Method for extracting flavonoids and polyphenols from basil

Abstract

The application discloses a method for extracting flavonoids and polyphenols from basil, which comprises the following steps: immersing basil dry leaf powder in an ethanol aqueous solution, and separating the solid and the liquid to obtain an immersion liquid after the immersion; adding aluminum chloride to the immersion liquid, stirring, and collecting the precipitate after sufficient reaction to obtain polyphenol-aluminum complex and flavonoid-aluminum complex; dissolving the polyphenol-aluminum complex and the flavonoid-aluminum complex in a beta-cyclodextrin aqueous solution, and obtaining a clathrate solution of beta-cyclodextrin clathrated polyphenol-aluminum complex and beta-cyclodextrin clathrated flavonoid-aluminum complex after the reaction; stirring the clathrate solution, adding alkali salt while stirring, maintaining the pH value at 10-12, and making the alkali salt and aluminum ions in the clathrate produce a precipitation reaction; and obtaining beta-cyclodextrin clathrated polyphenol and beta-cyclodextrin clathrated flavonoid extraction products after the reaction and the precipitation. The method can not only remove volatile oil, but also improve the stability and antioxidant activity of the extraction products.

Description

Technical Field

[0001] This invention relates to the field of natural plant extraction technology, and more specifically, to a method for extracting flavonoids and polyphenols from basil. Background Technology

[0002] Basil, also known as nine-layered pagoda, gold-invaluable, and orchid fragrance, is a plant used for both medicinal and culinary purposes. It is often used as a spice in cooking and has the effects of dispelling dampness and aiding digestion, relieving exterior symptoms and dispersing blood stasis, and dispelling wind and promoting blood circulation.

[0003] Basil mainly contains volatile oils, flavonoids, and polyphenols. Among them, polyphenols and flavonoids, due to their phenolic hydroxyl groups, can play a role in anti-oxidation, boosting immunity, and fighting infection. However, the volatile oil components are mainly used as fragrances and can easily cause allergies.

[0004] Among the existing methods for extracting flavonoids and polyphenols from basil, ethanol extraction is the most widely used and lowest-cost method. Basil powder is soaked in ethanol solution, and then the solid and liquid are separated to obtain an extract. The extract is then concentrated to obtain a paste. However, in addition to flavonoids and polyphenols, the paste also contains volatile oils, which pose a safety risk to sensitive populations. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects in the prior art and provide a method for extracting flavonoids and polyphenols from basil, which not only removes volatile oils but also improves the stability and antioxidant activity of the extracted product.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] A method for extracting flavonoids and polyphenols from basil includes the following steps:

[0008] Basil leaf powder was extracted with an aqueous ethanol solution. After extraction, the solid and liquid were separated to obtain the extract.

[0009] Aluminum chloride was added to the extract, stirred, and after the reaction was complete, the precipitate was collected by standing to obtain polyphenol-aluminum complex and flavonoid-aluminum complex.

[0010] The polyphenol-aluminum complex and the flavonoid-aluminum complex were dissolved in an aqueous solution of β-cyclodextrin. After the reaction was completed, an inclusion complex solution containing β-cyclodextrin containing the polyphenol-aluminum complex and β-cyclodextrin containing the flavonoid-aluminum complex was obtained.

[0011] The inclusion complex solution was stirred while adding an alkaline salt to maintain the pH at 10-12, allowing the alkaline salt to react with the aluminum ions in the inclusion complex to produce a precipitation reaction. After the reaction was completed, the mixture was allowed to stand to produce a precipitate. The supernatant was taken to obtain the extracts of β-cyclodextrin-included polyphenols and β-cyclodextrin-included flavonoids.

