Method for extracting polyphenol from oriental wormwood

Abstract

The invention belongs to the technical field of plant extraction, and particularly relates to a method for extracting polyphenol from oriental wormwood. The method comprises the following steps: adding herba artemisiae scopariae powder into an ethanol solution with the volume fraction of 30-60%, extracting at the extraction temperature of 50-80 DEG C for 45-90 minutes, filtering the extracting solution, and collecting filtrate; the dosage ratio of the herba artemisiae scopariae powder to the ethanol solution is 1g: (20-50ml). The method is simple in extraction and easy to operate, and the polyphenol extraction rate can reach 1.85%.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extraction, and particularly relates to a method for extracting polyphenols from Artemisia capillaris. Background Art

[0002] Artemisia capillaris is a plant of the genus Artemisia in the Asteraceae family. It is mainly distributed in Henan, Shandong, Shanxi, Shaanxi and other places in my country. It grows mostly on hillsides, river banks, and gravel land. It likes warm and humid climates and has strong adaptability. Artemisia capillaris has a strong aroma and is a natural plant with a long history of consumption and good medicinal effects. More than four hundred years ago, Li Shizhen discovered that "Artemisia capillaris was often planted as a vegetable in the past." In some areas, Artemisia capillaris is made into "Qingming Cake", a traditional food for Qingming Festival. In addition, Artemisia capillaris is also made into tea, cakes, etc. The Chinese also recognized the medicinal effects of Artemisia capillaris relatively early. The "Shennong Bencao Jing" written 1,800 years ago believed that "long-term use will make the body light and benefit the qi, and the face white for a long time." It has the effects of clearing heat and dampness, protecting the liver and promoting bile secretion; further research in modern medicine believes that Artemisia capillaris also has antioxidant, anti-inflammatory, antiviral, and lipid metabolism regulating effects.

[0003] The significant food and medicinal effects of Artemisia capillaris are due to the various active ingredients it contains, such as organic acids, volatile oils, terpenes, polyphenols, etc. Among them, polyphenols are important secondary metabolites in the process of plant growth and are commonly found in fruits, vegetables and other foods. In terms of structure, many of its hydrogen atoms are replaced by hydroxyl groups. These phenolic hydroxyl groups can react with free radicals in the human body, thereby preventing free radicals from harming the human body. Therefore, polyphenols have strong antioxidant capacity and play an important role in the prevention and treatment of chronic diseases such as cardiovascular disease, cancer, and Alzheimer's disease. In addition, research on polyphenols has been found in many plants, such as Rosa laevigata, tamarind, tulip, Zanthoxylum bungeanum, Momordica grosvenori, Phragmites australis, reed, and Zongye, but research on polyphenols in Artemisia capillaris is rare. Summary of the invention

[0004] In view of the problems and shortcomings in the prior art, the object of the present invention is to provide a method for extracting polyphenols from Artemisia capillaris.

[0005] Based on the above purpose, the present invention adopts the following technical solution:

[0006] The invention provides a method for extracting polyphenols from Artemisia capillaris, comprising the following steps: adding Artemisia capillaris powder to an ethanol solution with a volume fraction of 30% to 60% for extraction, the extraction temperature is 50 to 80°C, the extraction time is 45 to 90 minutes, filtering the extract to collect the filtrate; the usage ratio of the Artemisia capillaris powder to the ethanol solution is 1g: (20 to 50ml).

[0007] Preferably, the extraction temperature is 63°C.

[0008] Preferably, the extraction time is 72 min.

[0009] Preferably, the volume fraction of the ethanol solution is 50%.

[0010] Preferably, the usage ratio of the Artemisia capillaris powder to the ethanol solution is 1 g:30 ml.

[0011] Furthermore, the method for extracting polyphenols from Artemisia capillaris also includes a step of detecting the filtrate: adding sodium carbonate reagent and folin phenol reagent with a mass percentage concentration of 10% in sequence to the filtrate and diluting and mixing evenly, subjecting the mixed solution to a color development reaction at 25°C in the dark, and measuring the absorbance of the solution; the volume ratio of the sodium carbonate reagent with a mass percentage concentration of 10% and the folin phenol reagent is (0.5-4): (0.1-1); and the color development time is 10-60 min.

[0012] Furthermore, the filtrate needs to be treated with constant volume before testing.

[0013] Preferably, the volume ratio of the sodium carbonate reagent to the Folin phenol reagent is 1:0.5.

