Method for determining content of mulberry root ketone type compounds

Abstract

The application discloses a content determination method of mulberry root ketone type compounds. The method comprises the following steps: adopting high performance liquid chromatography to determine the content of SGF compounds and / or SGH compounds in a test solution; a C18 column is used in the high performance liquid chromatography; mobile phase A and mobile phase C are used in the high performance liquid chromatography, the mobile phase A is 0.1% phosphoric acid-water, and the mobile phase C is acetonitrile; a gradient elution mode of 0-12min, 40% A; 12-13min, 40%-30% A; 13-26min, 30% A is used in the high performance liquid chromatography. The content determination method has the characteristics of simple operation; the established content determination method has the characteristics of accurate results and convenient operation, and has the characteristics of strong specificity, linear relationship, precision and sample recovery rate, and meets the requirements of drug analysis.

Description

Technical Field

[0001] This invention relates to a method for determining the content of morula-type compounds. Background Technology

[0002] Black mulberry (Morus nigra L.) is a plant belonging to the genus *Morus* of the family Moraceae. In Xinjiang, it is known as medicinal mulberry. In Uyghur medicine, medicinal mulberry is primarily used to invigorate the brain and calm the nerves, benefit the spleen and stomach, and nourish the blood and liver; it is used for palpitations, insomnia, forgetfulness, spleen and stomach deficiency, jaundice, and hepatitis. Black mulberry has rich medicinal and edible value. The fruit, black mulberry, is a nutrient-rich fruit containing various nutrients; the leaves and stems contain abundant active substances, such as isopentenyl flavonoids, arbutin, alkaloids, stilbenes, and coumarins. Modern pharmacological studies have shown that these active substances give it various effects, including whitening, lowering blood sugar, and anti-inflammation. Morus root flavonoids, a special type of dihydroflavonoid component in black mulberry, represented by the compound SGF, have been reported to promote adipocyte differentiation, increase insulin sensitivity, promote glucose transport, and lower blood lipids. They can be further processed into insulin sensitizers and hypoglycemic drugs. Preliminary experiments have shown that SGF and SGH are present in relatively high amounts in the stems and branches of black mulberry. Currently, there is no evidence to control the quality of these two mulberry root ketone compounds in the black mulberry, a traditional medicinal plant.

[0003] Summary of the Invention

[0004] The technical problem this invention aims to solve is to provide a method for determining the content of mulberry root ketone-type compounds. This invention employs HPLC, optimizing the extraction method and chromatographic conditions to determine the content of active ingredients in black mulberry medicinal materials, providing a research basis for the formulation of quality standards and the rational development and utilization of this resource. The determination method is accurate, highly sensitive, has good repeatability, and reliable results, providing a basis for the quality control and evaluation of Uyghur black mulberry.

[0005] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0006] This invention provides a method for determining the content of mulberry root ketone-type compounds, which includes the following steps: using high performance liquid chromatography to determine the content of SGF compounds and / or SGH compounds in the test sample solution;

[0007]

[0008] The chromatographic column used in the high-performance liquid chromatography method is a C18 column;

[0009] The high-performance liquid chromatography method uses mobile phase A and mobile phase C, wherein mobile phase A is 0.1% phosphoric acid-water and mobile phase C is acetonitrile;

[0010] Elution was performed using the following gradient elution program: 0–12 min, 40% A; 12–13 min, 40%–30% A; 13–26 min, 30% A.

[0011] In some embodiments, the morula-type compounds refer to SGF compounds and / or SGH compounds.

[0012] In some embodiments, the test solution is obtained by extracting the black mulberry stems and branches in an alcohol solution using ultrasonic extraction.

[0013] In some embodiments, the alcohol solvent is methanol and / or ethanol, such as anhydrous methanol, 90% methanol, 70% methanol, anhydrous ethanol, 90% ethanol or 70% ethanol, preferably anhydrous methanol.

[0014] In some embodiments, the black mulberry stems and branches are dried powder of black mulberry stems and branches.

[0015] In some embodiments, the liquid-to-solid ratio in the ultrasonic extraction method is (1:10)-(1:40) g / mL, for example (1:10) g / mL, (1:20) g / mL or (1:30) g / mL, preferably (1:20) g / mL.

