Scutellaria baicalensis georgi protein high extraction rate extraction method and scutellaria baicalensis extract

Abstract

This invention provides a high-yield extraction method for baicalein, comprising the following steps: (1) pulverizing baicalein and extracting it in a reverse micelle extractant to obtain an extract; (2) mixing the extract with a demulsifier for reverse-phase extraction to obtain an extract; (3) filtering the extract and allowing it to stand for phase separation to obtain an aqueous extract; (4) filtering and drying the aqueous extract to obtain the baicalein extract. The high-yield extraction method for baicalein provided by this invention significantly improves the extraction rate of baicalein by using a reverse-phase micelle system for extraction and a demulsifier for reverse-phase extraction.

Description

Technical Field

[0001] This invention belongs to the field of skin care technology, specifically relating to an extraction method for high extraction rate of scutellaria baicalensis protein and the scutellaria baicalensis extract. Background Technology

[0002] Scutellaria baicalensis Georgieta is a perennial herb belonging to the Lamiaceae family and the Scutellaria genus. As a distinctive traditional Chinese medicine, it has wide applications in skin care products. Existing research focuses primarily on the unique components of Scutellaria baicalensis, such as baicalin and baicalein. For example, patent CN107513086B, entitled "A method for extracting and separating high-purity baicalin from the stems and leaves of Scutellaria baicalensis and baicalin," discloses a method for extracting baicalin with a high extraction rate by heating an ethanol extraction solution and then concentrating it under reduced pressure at 60-70℃ and 0.08MPa to 0.10MPa. Besides baicalin and baicalein, Scutellaria baicalensis also contains other unique natural components, such as baicalin peptides and baicalin amino acids. These components also possess excellent bioactivity and are expected to have good application value in skin care products.

[0003] In recent years, studies have confirmed that proteins derived from plant-based traditional Chinese medicines possess a wide range of biological activities, including antitumor, antiviral, and antibacterial effects. Among these, the biological activities related to cosmetics are mainly antibacterial and antioxidant. However, current research on baicalin, especially its extraction, has received little attention. Existing extraction methods such as ultrasound, microwave, and water extraction generally result in low extraction rates of baicalin, directly limiting its development and application.

[0004] Therefore, in order to fully explore and utilize the medicinal resources of Scutellaria baicalensis, how to efficiently extract Scutellaria baicalensis protein is a technical problem that urgently needs to be solved. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defect that the content of baicalin in the extract of Scutellaria baicalensis obtained by the existing extraction methods is low. The present invention provides an extraction method with high extraction rate of baicalin and the resulting extract. The baicalin content in the extract obtained by the extraction method is not less than 10%, which provides a foundation for the development and application of baicalin.

[0006] Therefore, the present invention provides the following technical solution:

[0007] A method for extracting scutellaria baicalensis protein with a high extraction rate includes the following steps:

[0008] (1) Scutellaria baicalensis was pulverized and extracted with reverse micelle extractant to obtain an extract;

[0009] (2) The extract is mixed with the demulsifier and subjected to reverse phase extraction to obtain the extract;

[0010] (3) After filtering the extract, allow it to stand and separate to obtain an aqueous extract.

[0011] (4) The aqueous extract was filtered and dried to obtain Scutellaria baicalensis extract.

[0012] The present invention provides a high-yield extraction method for baicalein, which involves extraction using a reverse-phase micelle system followed by reverse-phase extraction with a demulsifier. This method demulsifies the reverse micelle system containing baicalein, releasing it into the aqueous phase. Furthermore, it allows for the re-extraction of unencapsulated baicalein, significantly increasing the baicalein extraction rate: the baicalein content in the baicalein extract is not less than 10%. Reverse-phase micelle system: When surfactants form micelles or micromicelles in a non-polar solution, the arrangement of the surfactant is exactly the opposite of that in a polar solvent; that is, the hydrophobic groups face outwards, while the hydrophilic groups face inwards, forming a polar core region, known as reverse-phase micelles (or simply reverse micelles).

[0013] Optionally, in the high extraction rate extraction method of Scutellaria baicalensis protein provided by the present invention, in step (1), the Scutellaria baicalensis is pulverized to pass through a 40-mesh sieve, and the reverse micelle extractant is an AOT-isooctane solution. The material-liquid ratio of the extracted Scutellaria baicalensis to the reverse micelle extractant and the extraction parameters are not specifically limited, and conventional methods in the industry can be used. The material-liquid ratio of the extracted Scutellaria baicalensis to the reverse micelle extractant recommended by the present invention is 1:10-15 (m / m). The extraction is carried out at 25-85℃, and the stirring is performed with a stirrer at a speed of 100r / min-500r / min. The extraction time is 60-300min, which can be adjusted according to the actual situation.