[0014] Preferably, the color development time is 30 minutes.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) Using the method for extracting polyphenols from Artemisia capillaris provided by the present invention, by optimizing the extraction conditions, the solid-liquid ratio (the ratio of Artemisia capillaris powder to ethanol solution) is 1g:30ml, the volume fraction of ethanol is 50%, the extraction time is 72min, and the extraction temperature is 63°C. Under these conditions, the polyphenol extraction rate can reach 1.85%.

[0017] (2) The present invention adopts the colorimetric method to determine polyphenols, which has the advantages of higher polyphenol extraction rate, low cost, no need for complex equipment, and short detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effect of the extraction temperature on the extraction rate of polyphenols in Artemisia capillaris in the present invention;

[0019] Figure 2 The effect of the solid-liquid ratio on the extraction rate of polyphenols in Artemisia capillaris in the present invention;

[0020] Figure 3 The effect of the volume fraction of ethanol on the extraction rate of polyphenols in Artemisia capillaris of the present invention;

[0021] Figure 4 The effect of the extraction time on the polyphenol extraction rate in Artemisia capillaris of the present invention;

[0022] Figure 5 A three-dimensional surface map and contour map of the interaction between time and temperature of the present invention;

[0023] Figure 6 It is a three-dimensional surface map and contour map of the interaction between ethanol volume fraction and temperature of the present invention;

[0024] Figure 7 It is a three-dimensional surface diagram and contour diagram of the interaction between the material-liquid ratio and the temperature of the present invention;

[0025] Figure 8 The effect of the amount of Folin phenol added on the absorbance of the solution of the present invention;

[0026] Fig. 9 The effect of the addition amount of 10% sodium carbonate on the absorbance of the solution of the present invention;

[0027] Fig.10 The effect of the color development time of the present invention on the absorbance of the solution;

[0028] Fig.11 It is the standard curve of the gallic acid solution of the present invention. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below through embodiments in combination with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] 1. Materials and instruments

[0031] Gallic acid, purity greater than 98%, Sigma;

[0032] Anhydrous sodium carbonate, analytical grade, Tianjin Kemeiou Chemical Reagent Co., Ltd.;

[0033] Anhydrous ethanol, analytical grade, Tianjin Kemeiou Chemical Reagent Co., Ltd.;

[0034] Folinol, 1 mol / L, Shanghai Yuanye Biotechnology Co., Ltd.;

[0035] Constant temperature water bath, Beijing Kewei Yongxing Instrument Co., Ltd.;

[0036] UV-2700 UV-visible spectrophotometer, Shimadzu Corporation.

[0037] (II) Optimization of polyphenol extraction conditions from Artemisia capillaris

[0038] A method for extracting polyphenols from Artemisia capillaris comprises the following steps: weighing 0.5 g of dried Artemisia capillaris powder into a 250 ml conical flask, adding a certain volume and a certain volume fraction of an ethanol solution, placing the flask in a constant temperature water bath, setting the extraction temperature, and accurately timing the extraction. After the extraction is completed, filtering the extract into a 50 ml volumetric flask, rinsing the conical flask with a certain volume fraction of an ethanol solution for multiple times, filtering the flask for multiple times, combining the filtrates, and fixing the volume to 50 ml.

[0039] Take out 0.5ml of the filtrate and place it in a 25ml volumetric flask, add 10% sodium carbonate reagent and Folin phenol reagent in turn, use ultrapure water to make up to 25ml, shake well, place at 25℃ away from light, use the reagent blank as a reference, use a 1cm colorimetric dish, measure the absorbance of the solution at 760nm, calculate the mass of polyphenols in the solution based on the regression equation, and calculate the polyphenol extraction rate according to the following formula:

[0040] The optimization process was carried out by combining single factor optimization and response surface optimization. In the single factor experiment, the effects of extraction temperature (50, 60, 70, 80) (℃), solid-liquid ratio (1:20, 1:30, 1:40, 1:50) (m:V), ethanol volume fraction (30%, 40%, 50%, 60%), and extraction time (45, 60, 75, 90 min) on the polyphenol extraction rate were investigated in turn. On this basis, the Box-Behnken response surface design method was adopted, with solid-liquid ratio (A), ethanol volume fraction (B), extraction time (C), and extraction temperature (D) as the investigation factors and polyphenol extraction rate as the response value, and a four-factor three-level experiment was carried out. The design scheme is shown in Table 1.