[0012] Implementing the embodiments of the present invention will have the following beneficial effects:

[0013] In this embodiment of the invention, aluminum chloride is added to the extract, causing aluminum ions to form complexes with the phenolic hydroxyl groups of polyphenols and flavonoids in the extract through metal-metal complexation, resulting in precipitation. This separates the polyphenols and flavonoids from the volatile oil phase in the extract, removing the volatile oil. The water solubility of the complex is improved by using β-cyclodextrin to encapsulate it, thus enhancing the water solubility of the encapsulated polyphenols and flavonoids. Finally, an alkaline salt is added to the complex solution to produce aluminum hydroxide precipitate, which dissociates the polyphenol-aluminum complex and the flavonoid-aluminum complex, restoring the phenolic hydroxyl groups. The activity is enhanced, and under alkaline conditions, the stability of β-cyclodextrin is maintained, that is, the stability of the inclusion complexes of β-cyclodextrin with polyphenols and β-cyclodextrin with flavonoids is maintained. The inclusion of β-cyclodextrin with polyphenols and flavonoids can improve the water solubility of polyphenols and flavonoids, so that the inclusion complexes of β-cyclodextrin with polyphenols and β-cyclodextrin with flavonoids remain in the upper layer of the solution, thus obtaining the extract of β-cyclodextrin with polyphenols and β-cyclodextrin with flavonoids. The inclusion of β-cyclodextrin can also improve the stability and antioxidant activity of polyphenols and flavonoids. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] in:

[0016] Figure 1 This is the standard curve of polyphenol absorbance.

[0017] Figure 2 This is the curve showing the effect of extraction temperature on the extraction rate of basil polyphenols.

[0018] Figure 3 This is the curve showing the effect of ethanol concentration on the extraction rate of basil polyphenols.

[0019] Figure 4 This is the curve showing the effect of extraction time on the extraction rate of basil polyphenols.

[0020] Figure 5 This is the curve showing the effect of the liquid-to-solid ratio on the extraction rate of basil polyphenols.

[0021] Figure 6 This is a graph showing the DPPH· scavenging ability of flavonoids and polyphenols in basil.

[0022] Figure 7 This is a graph showing the scavenging ability of flavonoids and polyphenols in basil against ·OH. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] This invention discloses a method for extracting polyphenols and flavonoids from basil, comprising the following steps:

[0025] 1) Extract the dried basil leaf powder with an aqueous ethanol solution. After extraction, separate the solid and liquid to obtain the extract.

[0026] Basil's main components include volatile oils, flavonoids, and polyphenols. The volatile oils comprise approximately 17 components, such as eugenol, geraniol, linalool, methyl chamomile, ocimene, 1,8-cineole, limonene, Δ3-carene, pinene, bicyclic sesquicaprene, 1-epibripycepin, eugenol methyl ether, methyl cinnamate, 3-hexen-1-ol, 3-octanone, anethole, and furfural. Most of these volatile oil components are used as fragrances, have virtually no antioxidant activity, and are prone to causing allergies; therefore, they are components to be removed in this invention. Flavonoids are a series of compounds formed by two benzene rings (A and B rings) with phenolic hydroxyl groups linked by a central three-carbon atom. Flavonoids have antioxidant, blood circulation-improving, cholesterol-lowering, and anti-inflammatory effects; therefore, they are effective components to be retained in this invention. The polyphenols in basil mainly include rutin, isoquercitrin, rosmarinic acid, etc., which have antioxidant effects and are also the effective components to be retained in this invention.

[0027] Ethanol extraction can maximize the extraction of volatile oils, flavonoids and polyphenols from basil. In a preferred embodiment, ultrasound is applied simultaneously during the ethanol extraction process to assist extraction and further improve the extraction rate and efficiency.

[0028] This invention obtains the preferred parameter range for the ultrasonic-assisted ethanol extraction method through single-factor experiments, as follows.

[0029] Basil leaves were pulverized, and 1.0000 g of the powder was extracted using ethanol and water as solvents, with the aid of ultrasound (480 W) to obtain an extract. After extraction, the supernatant was taken, diluted with ethanol, and centrifuged again. 1 mL of the supernatant from the second centrifugation was added, along with 5 mL of distilled water and 0.7 mL of Folin-Ciocalteu stock solution. The mixture was shaken well, allowed to stand for 3 min, and then 3 mL of 7.5% sodium carbonate solution was added. Finally, 0.3 mL of distilled water was added, and the mixture was mixed and kept in the dark for 50 min. The absorbance was measured at 760 nm. Three parallel experiments were conducted, and the polyphenol extraction rate was calculated. The effects of extraction temperature, extraction time, ethanol-water solution concentration, and liquid-to-solid ratio on the basil polyphenol extraction rate were investigated. The flavonoids in basil mainly include quercetin and kaempferol, which contain multiple hydroxyl groups on their benzene rings and are also polyphenols. Therefore, this invention uses the polyphenol extraction rate to calculate the extraction rate.