[0014] In the extraction method of Scutellaria baicalensis extract provided by this invention, AOT refers to sodium dioctyl dibutyrate sulfonate. The AOT-isooctane solution is AOT dissolved in isooctane solution. The concentration of the AOT-isooctane solution is not specifically limited because it does not affect the content of baicalin in Scutellaria baicalensis extract. However, considering factors such as cost and extraction efficiency, the concentration of the AOT-isooctane solution recommended in this invention is 10-80 mg / mL.

[0015] Optionally, in the high extraction rate extraction method of scutellaria baicalensis protein provided by the present invention, in step (2), the demulsifier is a potassium chloride solution with a concentration of 0.1-0.5 mol / L, and the mass ratio of the potassium chloride solution to the reverse micelle system is 1:1. The parameters of the reverse phase extraction are not specifically limited and can be those commonly used in the industry. The reverse phase extraction recommended by the present invention is carried out at 25-105℃, with stirring by a stirrer at a speed of 100 r / min-500 r / min, and the reverse phase extraction time is 0.5-4.5 h, which can be adjusted according to the actual situation.

[0016] The reverse micelles of AOT-isooctane solution used in this invention can encapsulate biomacromolecules such as Scutellaria baicalensis protein in the polar core region. Then, a potassium chloride solution of a specific concentration is used to demulsify the reverse micelles containing biomacromolecules to release the Scutellaria baicalensis protein. The steps and parameters are coordinated with each other, thereby significantly improving the extraction rate of Scutellaria baicalensis protein from Scutellaria baicalensis.

[0017] Optionally, in the high extraction rate extraction method of scutellaria baicalensis protein provided by the present invention, in step (3), the extract is cooled to below 35°C and then filtered under reduced pressure 3-5 times, and then the aqueous phase extract is separated using a separatory funnel.

[0018] Optionally, in the extraction method for high extraction rate of baicalin provided by the present invention, step (4) can be carried out using conventional filtration and drying methods. The present invention recommends that the aqueous extract be subjected to vacuum filtration 3-5 times and then vacuum dried to obtain baicalin extract. The parameters of the vacuum drying are not specifically limited and can be conventional in the industry. The present invention recommends that the vacuum drying temperature be 70-75℃, the pressure be 0.1 MPa, and the time be 24-48 h. The content of baicalin in the final baicalin extract is 10%-13.5%.

[0019] This invention also provides a Scutellaria baicalensis extract, obtained according to the above-described high-extraction method for baicalin, wherein the baicalin content in the extract is 10%-13.5%. Currently, baicalin is primarily used in skincare products for its antibacterial and antioxidant effects. Detailed Implementation

[0020] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0021] Example 1

[0022] This embodiment provides a method for extracting Scutellaria baicalensis extract, the specific steps of which are as follows:

[0023] (1) After pulverizing Scutellaria baicalensis, pass it through a 40-mesh sieve to obtain Scutellaria baicalensis powder. Weigh 16g of Scutellaria baicalensis powder and add it to 200g of 10mg / mL LAOT-isooctane solution. Then extract it at 45℃ and 300r / min for 120min to obtain the extract.

[0024] (2) Add the extract obtained in step (1) to 200g of potassium chloride solution with a concentration of 0.1mol / L, and then perform reverse phase extraction at 25℃ and 300r / min for 0.5h to obtain the extract;

[0025] (3) After cooling the extract obtained in step (2) to 25-30℃, filter it under reduced pressure 3 times, then transfer it to a separatory funnel to stand and separate the phases, and separate the aqueous phase extract.

[0026] (4) The aqueous extract obtained in step (3) was subjected to vacuum filtration three times, and then vacuum dried at 70-75℃ and 0.1Mpa for 36h to obtain Scutellaria baicalensis extract.

[0027] Example 2

[0028] This embodiment provides a method for extracting Scutellaria baicalensis extract, the specific steps of which are as follows:

[0029] (1) After pulverizing Scutellaria baicalensis, pass it through a 40-mesh sieve to obtain Scutellaria baicalensis powder. Weigh 16g of Scutellaria baicalensis powder and add it to 200g of 40mg / mL LAOT-isooctane solution. Then extract it at 45℃ and 300r / min for 120min to obtain the extract.