[0041] Table 1 Response surface design scheme

[0042]

[0043] (III) Optimization of conditions for colorimetric determination of polyphenols

[0044] The effects of the amount of forlin phenol added (ml), the amount of 10% sodium carbonate added (ml), and the color development time (min) on the absorbance of a certain concentration of gallic acid standard solution were investigated in turn, and the correlation coefficient of the standard curve under the optimal conditions was investigated. The experimental design is shown in Table 2.

[0045] Table 2 Condition optimization experiment for polyphenol determination

[0046]

[0047] (IV) Determination and analysis of polyphenol content in Artemisia capillaris in different urban areas of Henan

[0048] The extraction and determination methods optimized in the previous article were used to process the samples of Artemisia capillaris in Henan Province. The samples of Artemisia capillaris were collected from 18 cities in Henan Province, and they were scientifically collected and widely distributed. The number of samples is shown in Table 3.

[0049] Table 3 Number and distribution areas of Artemisia capillaris samples

[0050]

[0051] All experimental data were repeated three times, origin2018 was used for data processing and graphics, and Design-Expert12 was used for response surface design and analysis.

[0052] Example 1 Extraction of polyphenols from Artemisia capillaris

[0053] Example 1.1 Determination of the optimal temperature in a single factor experiment

[0054] The polyphenols were extracted at 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, and 80°C for 72 min, respectively. The solid-liquid ratio (the ratio of Artemisia capillaris powder to ethanol solution) was 1 g:30 ml, and 50% volume fraction of ethanol was selected as the extracting solution. The extraction results are shown in Figure 1 As the extraction temperature increases, the polyphenol extraction rate increases first and then decreases, reaching a maximum at 60°C. This is because, on the one hand, the increase in temperature promotes the dissolution and diffusion of polyphenols, but on the other hand, too high a temperature can change the stability of polyphenols. Therefore, the optimal extraction temperature was determined to be 60°C in the single factor experiment.

[0055] Example 1.2 Determination of the optimal material-liquid ratio in single factor experiment

[0056] When the solid-liquid ratio (ratio of Artemisia capillaris powder to ethanol solution) was set to 1:20, 1:30, 1:40, and 1:50, 50% volume fraction of ethanol was selected as the extraction solution, and the polyphenols in Artemisia capillaris were extracted at 63°C for 72 minutes. The extraction results are shown in Figure 2 . With the increase of the solid-liquid ratio, the extraction rate of polyphenols in Artemisia capillaris showed a trend of first increasing and then decreasing. This is because the increase of ethanol solvent increases the cell osmotic pressure of the Artemisia capillaris sample, which is conducive to the dissolution of polyphenols and improves its extraction rate. When the solid-liquid ratio is 1:30, the extraction rate of polyphenols is the highest. Further increasing the ethanol solvent is equivalent to diluting the polyphenols. Therefore, from the perspective of improving experimental efficiency and reducing experimental costs, 1:30 is selected as the optimal solid-liquid ratio.

[0057] Example 1.3 Determination of the optimal ethanol volume fraction in a single factor experiment

[0058] 30%, 35%, 40%, 45%, 50%, 55%, 60% ethanol solution were selected to extract polyphenols from Artemisia capillaris at 63°C for 72 min, where the solid-liquid ratio (the ratio of Artemisia capillaris powder to ethanol solution) was 1 g: 30 ml. The extraction results are shown in Figure 3 . With the increase of ethanol volume fraction, polyphenol extraction first increases and then decreases, and 50% ethanol extracts the most polyphenols. This is because different ethanol volume fractions result in different polarities of the extraction solvent, which will affect the dissolution of active ingredients. When there is more water phase in the extraction solvent, the dissolution of polyphenols will be inhibited, and when there is more ethanol in the extraction solvent, the dissolution of other fat-soluble components will increase, forming a competitive inhibition of polyphenol dissolution. Therefore, 50% ethanol is the optimal extraction concentration.

[0059] Example 1.4 Determination of the optimal extraction time in a single factor experiment

[0060] The extraction time was set to 45min, 50min, 55min, 60min, 65min, 70min, 75min, 80min, 85min, 90min, and 95min respectively to extract Artemisia capillaris polyphenols at 63°C, with a solid-liquid ratio of 1g:30ml. The extraction results are shown in Figure 4 . As time goes by, the extraction rate of polyphenols increases continuously, reaching the highest extraction rate at 75 minutes, but further extension of time reduces the extraction rate of polyphenols. This is because the longer extraction time causes the dissolved polyphenols to be oxidized in the air, and the structure changes, so it cannot be detected during the determination. Therefore, 75 minutes is selected as the optimal extraction time.