[0016] In some embodiments, the ultrasonic temperature in the ultrasonic extraction method is 25°C-60°C, for example 25°C, 40°C or 50°C, preferably 50°C.

[0017] In some embodiments, the ultrasonic power in the ultrasonic extraction method is 50-100W, for example 50W, 70W or 100W, preferably 100W.

[0018] In some embodiments, the ultrasonic extraction time is 10 min to 40 min, for example 10 min, 20 min, 30 min or 40 min, preferably 30 min.

[0019] In some embodiments, the ultrasonic extraction method may further include filtration after ultrasonic extraction. The filtration may involve replenishing the weight with the alcohol solvent and filtering through a 0.45 μm microporous membrane.

[0020] In some embodiments, the detection wavelength in the high-performance liquid chromatography is 254–310 nm, preferably 310 nm.

[0021] In some embodiments, the C18 column may be a Welch column. XB-C18 column (4.6×250mm, 5μm), Agilent Eclipse XDB-C18 (250mm×4.6mm, 5μm) column, or Waters column. Hss T3 column (4.6×250mm, 5μm).

[0022] In some embodiments, the flow rate in the high-performance liquid chromatography is 0.8–1.2 mL / min. -1 The preferred flow rate is 1 mL·min -1 .

[0023] In some embodiments, the column temperature in the high-performance liquid chromatography is 25–35°C, preferably 30°C.

[0024] In some embodiments, the injection volume in the high-performance liquid chromatography is 5–20 μL, preferably 10 μL.

[0025] In some embodiments, the content determination method simultaneously determines the content of SGF compound and SGH compound.

[0026] In some embodiments, the content of SGF compound and / or SGH compound in the test sample solution is determined using the external standard method.

[0027] In some embodiments, the concentration range of the SGF compound reference stock solution in the external standard method is 0.02612 mg / mL. -1 ~0.836 mg·mL -1 .

[0028] In some embodiments, the concentration range of the SGH compound reference stock solution in the external standard method is 0.0056 mg / mL. -1 ~0.1111 mg·mL -1 .

[0029] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0030] The reagents and raw materials used in this invention are all commercially available.

[0031] The positive and progressive effects of this invention are as follows: This invention provides a method for determining the content of black mulberry ketone-type compounds, which is simple and easy to operate; the established content determination method is not only highly specific, but also meets the requirements of drug analysis in terms of linearity, precision, and recovery rate, and has the characteristics of accurate results and convenient operation. Detailed Implementation

[0032] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0033] The chromatographic conditions for the content determination methods in Examples 1-5 were as follows: Agilent Eclipse XDB-C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: 0.1% phosphoric acid-water (A)-acetonitrile (C), gradient elution (0–12 min, 40% A; 12–13 min, 40%–30% A; 13–26 min, 30% A); wavelength: 310 nm; column temperature: 30 °C; flow rate: 1 mL / min. -1 The injection volume is 10 μL.

[0034] Example 1

[0035] The extraction method for two mulberry root flavonoids, SGF and SGH, from black mulberry stems and branches includes the following steps: Six 0.5g portions of black mulberry stem powder (passed through a No. 3 sieve) are accurately weighed and placed in stoppered conical flasks. Each of the six portions of black mulberry stem powder corresponds to one of six solvents. Three parallel samples are weighed for each solvent, and 10mL of anhydrous methanol, 90% methanol, 70% methanol, anhydrous ethanol, 90% ethanol, and 70% ethanol are added respectively. The samples are weighed, and the mixture is ultrasonically extracted at 50℃ for 30min. After cooling, the lost weight is replenished with the corresponding solvent, the mixture is shaken well, filtered, and the filtrate is collected. 10μL of the extract is injected into a high-performance liquid chromatograph for determination. The SGF and SGH contents obtained under various conditions are shown in the table below.