[0030] (2) Add the extract obtained in step (1) to 200g of potassium chloride solution with a concentration of 0.25mol / L, and then perform reverse phase extraction at 65℃ and 300r / min for 2.5h to obtain the extract;

[0031] (3) After cooling the extract obtained in step (2) to 25-30℃, filter it under reduced pressure 3 times, then transfer it to a separatory funnel to stand and separate the phases, and separate the aqueous phase extract.

[0032] (4) The aqueous extract obtained in step (3) was subjected to vacuum filtration three times, and then vacuum dried at 70-75℃ and 0.1Mpa for 36h to obtain Scutellaria baicalensis extract.

[0033] Example 3

[0034] This embodiment provides a method for extracting Scutellaria baicalensis extract, the specific steps of which are as follows:

[0035] (1) After pulverizing Scutellaria baicalensis, pass it through a 40-mesh sieve to obtain Scutellaria baicalensis powder. Weigh 16g of Scutellaria baicalensis powder and add it to 200g of 80mg / mL LAOT-isooctane solution. Then extract it at 45℃ and 300r / min for 120min to obtain the extract.

[0036] (2) Add the extract obtained in step (1) to 200g of potassium chloride solution with a concentration of 0.5mol / L, and then perform reverse phase extraction at 105℃ and 300r / min for 4.5h to obtain the extract;

[0037] (3) After cooling the extract obtained in step (2) to 25-30℃, filter it under reduced pressure 3 times, then transfer it to a separatory funnel to stand and separate the phases, and separate the aqueous phase extract.

[0038] (4) The aqueous extract obtained in step (3) was subjected to vacuum filtration three times, and then vacuum dried at 70-75℃ and 0.1Mpa for 36h to obtain Scutellaria baicalensis extract.

[0039] Example 4

[0040] This embodiment provides a method for extracting Scutellaria baicalensis extract, the specific steps of which are as follows:

[0041] (1) After pulverizing Scutellaria baicalensis, pass it through a 40-mesh sieve to obtain Scutellaria baicalensis powder. Weigh 16g of Scutellaria baicalensis powder and add it to 200g of 20mg / mL LAOT-isooctane solution. Then extract it at 45℃ and 300r / min for 120min to obtain the extract.

[0042] (2) Add the extract obtained in step (1) to 200g of potassium chloride solution with a concentration of 0.2mol / L, and then perform reverse phase extraction at 45℃ and 300r / min for 1.5h to obtain the extract;

[0043] (3) After cooling the extract obtained in step (2) to 25-30℃, filter it under reduced pressure 3 times, then transfer it to a separatory funnel to stand and separate the phases, and separate the aqueous phase extract.

[0044] (4) The aqueous extract obtained in step (3) was subjected to vacuum filtration three times, and then vacuum dried at 70-75℃ and 0.1Mpa for 36h to obtain Scutellaria baicalensis extract.

[0045] Example 5

[0046] This embodiment provides a method for extracting Scutellaria baicalensis extract, the specific steps of which are as follows:

[0047] (1) After pulverizing Scutellaria baicalensis, pass it through a 40-mesh sieve to obtain Scutellaria baicalensis powder. Weigh 16g of Scutellaria baicalensis powder and add it to 200g of 60mg / mL LAOT-isooctane solution. Then extract it at 45℃ and 300r / min for 120min to obtain the extract.

[0048] (2) Add the extract obtained in step (1) to 200g of potassium chloride solution with a concentration of 0.3mol / L, and then perform reverse phase extraction at 85℃ and 300r / min for 4h to obtain the extract;

[0049] (3) After cooling the extract obtained in step (2) to 25-30℃, filter it under reduced pressure 3 times, then transfer it to a separatory funnel to stand and separate the phases, and separate the aqueous phase extract.

[0050] (4) The aqueous extract obtained in step (3) was subjected to vacuum filtration three times, and then vacuum dried at 70-75℃ and 0.1Mpa for 36h to obtain Scutellaria baicalensis extract.

[0051] Comparative Example 1

[0052] This comparative example provides a method for extracting Scutellaria baicalensis extract, the specific steps of which are as follows:

[0053] (1) After pulverizing Scutellaria baicalensis, pass it through a 40-mesh sieve to obtain Scutellaria baicalensis powder. Weigh 16g of Scutellaria baicalensis powder and add it to 200g of 5mg / mL LAOT-isooctane solution. Then extract it at 45℃ and 300r / min for 120min to obtain the extract.

[0054] (2) Add the extract obtained in step (1) to 200g of potassium chloride solution with a concentration of 0.05mol / L, and then perform reverse phase extraction at 15℃ and 300r / min for 0.25h to obtain the extract;

[0055] (3) After cooling the extract obtained in step (2) to 25-30℃, filter it under reduced pressure 3 times, then transfer it to a separatory funnel to stand and separate the phases, and separate the aqueous phase extract.