[0061] Example 1.5 Optimization of extraction conditions of polyphenols from Artemisia capillaris by response surface method

[0062] On the basis of single factor experiment, the polyphenol extraction rate was taken as the response value, and the solid-liquid ratio (A), ethanol volume fraction (B), extraction time (C) and extraction temperature (D) were taken as the investigation factors. The Design-Expert 12.0 software was used to design a Box-Behnken response surface experiment with 4 factors and 3 levels. The experimental design and results are shown in Table 4, and the variance analysis is shown in Table 5.

[0063] Table 4 Box-Behnken response surface experimental design and results

[0064]

[0065]

[0066] Table 5 Analysis of variance in Box-Behnken response surface experiment

[0067]

[0068] The above results were analyzed by multiple regression fitting using Design Expert software, and the fitting equations for the polyphenol extraction rate of Artemisia capillaris for the four single factors in the experiment were obtained as follows:

[0069] Y=1.83-0.0203A-0.0312B+0.0611C+0.0632D+0.0156AB+0.0235AC-0.0021A D-0.0064BC+0.0384BD+0.0286CD-0.0590A2-0.0665B2-0.0833C2-0.1129D2;

[0070] From the variance analysis in Table 5, it can be seen that the model is extremely significant (P<0.01) and the lack of fit term is not significant (P>0.05), indicating that the equation can well reflect the relationship between the polyphenol extraction rate and the four experimental factors. 2 =0.9807, indicating that the correlation of the fitting equation is very strong; R 2 and R adj 2 The difference is less than 0.2, which is within a reasonable range. The linear coefficients A, B, C, D and the quadratic terms AC, BD, CD, A 2 , B 2 , C 2 , D 2 There is a significant difference (P<0.01 or P<0.05). From the F value, the larger the F value, the greater the influence of the factor on the response value. Therefore, the order of influence on the polyphenol extraction rate in Artemisia capillaris is D>C>B>A, that is, temperature>time>ethanol volume fraction>solid-liquid ratio.

[0071] Through response surface interaction analysis, the effects of two factors can be obtained. Its three-dimensional surface graph and contour graph are as follows: Figures 5 to 7 shown.

[0072] The three-dimensional surface graph and contour graph can intuitively reflect the degree of influence of the interaction between factors on the response value. The steep slope of the surface and the dense contour lines indicate that the interaction between the two factors is significant. Figures 5 to 7 The three-dimensional surface graphs are all convex surfaces with the highest point, indicating that the extraction rate of Artemisia capillaris polyphenols has a maximum value within the factor design range. Figures 5 to 7 From the contour map, we can see that CD and BD are both elliptical, indicating that the interaction between time and temperature, ethanol volume fraction and temperature is significant, while AD is nearly circular, indicating that the interaction between solid-liquid ratio and temperature is not significant. This is consistent with the results of variance analysis.

[0073] Example 1.6 Verification experiment

[0074] The optimal extraction conditions of polyphenols in Artemisia capillaris were obtained by Design-expert software: solid-liquid ratio 1:28.80, ethanol volume fraction 48.18%, time 72.25min, temperature 63.01℃, and total polyphenol content 1.86%. Considering the feasibility of experimental operation, it was revised to solid-liquid ratio 1:30, ethanol volume fraction 50%, time 72min, and temperature 63℃. According to the optimized experimental parameters, Artemisia capillaris was measured, and the average value of total polyphenols measured three times was 1.85%, which was very close to the theoretical value, indicating that the model was stable and reliable.

[0075] Example 2 Colorimetric detection of polyphenol content

[0076] Example 2.1 Optimal amount of Folin phenol added

[0077] Take 10 mg of gallic acid, dissolve it to 100 ml, and make a 0.1 mg / ml gallic acid standard solution. Take 2 ml of the standard solution to a 25 ml volumetric flask, add 1 ml of 10% Na2CO3, add different volumes (ml) of Folin phenol reagent (0.1, 0.2, 0.5, 1), make the volume to 25 ml, react in the dark for 1 hour, and then measure the absorbance of the solution at 760 nm. Do 3 parallels for each treatment and take the average value. See the results. Figure 8 .