[0030] Gallic acid is used as a standard in this invention. Gallic acid is dissolved in 95% ethanol, and 0-1 mL of the solution is taken out, with an interval of 0.2 mL between each batch. The solution is then diluted to 1 mL with 95% ethanol and mixed thoroughly. 0.7 mL of Folin-Ciocalteu stock solution and 5 mL of distilled water are added. The mixture is shaken well and allowed to stand for 3 min. 3 mL of sodium carbonate solution and 0.3 mL of distilled water are added, and the mixture is mixed and kept in the dark for 50 min. The absorbance is measured at 760 nm. To reduce error, three parallel experiments are performed, yielding the standard curve: Y = 0.01075x - 0.0054(R). 2 =0.99934), where x: gallic acid solution concentration, μg / mL; Y: absorbance, L / (g·cm), and the standard curve is as follows: Figure 1 As shown.

[0031] refer to Figure 2 The effect of extraction temperature on the extraction rate of basil polyphenols was investigated. Figure 2 It is evident that increasing the extraction temperature from 30℃ to 50℃ increases the extraction rate of basil polyphenols, with a peak extraction rate of 124.3 mg / g at 50℃. Further increasing the extraction temperature from 50℃ to 70℃ leads to a decrease in the extraction rate of basil polyphenols. Therefore, the preferred extraction temperature range is 40℃–60℃, with 50℃ being the optimal range.

[0032] refer to Figure 3 The effect of ethanol concentration on the extraction rate of basil polyphenols was investigated. Figure 3 It is evident that the extraction rate peaks at 132 mg / g when the ethanol concentration is increased from 40% to 60%, indicating that this is the optimal ethanol concentration. As the ethanol concentration increases from 60% to 80%, the extraction rate gradually decreases, although the rate of decrease slows slightly. Therefore, the preferred range for ethanol concentration is 50 wt%–70 wt%, with 60 wt% being the most preferred.

[0033] refer to Figure 4The effect of extraction time on the extraction rate of basil polyphenols was discussed. Figure 4 It can be seen that when the extraction time is extended from 20 min to 40 min, the extraction rate reaches a peak of 132 mg / g. When the extraction time is increased from 40 min to 60 min, the extraction rate no longer increases, but instead decreases slightly. Therefore, the optimal range for extraction time is 30 min to 50 min, with the optimal value being 45 min.

[0034] refer to Figure 5 The effect of the liquid-to-solid ratio on the extraction rate of basil polyphenols was observed. Figure 5 It is evident that when the liquid-to-solid ratio is greater than 10:1 mL / g and less than 30:1 mL / g, the extraction rate of basil polyphenols increases rapidly. The extraction rate reaches its highest value of 132 mg / g at a liquid-to-solid ratio of 30:1 mL / g. Further increasing the liquid-to-solid ratio to 50:1 mL / g results in a slight increase in extraction rate. Considering the amount of solution used, the optimal range for the liquid-to-solid ratio is determined to be 20:1 mL / g to 40:1 mL / g, with the most optimal value being 30:1 mL / g.

[0035] In one specific embodiment, the power of the ultrasonic wave is 450W to 500W.

[0036] 2) Add aluminum chloride to the extract, stir, and after the reaction is complete, let it stand and collect the precipitate to obtain polyphenol-aluminum complex and flavonoid-aluminum complex.

[0037] The purpose of this step is to remove volatile oils from the extract. This step utilizes the principle that aluminum chloride can form metal complexes with the 4-oxygen, 5-carbonyl, and ortho-phenolic hydroxyl groups in flavonoids to convert flavonoids into flavonoid-aluminum complexes. Similarly, it utilizes the principle that aluminum chloride can form metal complexes with the ortho-phenolic hydroxyl groups of polyphenols to convert polyphenols other than flavonoids into polyphenol-aluminum complexes.

[0038] In this step, excess aluminum chloride is used to convert as much of the flavonoids and polyphenols as possible into precipitates that are collected.