[0036]

[0037]

[0038] Example 2

[0039] The extraction method for two mulberry root flavonoids, SGF and SGH, from black mulberry stems and branches includes the following steps: Accurately weigh 0.5g of black mulberry stem powder (passed through a No. 3 sieve) into four portions and place them in stoppered conical flasks. Each of the four portions of black mulberry stem powder corresponds to one of the four extraction times, and three samples are weighed in parallel for each extraction time. Add 10mL of anhydrous methanol at a liquid-to-solid ratio of 1:20, weigh the mixture, and ultrasonically extract at 50℃ for 10min, 20min, 30min, and 40min respectively. After cooling, replenish the lost weight with anhydrous methanol, shake well, filter, and collect the filtrate. Inject 10μL into a high-performance liquid chromatograph (HPLC) for determination. The SGF and SGH contents obtained under each condition are shown in the table below. Observation revealed that the contents of both components first increased and then leveled off with increasing extraction time; therefore, 30min was selected as the extraction time.

[0040] Extraction time SGF content (mg / g) SGH content (mg / g) SD 10min 2.345 0.536 0.0001 20min 2.364 0.541 0.011 30min 2.418 0.555 0.0038 40min 2.412 0.549 0.021

[0041] Example 3

[0042] The extraction method for two mulberry root flavonoids, SGF and SGH, from black mulberry stems and branches includes the following steps: Accurately weigh three 0.5g portions of black mulberry stem powder (passed through a No. 3 sieve) and place them in stoppered conical flasks. The three portions of black mulberry stem powder correspond to three different liquid-to-solid ratios, and three parallel samples are weighed for each ratio. Anhydrous methanol is added at liquid-to-solid ratios of 1:10, 1:20, and 1:30, and the samples are weighed. The mixture is ultrasonically extracted at 50℃ for 30 min, cooled, and the lost weight is replenished with the corresponding solvent. The mixture is shaken well, filtered, and the filtrate is collected. 10 μL of the extract is injected into a high-performance liquid chromatograph (HPLC) for determination. The SGF and SGH contents obtained under each condition are shown in the table below. Observation revealed that the extraction rate initially increased and then decreased with increasing liquid-to-solid ratio; therefore, the liquid-to-solid ratio of 1:20, which yielded the highest extraction rate, was selected as the optimal extraction ratio.

[0043] Liquid-to-solid ratio SGF content (mg / g) SGH content (mg / g) SD 1:10 2.380 0.555 0.0011 1:20 2.382 0.547 0.0010 1:30 2.305 0.535 0.0084

[0044] Example 4

[0045] Extraction methods for two mulberry root flavonoids, SGF and SGH, from black mulberry stems and branches include the following steps: Accurately weigh 0.5g of black mulberry stem powder (passed through a No. 3 sieve) into four portions and place them in stoppered conical flasks. Each of the four portions of black mulberry stem powder corresponds to one of the four extraction temperatures. For each extraction temperature, three portions are weighed in parallel. Add 10ml of anhydrous methanol at a liquid-to-solid ratio of 1:20, weigh the mixture, and ultrasonically extract at 25℃, 40℃, 50℃, and 60℃ for 30min respectively. After cooling, replenish the lost weight with the appropriate solvent, shake well, filter, and collect the filtrate. Inject 10μL into a high-performance liquid chromatograph (HPLC) for determination. The SGF and SGH contents obtained under each condition are shown in the table below. Observation revealed that the extraction rate gradually increased with increasing temperature, but excessive solvent evaporation occurred at 60℃. Therefore, 50℃ was selected as the most suitable extraction temperature.

[0046]

[0047] Example 5

[0048] Extraction methods for two mulberry root flavonoids, SGF and SGH, from black mulberry stems and branches include the following steps: Accurately weigh 0.5g of black mulberry stem powder (passed through a No. 3 sieve) in three portions and place them in stoppered conical flasks. The three portions of black mulberry stem powder correspond to three different power levels. For each power level, three samples are weighed in parallel. Add 10mL of anhydrous methanol at a liquid-to-solid ratio of 1:20, weigh the samples, and extract using ultrasound at 50W, 70W, and 100W for 30min respectively. After cooling, replenish the lost weight with the appropriate solvent, shake well, filter, and collect the filtrate. Inject 10μL into a high-performance liquid chromatograph for determination. The SGF and SGH contents obtained under each condition are shown in the table below. Observation showed that the extraction rate was highest at 100W ultrasound extraction.