[0078] Depend on Figure 8 It can be seen that for a certain concentration of gallic acid standard solution, as the amount of Folin phenol added increases, the absorbance of the solution first increases and then decreases. When 0.5 ml of Folin phenol is added, the absorbance of the solution is the largest. Therefore, 0.5 ml of Folin phenol is selected as the optimal amount for the experiment.

[0079] Example 2.2 Optimal addition amount of 10% sodium carbonate

[0080] Take 2ml of the standard solution to a 25ml volumetric flask, add different volumes (ml) of 10% Na2CO3 reagent (0.5, 1, 2, 4), add 0.5ml of Folin phenol reagent, dilute to 25ml, react in the dark for 1h, and then measure the absorbance of the solution at 760nm. Do 3 parallels for each treatment and take the average value. Fig. 9 .

[0081] Depend on Fig. 9 It can be seen that with the addition of different volumes of 10% Na2CO3 reagent, the absorbance of the gallic acid standard solution first increases and then decreases. When 1 ml of the reagent is added, the absorbance of the solution is the largest. This is because the Folin phenol reagent must react with polyphenols in a weakly alkaline environment and stably develop color. If the Na2CO3 reagent is insufficient, the color development is incomplete, and if it is excessive, the color development system is unstable. Therefore, the present invention chooses to add 1 ml of 10% Na2CO3 reagent.

[0082] Example 2.3 Determination of the optimal color development time

[0083] Take 2ml of the standard solution to a 25ml volumetric flask, add 1ml of 10% Na2CO3 reagent, add 0.5ml of Folin phenol reagent, dilute to 25ml, place in the dark for different time periods (10min, 20min, 30min, 60min), and then measure the absorbance of the solution on a computer. The results are shown in Fig.10 .

[0084] like Fig.10 As shown in the figure, before 30 minutes, the absorbance of the gallic acid standard solution increased with time, but after 30 minutes, the absorbance of the solution remained stable. In order to improve the efficiency of the experiment, 30 minutes was selected as the optimal color development time.

[0085] Example 2.4 Standard curve under optimal experimental conditions

[0086] like Fig.11 As shown, the absorbance of a series of standard solutions was determined under optimized conditions, and the correlation coefficient of the generated standard curve was 0.9993, which means that it has a good linear relationship and can meet the needs of the experiment.

[0087] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. A method for extracting polyphenols from Artemisia capillaris, characterized in that: The following steps are involved: The Artemisia capillaris powder is added to an ethanol solution with a volume fraction of 30% to 60% for extraction. The extraction temperature is 50 to 80°C and the extraction time is 45 to 90 minutes. The extract is filtered and the filtrate is collected. The usage ratio of the Artemisia capillaris powder to the ethanol solution is 1g: (20 to 50ml).

2. The method for extracting polyphenols from Artemisia capillaris according to claim 1, characterized in that: The extraction temperature is 63°C.

3. The method for extracting polyphenols from Artemisia capillaris according to claim 1, characterized in that: The extraction time is 72 min.

4. The method for extracting polyphenols from Artemisia capillaris according to claim 1, characterized in that: The volume fraction of the ethanol solution is 50%.

5. The method for extracting polyphenols from Artemisia capillaris according to claim 1, characterized in that: The usage ratio of the Artemisia capillaris powder to the ethanol solution is 1g:30ml.

6. The method for extracting polyphenols from Artemisia capillaris according to claim 1, characterized in that: The method also includes a step of detecting the filtrate: adding sodium carbonate reagent and Folin phenol reagent with a mass percentage concentration of 10% in sequence to the filtrate, diluting and mixing evenly, subjecting the mixed solution to a color development reaction at 25° C. in the dark, and measuring the absorbance of the solution; the volume ratio of the sodium carbonate reagent to the Folin phenol reagent is (0.5-4): (0.1-1); and the color development time is 10-60 minutes.

7. The method for extracting polyphenols from Artemisia capillaris according to claim 1, characterized in that: The filtrate needs to be fixed to a constant volume before testing.

8. The method for extracting polyphenols from Artemisia capillaris according to claim 6, characterized in that: The volume ratio of the sodium carbonate reagent to the Folin phenol reagent is 1:0.

5.

9. The method for extracting polyphenols from Artemisia capillaris according to claim 6, characterized in that: The color development time is 30 minutes.

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