[0039] 3) Dissolve the polyphenol-aluminum complex and the flavonoid-aluminum complex in an aqueous solution of β-cyclodextrin. After the reaction is complete, an inclusion complex solution containing the polyphenol-aluminum complex and the flavonoid-aluminum complex containing the β-cyclodextrin is obtained.

[0040] In this step, the inclusion effect of the β-cyclodextrin cavity is used to encapsulate flavonoid-aluminum complexes and polyphenol-aluminum complexes, respectively. The main purpose of this step is to improve the water solubility of various flavonoid and polyphenol components using β-cyclodextrin. Since most flavonoids in basil are poorly soluble in water, while polyphenols are readily soluble, this step utilizes the inclusion of β-cyclodextrin to make both flavonoids and polyphenols water-soluble, facilitating the removal of aluminum through precipitation in the subsequent decomplexing step while retaining both flavonoids and polyphenols.

[0041] Specifically, in one embodiment, the total mass ratio of the polyphenol-aluminum complex and the flavonoid-aluminum complex to the β-cyclodextrin is 1:5 to 1:10, and the concentration of the β-cyclodextrin aqueous solution is 0.05 g / mL to 0.1 g / mL.

[0042] 4) Stir the inclusion complex solution while adding the alkaline salt, maintaining the pH at 10-12, so that the alkaline salt reacts with the aluminum ions in the inclusion complex to produce a precipitation reaction. After the reaction is complete, let it stand to produce a precipitate, and take the upper layer solution to obtain the extract of β-cyclodextrin-included polyphenols.

[0043] In this step, the precipitation reaction between alkali and aluminum is used to decomplex the polyphenols, restoring their activity and simultaneously separating the inclusion complex from the precipitate. Furthermore, an alkaline environment with a pH of 10–12 is beneficial for maintaining the structural stability of β-cyclodextrin, promoting the precipitation reaction, and facilitating decomplexation. The inclusion of β-cyclodextrin also enhances the stability and antioxidant activity of polyphenols and flavonoids.

[0044] Specifically, alkaline salts include, but are not limited to, sodium hydroxide and / or potassium hydroxide.

[0045] In summary, the extraction product obtained by the method of the present invention not only removes volatile oils, but also improves the stability and activity of polyphenols and flavonoids.

[0046] The following are specific examples.

[0047] Example 1

[0048] A method for extracting polyphenols and flavonoids from basil includes the following steps:

[0049] 1) After pulverizing dried basil leaves, take 1.0000g of powder, use 30mL of 60wt% ethanol-water as solvent, and simultaneously use ultrasonic extraction (power 480w) at 50℃ for 45min. After extraction, take the upper layer of extract, dilute with ethanol, and centrifuge again to obtain the extract.

[0050] Take 1 mL of the extract, add 5 mL of distilled water, add 0.7 mL of Folin-Ciocalteu stock solution, shake well, let stand for 3 min, add 3 mL of 7.5% sodium carbonate solution, and finally add 0.3 mL of distilled water. Mix well and protect from light for 50 min, then measure the absorbance at 760 nm. Perform three parallel experiments and calculate the average polyphenol extraction rate as 134.506 mg / g.

[0051] 2) Add 0.5g of aluminum chloride to the extract, stir, and after the reaction is complete, let it stand and collect the precipitate to obtain polyphenol-aluminum complex and flavonoid-aluminum complex. Concentrate the supernatant to obtain volatile oil paste.

[0052] 3) Dissolve the polyphenol-aluminum complex and flavonoid-aluminum complex obtained in step 2) in 30 mL of 0.1 g / mL β-cyclodextrin aqueous solution. After thorough stirring, the inclusion complex solution is obtained.

[0053] 4) Stir the inclusion complex solution while adding sodium hydroxide to maintain the pH at 10-12. After the reaction is complete, let it stand to produce a precipitate. Take the upper layer solution to obtain the extracts of β-cyclodextrin-included polyphenols and β-cyclodextrin-included flavonoids.

[0054] Take 1 mL of the upper layer solution obtained in step 4), add 5 mL of distilled water, add 0.7 mL of Folin-Ciocalteu stock solution, shake well, let stand for 3 min, add 3 mL of 7.5% sodium carbonate solution, and finally add 0.3 mL of distilled water. Mix well and protect from light for 50 min, then measure the absorbance at 760 nm. Three parallel experiments were conducted, and the average extraction rate of polyphenols was calculated to be 129.342 mg / g. It can be seen that the method of the present invention can convert the extracted polyphenols and flavonoids into β-cyclodextrin-encapsulated polyphenols and β-cyclodextrin-encapsulated flavonoids.