[0049] Extraction power SGF content (mg / g) SGH content (mg / g) SD 50W 2.414 0.554 0.0155 70W 2.364 0.545 0.0157 100W 2.424 0.555 0.0046

[0050] Preparation method of test solution: The final extraction method of the test sample was determined to be ultrasonic extraction with anhydrous methanol at 50℃ for 30 min, with a liquid-to-solid ratio of 1:20 and a power of 100W.

[0051] Example 6: Method for determining the content of SGF and SGH

[0052] (1) Selection of detection wavelength

[0053] The absorbance of SGF and SGH reference standard solutions was measured at wavelengths of 254 nm, 285 nm, and 310 nm. It was found that the absorbance of SGF and SGH was the highest at 310 nm, so 310 nm was selected as the detection wavelength.

[0054] (2) Chromatographic conditions

[0055] The chromatographic column was an Agilent Eclipse XDB-C. 18 (250mm × 4.6mm, 5μm); Mobile phase: 0.1% phosphoric acid-water (A)-acetonitrile (C), gradient elution (0–12 min, 40% A; 12–13 min, 40%–30% A; 13–26 min, 30% A), wavelength 310 nm; column temperature 30℃; flow rate 1 mL·min -1 The injection volume is 10 μL.

[0056] (3) Linear relationship

[0057] Establishment of SGF and SGH standard curves: Accurately weigh 16.72 mg of SGF reference standard into a 10 mL volumetric flask to prepare a concentration of 1.672 mg / mL. -1 The reference stock solution was diluted to the mark with anhydrous methanol and set aside. It was then diluted six times with anhydrous methanol at a dilution factor of 1 / 2, and each subsequent dilution was brought to the mark to prepare a solution with a concentration of 0.836 mg / mL. -1 0.418 mg·mL-1 0.209 mg·mL -1 0.1045 mg·mL -1 0.0522 mg·mL -1 0.02612 mg·mL -1 Reference standard stock solution.

[0058] Accurately weigh 11.11 mg of SGH reference standard into a 10 mL volumetric flask, and dilute to the mark with anhydrous methanol to prepare the stock solution. Take 5 mL of this stock solution and dilute it into a 25 mL volumetric flask to prepare a solution of 0.2222 mg / mL. -1 The reference stock solution was then prepared into 0.1111 mg / mL solutions. -1 0.0555 mg·mL -1 0.0278 mg·mL -1 0.01111 mg·mL -1 0.0056 mg·mL -1 Reference standard stock solution.

[0059] The SGF and SGH reference stock solutions were determined under the chromatographic conditions described above, with an injection volume of 10 μL. Regression curves for the two components were plotted with peak area as the ordinate (Y) and reference concentration as the abscissa (X). The linear range and regression coefficients are shown in the table below.

[0060] Compound Name Regression curve <![CDATA[Regression coefficient (R 2 )]]> Linear range (mg / mL) SGF Y = 16747584.0781x - 97034.0747 1.0000 0.026～0.836 SGH Y = 12190710.7646x - 14788.6779 0.9999 0.0056～0.1111

[0061] (4) Repeatable experiments

[0062] Six parallel portions (0.5 g each) of the same batch of black mulberry stem and branch powder were weighed and prepared according to the method for preparing the test solution. Analysis was performed under the aforementioned chromatographic conditions, and the average contents of SGF and SGH were found to be 2.54 mg·g⁻¹. -1 and 0.73 mg·g -1 The calculated RSDs for the peak areas of SGF and SGH were 1.9% and 1.8%, respectively.

[0063] (5) Stability test

[0064] 0.5 g of black mulberry stem and branch powder was weighed and prepared according to the method for preparing the test solution. The solution was analyzed under the chromatographic conditions described above. Its stability within 24 hours was investigated, and the RSDs of the peak areas of SGF and SGH were calculated to be 1.2% and 1.2%, respectively. This indicates that the test sample has good stability within 24 hours.

[0065] (6) Precision test

[0066] SGF and SGH reference solutions were injected six times consecutively under the chromatographic conditions described above for HPLC analysis. The RSDs of the peak areas of SGF and SGH were calculated to be 0.64% and 0.52%, respectively, indicating that the instrument has good precision.