[0056] (4) The aqueous extract obtained in step (3) was subjected to vacuum filtration three times, and then vacuum dried at 70-75℃ and 0.1Mpa for 36h to obtain Scutellaria baicalensis extract.

[0057] Comparative Example 2

[0058] This comparative example provides a method for extracting Scutellaria baicalensis extract, the specific steps of which are as follows:

[0059] (1) After pulverizing Scutellaria baicalensis, pass it through a 40-mesh sieve to obtain Scutellaria baicalensis powder. Weigh 16g of Scutellaria baicalensis powder and add it to 200g of 100mg / mL LAOT-isooctane solution. Then extract it at 45℃ and 300r / min for 120min to obtain the extract.

[0060] (2) Add the extract obtained in step (1) to 200g of potassium chloride solution with a concentration of 0.75mol / L, and then perform reverse phase extraction at 125℃ and 300r / min for 5h to obtain the extract;

[0061] (3) After cooling the extract obtained in step (2) to 25-30℃, filter it under reduced pressure 3 times, then transfer it to a separatory funnel to stand and separate the phases, and separate the aqueous phase extract.

[0062] (4) The aqueous extract obtained in step (3) was subjected to vacuum filtration three times, and then vacuum dried at 70-75℃ and 0.1Mpa for 36h to obtain Scutellaria baicalensis extract.

[0063] Comparative Example 3 (Water Extraction Control)

[0064] This comparative example provides a method for extracting Scutellaria baicalensis extract, the specific steps of which are as follows:

[0065] (1) Scutellaria baicalensis was pulverized and passed through a 40-mesh sieve to obtain Scutellaria baicalensis powder. 16g of Scutellaria baicalensis powder was weighed and added to 200g of water. Then, it was extracted at 45℃ and 300r / min for 120min to obtain the extract.

[0066] (2) After cooling the extract obtained in step (1) to 25-30℃, filter under reduced pressure three times, and dry under vacuum at 70-75℃ and 0.1Mpa for 36h to obtain Scutellaria baicalensis extract.

[0067] Comparative Example 4 (Enzyme Extraction Control)

[0068] This comparative example provides a method for extracting Scutellaria baicalensis extract, the specific steps of which are as follows:

[0069] (1) After pulverizing Scutellaria baicalensis, pass it through a 40-mesh sieve to obtain Scutellaria baicalensis powder. Weigh 16g of Scutellaria baicalensis powder and add it to 200g of water. Then add 30u / g of cellulase (0.024g) and 30u / g of protease (0.024g) of herb. Extract at 45℃ and 300r / min for 120min to obtain the extract.

[0070] (2) After cooling the extract obtained in step (1) to 25-30℃, filter under reduced pressure three times, and dry under vacuum at 70-75℃ and 0.1Mpa for 36h to obtain Scutellaria baicalensis extract.

[0071] Comparative Example 5 (Other antimicelle systems as a reference)

[0072] This comparative example provides a method for extracting Scutellaria baicalensis extract, the specific steps of which are as follows:

[0073] (1) After pulverizing Scutellaria baicalensis, pass it through a 40-mesh sieve to obtain Scutellaria baicalensis powder. Weigh 16g of Scutellaria baicalensis powder and add it to 200g of CTAB-isooctane solution with a concentration of 40mg / mL (CTAB is hexadecyltrimethylammonium bromide). Then extract it at 45℃ and 300r / min for 120min to obtain the extract.

[0074] (2) Add the extract obtained in step (1) to 200g of potassium chloride solution with a concentration of 0.25mol / L, and then perform reverse phase extraction at 65℃ and 300r / min for 2.5h to obtain the extract;

[0075] (3) After cooling the extract obtained in step (2) to 25-30℃, filter it under reduced pressure 3 times, then transfer it to a separatory funnel to stand and separate the phases, and separate the aqueous phase extract.

[0076] (4) The aqueous extract obtained in step (3) was subjected to vacuum filtration three times, and then vacuum dried at 70-75℃ and 0.1Mpa for 36h to obtain Scutellaria baicalensis extract.