[0055] Comparative Example 1

[0056] The only difference between Comparative Example 1 and Example 1 is that step 3) is missing. After obtaining the polyphenol-aluminum complex and flavonoid-aluminum complex in step 2), cyclodextrin was not used for inclusion. Instead, sodium hydroxide was added to dissolve the complex after dissolving it in water and stirring while maintaining the pH at 10-12. After the reaction was completed, the mixture was allowed to stand to produce a precipitate, and the upper layer solution was taken to obtain the extraction product.

[0057] Take 1 mL of the extract from the supernatant, add 5 mL of distilled water, add 0.7 mL of Folin-Ciocalteu stock solution, shake well, let stand for 3 min, add 3 mL of 7.5% sodium carbonate solution, and finally add 0.3 mL of distilled water. Mix well and protect from light for 50 min, then measure the absorbance at 760 nm. Three parallel experiments were performed, and the average extraction rate of polyphenols was calculated to be 14.029 mg / g. The low extraction rate is due to insufficient decomplexation, resulting in precipitation.

[0058] Comparative Example 2

[0059] Comparative Example 2 is the prior art, which only includes the ultrasonic extraction step of ethanol, as detailed below:

[0060] After pulverizing dried basil leaves, take 1.0000g of powder, use 30mL of 60wt% ethanol-water as solvent, and simultaneously use ultrasound (power 480w) to extract at 50℃ for 45min. After extraction, take the upper extract, dilute with ethanol, and centrifuge again to obtain the extract.

[0061] The extract was rotary evaporated to obtain an extract.

[0062] Oxidizing property test of the extract

[0063] The extract obtained from the upper layer solution of Example 1 was obtained by rotary evaporation, diluted at a certain ratio, and its scavenging rate of DPPH· and ·OH was measured. It was compared with the extract prepared by the prior art in Comparative Example 2.

[0064] refer to Figure 6 and Figure 7 The values ​​represent the scavenging abilities of flavonoids and polyphenols in basil for DPPH· and ·OH, respectively. Figure 6 It is evident that the extract with cyclodextrin inclusion complex of the present invention exhibits a higher scavenging rate of DPPH· and ·OH compared to the extract without cyclodextrin inclusion complex of Comparative Example 2.

[0065] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for extracting flavonoids and polyphenols from basil, characterized in that, The process includes the following: 1) Basil leaf powder is extracted with an ethanol aqueous solution. Ultrasonic waves are applied during the extraction process. The extraction time is 30 min to 50 min and the extraction temperature is 40℃ to 60℃. After the extraction is completed, the solid and liquid are separated to obtain the extract. 2) Add excess aluminum chloride to the extract, stir, and after the reaction is complete, let it stand and collect the precipitate to obtain polyphenol-aluminum complex and flavonoid-aluminum complex; 3) Dissolve the polyphenol-aluminum complex and the flavonoid-aluminum complex in an aqueous solution of β-cyclodextrin with a concentration of 0.05 g / mL to 0.1 g / mL, wherein the total mass ratio of the polyphenol-aluminum complex and the flavonoid-aluminum complex to the β-cyclodextrin is 1:5 to 1:

10. After the reaction is completed, an inclusion complex solution containing the polyphenol-aluminum complex and the flavonoid-aluminum complex containing the β-cyclodextrin is obtained. 4) Stir the inclusion complex solution while adding alkaline salt to maintain the pH at 10-12, so that the alkaline salt reacts with the aluminum ions in the inclusion complex to produce a precipitation reaction. After the reaction is complete, let it stand to produce a precipitate, and take the upper layer solution to obtain the extracts of β-cyclodextrin-included polyphenols and β-cyclodextrin-included flavonoids.

2. The method for extracting flavonoids and polyphenols from basil according to claim 1, characterized in that, The power of the ultrasonic wave is 450W to 500W.

3. The method for extracting flavonoids and polyphenols from basil according to any one of claims 1 to 2, characterized in that, The alkaline salts include sodium hydroxide and / or potassium hydroxide.

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