] The characteristic parameters (induction time, oscillation period, maximum amplitude, oscillation lifetime, minimum potential, maximum potential and stop potential) of the electrochemical fingerprint spectra of tangerine peel from 13 different origins and the half-inhibitory concentration values ​​of ABTS free radical scavenging rate were imported into Origin 2021 software, and the Correlation Plot plug-in was used for Pearson correlation analysis.

[0035] Pearson correlation analysis was used to study the spectrum-effect relationship between the characteristic parameters of the electrochemical fingerprint and the half-inhibitory concentration of the ABTS free radical scavenging rate. When the Pearson correlation coefficient is positive and the larger it is, the weaker the ABTS free radical scavenging activity of the common peak is. The results are as follows: Figure 2 As shown in Table 3. The induction time and the lowest potential were negatively correlated with the half-inhibitory concentration of ABTS free radical scavenging rate, with correlation coefficients of -0.39 and -0.26, respectively. The oscillation period, maximum amplitude, oscillation life, maximum potential and stop potential were positively correlated with the half-inhibitory concentration of ABTS free radical scavenging rate, among which the correlation coefficients of oscillation life, maximum amplitude and maximum potential were all greater than 0.8, which were 0.97, 0.92 and 0.8, respectively, indicating that the larger the maximum amplitude, the longer the oscillation life and the higher the maximum potential, the smaller the ABTS antioxidant activity of tangerine peel from the corresponding origin.

[0036] Table 3 Pearson correlation coefficients between electrochemical fingerprint characteristic parameters and ABTS activity

[0037]

[0038] The oscillation life, maximum amplitude and highest potential with a correlation coefficient greater than 0.8 were selected to explore the relationship between the half inhibition concentration value of the ABTS free radical scavenging rate. It was observed in Tables 1 and 2 that as the antioxidant activity decreased, the oscillation life, maximum amplitude and highest potential of dried orange peels from different origins all showed a trend of gradual increase. Among them, the trend of oscillation life was the most obvious. This is consistent with the result of Pearson's correlation coefficient. At the same time, the antioxidant activity of dried orange peels from different origins can be intuitively reflected based on the length of the oscillation life in the electrochemical fingerprint combined with specific characteristic parameters.

Claims

1. A method for constructing spectrum-effect relationship of tangerine peel from different origins based on electrochemical fingerprint combined with antioxidant activity, characterized in that: The invention establishes the electrochemical fingerprints of tangerine peels from different origins, and uses Pearson correlation to analyze the spectrum-effect relationship between the characteristic parameters of the electrochemical fingerprints of tangerine peels from different origins and the half-inhibition concentration of ABTS free radical scavenging rate, so as to establish the spectrum-effect relationship of the electrochemical fingerprints of tangerine peels from different origins.

2. The method according to claim 1, wherein the test conditions of the electrochemical fingerprint are as follows: adding 0.1000+0.0003-0.0007g of Chinese medicinal material powder under a constant temperature of 35°C, measuring 12ml of 1mol / L sulfuric acid aqueous solution, 3mL of 0.005mol / L cerium ammonium sulfate aqueous solution, and 6mL of 0.45mol / L malonic acid aqueous solution in an electrolytic cell in sequence, carefully inserting a platinum electrode and a saturated calomel electrode, and then covering the electrolytic cell with a lid; taking 3mL of 0.3mol / L sodium bromate aqueous solution, preheating for 13min, and then quickly adding the sodium bromate aqueous solution into an open constant temperature double-layer electrolytic cell, and then clicking the start data collection button on the computer until the oscillation ends, and the electrochemical fingerprint is obtained.

3. The method according to claim 2, characterized in that: It is used to establish the electrochemical fingerprint of tangerine peel from 13 production areas including Shaoyang, Zhangjiajie, Jingzhou, Shiyan, Quzhou, Jiaxing, Yunnan, Sichuan, Chongqing, Jiangxi, Guangxi, Guangdong and Fujian.

4. The method according to claim 1, characterized in that: Using the ABTS free radical scavenging rate method with high sensitivity and stability, 88 μL of 7 mmol / L ABTS solution was added to 5 mL of 140 mmol / L potassium persulfate and reacted in the dark for 12 hours; 500 μL of different concentrations of citrus aurantium alcohol extract and ABTS were placed in a test tube, shaken well, and reacted in the dark for 6 minutes, then the absorbance was measured at 517 nm, with Vc as the positive control.

5. The method according to claim 1, characterized in that: Take an appropriate amount of tangerine peel powder that has passed through a No. 2 sieve from different origins, add 80% ethanol solution at a liquid-to-solid ratio of 20:1, reflux extraction time for 3 hours, filter while hot after extraction, use a rotary evaporator to concentrate the extract, and then freeze-dry to obtain an alcohol extract; accurately weigh an appropriate amount of the alcohol extract and dissolve it in 80% ethanol solution to obtain tangerine peel alcohol extracts of different concentrations for activity determination.

6. The method according to claim 1, characterized in that: Pearson correlation analysis was used to study the spectrum-effect relationship between the electrochemical fingerprint and the half-inhibitory concentration of ABTS free radical scavenging rate. The larger the Pearson correlation coefficient was, the weaker the free radical scavenging activity of the sample was. The induction time and the lowest potential were negatively correlated with the half-inhibitory concentration of ABTS free radical scavenging rate, with correlation coefficients of -0.39 and -0.26, respectively. The oscillation lifetime, maximum amplitude and highest potential were positively correlated with the half-inhibitory concentration of ABTS free radical scavenging rate, with correlation coefficients of 0.97, 0.92 and 0.88, respectively, all greater than 0.8, indicating that the larger the oscillation lifetime, maximum amplitude and highest potential, the larger the value of the half-inhibitory concentration of ABTS free radical scavenging rate, and the smaller the ABTS antioxidant activity of tangerine peel from the corresponding origin.

7. The method according to claim 1, characterized in that: The correlation coefficient between the oscillation lifetime and the half-inhibition concentration of ABTS free radical scavenging rate is the largest. By comparing the oscillation lifetime and antioxidant activity of tangerine peel from different origins, it was found that as the antioxidant activity decreased, the characteristic parameter oscillation lifetime showed an increasing trend.

8. The method according to claim 1, characterized in that: The antioxidant activity of tangerine peel from different origins can be intuitively reflected based on the length of the oscillation lifetime in the electrochemical fingerprint combined with specific characteristic parameters.

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