[0077] The content of the main active ingredients in the Scutellaria baicalensis extracts (dry powder) obtained from the above examples and comparative examples was measured. Specifically, the content of Scutellaria baicalensis protein was determined using the Folin-phenol method. The Folin-phenol reagent consists of reagent A and reagent B. Reagent A consists of sodium carbonate, sodium hydroxide, copper sulfate, and potassium sodium tartrate. Under alkaline conditions, the peptide bonds in the polypeptide react with the potassium sodium copper tartrate solution to form a purple-red complex. Reagent B consists of phosphomolybdic acid, phosphotungstic acid, sulfuric acid, and bromine. Under alkaline conditions, this reagent is easily reduced by the phenolic groups of tyrosine in the polypeptide, resulting in a blue reaction. The intensity of the color is directly proportional to the polypeptide content. The polyphenol concentration was quantified at a wavelength of 700 nm using bovine serum albumin as a calibration standard by a UV spectrophotometer (UV-1600).

[0078] The method for detecting polyphenols in Scutellaria baicalensis refers to GB / T 8313-2008, "Determination of Tea Polyphenols and Catechins in Tea". The -OH groups in the polyphenols are oxidized with Folin-Ciocalteu reagent to make them appear blue. The polyphenol concentration is then quantified at 765 nm using a UV-1600 ultraviolet spectrophotometer with gallic acid as a calibration standard.

[0079] The yield of flavonoids in Scutellaria baicalensis extract was determined using the NaNO2-Al(NO3)3 colorimetric method (GB / T 20574-2006 Determination of Total Flavonoid Content in Propolis - Spectrophotometric Method). Under neutral or weakly alkaline conditions and in the presence of NaNO2, flavonoids were reduced, then aluminum nitrate was added to form a chelate. Upon addition of sodium hydroxide solution under alkaline conditions, the flavonoids underwent ring-opening, generating 2-hydroxychalcone, which showed a red-orange color with an absorption peak at 510 nm, conforming to Beer's Law for quantitative analysis. Quantitative analysis can generally be performed by comparison with a series of rutin standards. In this experiment, rutin was used as a reference standard. The flavonoid content was determined using the NaNO2-Al(NO3)3 colorimetric method with a UV spectrophotometer (UV-1600). Linear regression was performed using the absorbance value and concentration of the rutin solution to obtain the regression equation, from which the concentration of total flavonoids in the analyte was derived.

[0080]

[0081]

[0082] Baicalein extraction rate = Baicalein content in dry powder × Extraction rate of dry powder × 100%

[0083]

[0084] Polyphenol extraction rate = Polyphenol content in dry powder × Dry powder extraction rate × 100%

[0085]

[0086] Flavonoid extraction rate = Flavonoid content in dry powder × Dry powder extraction rate × 100%

[0087] Table 1

[0088]

[0089] Note: The methods for determining the content of baicalin, polyphenols and flavonoids in Scutellaria baicalensis are the same as those for determining the content of each component in Scutellaria baicalensis extract.

[0090] As shown in the table above, compared with the comparative examples, the active substances in the dried powdered Scutellaria baicalensis extracts obtained in each example were increased. Among them, the optimal group had a Scutellaria baicalensis protein content of 18.63% and a flavonoid content of 2.05%, but a decrease in polyphenol content (16.68%). The results of the total extraction rate of active substances are shown in Table 1 above. Compared with the traditional process (comparative examples 3-5), the protein content (16.41%-18.63%) and protein extraction rate (4.22%-5.79%) of the Scutellaria baicalensis extracts (Examples 1-5) within the parameter range were significantly increased; the flavonoid and polyphenol contents decreased slightly, mainly due to the increase in the dry powder extraction rate (25.71%-34.06%).

[0091] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various specific technical features in any suitable manner. To avoid unnecessary repetition, the present invention will not describe the various possible combinations separately. However, these simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for extracting scutellaria baicalensis protein with a high extraction rate, characterized in that, It has the following steps: (1) After crushing Scutellaria baicalensis, pass it through a 40-mesh sieve to obtain Scutellaria baicalensis powder. Weigh 16g of Scutellaria baicalensis powder and add it to 200g of 80mg / mL LAOT-isooctane solution. Then extract it at 45℃ and 300r / min for 120min to obtain the extract. (2) Add the extract obtained in step (1) to 200g of potassium chloride solution with a concentration of 0.5mol / L, and then perform reverse phase extraction at 105℃ and 300r / min for 4.5h to obtain the extract; (3) After cooling the extract obtained in step (2) to 25-30℃, filter it under reduced pressure 3 times, then transfer it to a separatory funnel to stand and separate the phases, and separate the aqueous phase extract. (4) The aqueous extract obtained in step (3) was subjected to vacuum filtration three times, and then vacuum dried at 70-75℃ and 0.1Mpa for 36h to obtain Scutellaria baicalensis extract.

2. A Scutellaria baicalensis extract, characterized in that, The baicalein extract obtained by the high extraction rate extraction method according to claim 1 has a baicalein content of 18.63%.

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