[0067] (7) Recovery test

[0068] Accurately weigh 0.25 g of black mulberry extract with known content, and make nine parallel weighings. Place each sample in a stoppered conical flask, and accurately add appropriate amounts of SGF and SGH reference solutions. Prepare samples according to the preparation method of the test solution. Perform HPLC analysis under the above chromatographic conditions, and calculate the recovery rate and RSD of each component. The average recoveries of SGF and SGH were 98.97% and 98.38%, respectively, and the RSDs were 1.57% and 1.34%, respectively. This indicates that the method used has good accuracy (see table below).

[0069]

[0070]

[0071] (8) Durability test

[0072] The study investigated column temperature, flow rate, injection volume, different column types, and different instruments, and found that the method has good durability.

[0073] a. Weigh 0.5 g of black mulberry stem and branch powder, prepare it according to the method for preparing the test solution, and analyze it under the above chromatographic conditions. Investigate the robustness of the method at 25℃, 30℃ and 35℃ respectively. Calculate the relative RSDs of SGF and SGH contents to be 6.49% and 6.84% respectively. This indicates that the robustness of the test sample at different temperatures is generally poor.

[0074] b. Weigh 0.5 g of black mulberry stem and branch powder, prepare it according to the method for preparing the test solution, and analyze it under the above chromatographic conditions, investigating the results at 0.8 mL·min. -1 1.0 mL·min -1 and 1.2 mL·min -1 The robustness of the method was tested, and the relative RSDs of SGF and SGH contents were calculated to be 3.23% and 4.34%, respectively. This indicates that the test sample exhibits good robustness at different flow rates.

[0075] c. Weigh 0.5 g of black mulberry stem and branch powder, prepare the test solution according to the above method, and analyze it under the above chromatographic conditions. Examine the robustness of the method at 5 μL, 10 μL, and 20 μL, respectively. Calculate the relative RSDs of SGF and SGH contents to be 0.63% and 0.64%, respectively. This indicates that the test sample has good robustness at different injection volumes.

[0076] d. Weigh 0.5 g of black mulberry stem and branch powder, prepare it according to the method for preparing the test solution, and analyze it under the above chromatographic conditions. Investigate the Welch column. XB-C18(4.6×250mm,5μmcolumn)、Waters The robustness of the method under Hss T3 (4.6×250mm, 5μm column) and Agilent Eclipse C18 (4.6×250mm, 5μm column) was measured, and the relative RSDs of SGF and SGH contents were calculated to be 0.43% and 2.93%, respectively. This indicates that the test sample exhibits good robustness under different chromatographic columns (see table below).

[0077] e. Weigh 0.5 g of black mulberry stem and branch powder, prepare it according to the method for preparing the test solution, and analyze it under the above chromatographic conditions. The robustness of the method was investigated using the Ultimate 3000 instrument and Waters Acquity Arc 2695 and Waters 2998PDA. The relative RSDs of SGF and SGH contents were calculated to be 0.86% and 2.96%, respectively. This indicates that the test sample has good robustness under different instruments.

[0078] The relative RSD results of the peak areas for HPLC method robustness are shown in the table below.

[0079]

[0080] (9) Limit of detection and limit of quantitation

[0081] Take the reference solution and dilute it stepwise using the same method as when establishing the linear relationship. Inject 10 μL of the reference solution into the high-performance liquid chromatograph and record the peak area and signal-to-noise ratio.

[0082] The results showed that when the signal-to-noise ratio (SNR) was 3, the limits of detection (LOD) for SGF and SGH were 0.085 μg and 0.036 μg, respectively. When the SNR was 10, the limits of quantitation (LOQ) for SGF and SGH were 0.341 μg and 0.142 μg, respectively.

[0083] (10) Prepare the test solution of the black mulberry stem and branch test sample according to the preparation method of the test solution determined above. Perform high performance liquid chromatography analysis under the chromatographic conditions determined above. Calculate the content of each index component using the external standard method. The content of SGF is 2.596 mg / g and the content of SGH is 0.749 mg / g. The parameters of SGF and SGH in the obtained high performance liquid chromatogram are shown in the table below.

[0084]

Claims

1. A method for determining the content of mulberry root ketone-type compounds, comprising the following steps: The contents of SGF and SGH compounds in the test solution were determined by high performance liquid chromatography. ； The chromatographic column used in the high-performance liquid chromatography method is a C18 column; In the high-performance liquid chromatography method, the detection wavelength is 254~310nm; The high-performance liquid chromatography method uses mobile phase A and mobile phase C, wherein mobile phase A is 0.1% phosphoric acid-water and mobile phase C is acetonitrile; The high-performance liquid chromatography method employs the following gradient elution program: 0–12 min, 40% A; 12–13 min, 40%–30% A; 13–26 min, 30% A; The test solution was obtained by extracting the black mulberry stems and branches in an alcohol solvent using ultrasonic extraction.

2. The content determination method as described in claim 1, characterized in that, The content determination method meets one or more of the following conditions: (1) The C18 column is a Welch ultimate® XB-C18 column, an Agilent Eclipse XDB-C18 column, or a WatersXselect® Hss T3 column; (2) In the high performance liquid chromatography method, the flow rate is 0.8~1.2 mL. min -1 ; (3) In the high performance liquid chromatography method, the column temperature is 25~35℃; (4) In the high performance liquid chromatography method, the injection volume is 5~20μL.

3. The content determination method as described in claim 2, characterized in that, The content determination method meets one or more of the following conditions: (1) In the high performance liquid chromatography method, the detection wavelength is 310 nm; (2) In the high performance liquid chromatography method, the flow rate is 1 mL. min -1 ; (3) In the high performance liquid chromatography method, the column temperature is 30℃; (4) In the high performance liquid chromatography method, the injection volume is 10 μL; (5) The specifications of the Welch ultimate® XB-C18 column are 4.6×250mm, 5µm; (6) The specifications of the Agilent Eclipse XDB-C18 column are 250mm × 4.6mm, 5μm; (7) The specifications of the Waters Xselect® Hss T3 column are 4.6×250mm, 5µm.

4. The content determination method according to claim 1, characterized in that, The content determination method meets one or more of the following conditions: (1) The alcohol solvent is methanol and / or ethanol; (2) The black mulberry stems and branches are dried powder of black mulberry stems and branches; (3) The liquid-to-solid ratio in the ultrasonic extraction method is (1:10)-(1:40) g / mL; (4) The ultrasonic temperature in the ultrasonic extraction method is 25℃-60℃; (5) The ultrasonic power in the ultrasonic extraction method is 50-100W; (6) The ultrasonic extraction time is 10 min to 40 min; (7) The ultrasonic extraction method may also include filtration after ultrasonic extraction.

5. The content determination method as described in claim 4, characterized in that, The method described satisfies one or more of the following conditions: (1) The alcohol solvent is anhydrous methanol, 90% methanol, 70% methanol, anhydrous ethanol, 90% ethanol or 70% ethanol; (2) The liquid-to-solid ratio in the ultrasonic extraction method is (1:10) g / mL, (1:20) g / mL or (1:30) g / mL; (3) The ultrasonic temperature in the ultrasonic extraction method is 25℃, 40℃ or 50℃; (4) The ultrasonic power in the ultrasonic extraction method is 50W, 70W or 100W; (5) The ultrasonic extraction time is 10 min, 20 min, 30 min or 40 min; (6) The filtration is performed by replenishing the weight with the alcohol solvent and filtering with a 0.45μm microporous membrane.

6. The content determination method as described in claim 5, characterized in that, The method described satisfies one or more of the following conditions: (1) The alcohol solvent is anhydrous methanol; (2) The liquid-to-solid ratio in the ultrasonic extraction method is (1:20) g / mL; (3) The ultrasonic temperature in the ultrasonic extraction method is 50℃; (4) The ultrasonic power in the ultrasonic extraction method is 100W; (5) The ultrasonic extraction time is 30 min.

7. The content determination method according to claim 1, characterized in that, The content determination method described above uses the external standard method to determine the content of SGF and SGH compounds in the test sample solution.

8. The content determination method as described in claim 7, characterized in that, The content determination method described herein satisfies one or more of the following conditions: (1) In the external standard method, the concentration range of the reference stock solution of SGF compound is 0.02612 mg. mL -1 ~0.836mg mL -1 ; (2) In the external standard method, the concentration range of the reference stock solution of SGH compound is 0.0056 mg. mL -1 ~0.1111mg mL -1 .

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