A method for simultaneously detecting the contents of ligusticum glycoside, scutellaria baicalensis glycoside and ellagic acid in leaves of Rhizoma Cyperi

Abstract

The invention relates to a method for simultaneously detecting the contents of privet glycoside, wild sumac glycoside and ellagic acid in leaves of Rhizoma Cyperi. The method is suitable for detecting the contents of privet glycoside, wild sumac glycoside and ellagic acid in Rhizoma Cyperi medicinal materials, intermediates and related preparations of Rhizoma Cyperi. The method has the characteristics of high precision, high accuracy, greater convenience and speed, etc.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for simultaneously detecting the contents of privet glycoside, scutellaria baicalensis glycoside and ellagic acid in leaves of Scutellaria baicalensis. Background Art

[0002] Turpinia arguta is a plant of the genus Turpinia in the family Staphyleaceae. The family Staphyleaceae includes 5 genera and about 60 species, which are produced in tropical Asia, America and the northern temperate zone. There are 4 genera in my country, with more than 23 species, and all 4 genera are produced in Yunnan. There are about 30 to 40 species of Turpinia arguta in the world, distributed in Sri Lanka, Japan, Malaysia and the tropical areas of Central and Southern America. There are 16 species in China, which are produced from southwest my country to Taiwan. The main medicinal plants of this genus include Turpinia arguta and Turpinia ternate. The main compounds isolated from the plants of this genus are flavonoids (privet glycosides, wild sumac glycosides, etc.) and triterpenoids (ursolic acid, oleanolic acid, etc.), as well as organic acid compounds (ellagic acid, etc.) and sterol compounds.

[0003] Ligustroflavone, CAS: [260413-62-5], molecular formula: C 33 H 40 O 18 , molecular weight: 724.22.

[0004] Rhoifolin, CAS: [17306-46-6], molecular formula: C 27 H30O 14 , molecular weight: 578.52.

[0005]

[0006] Ellagic acid, CAS: [476-66-4], molecular formula: C 14 H6O8, molecular weight: 302.19.

[0007]

[0008] At present, the determination methods of Ligustrum lucidum and Rhus chinensis glycosides in the Chinese Pharmacopoeia are as follows:

[0009] Chromatographic conditions and system suitability: The test used octadecylsilane bonded silica gel as filler; methanol-0.5% formic acid solution (43:57) as mobile phase; detection wavelength was 336nm. The theoretical plate number calculated based on the peak of Ligustrum lucidum should not be less than 3000.

[0010] Preparation of reference solution: Take 10 mg each of the Ligustrum lucidum glycoside reference substance and the Rhus chinensis glycoside reference substance that have been dried under reduced pressure with phosphorus pentoxide for 24 hours, weigh them accurately, place them in 100 ml volumetric flasks, add 50% methanol to dissolve and dilute to the scale, and shake well; accurately measure 25 ml of the Ligustrum lucidum glycoside reference solution and 10 ml of the Rhus chinensis glycoside reference solution, place them in the same 50 ml volumetric flask, add 50% methanol to dilute to the scale, shake well, and obtain (each 1 ml contains 50 μg of Ligustrum lucidum glycoside and 20 μg of Rhus chinensis glycoside).

[0011] Preparation of test solution: Take about 0.3 g of the powder of the product (passed through No. 3 sieve), weigh it accurately, put it in a stoppered conical flask, accurately add 50 ml of 50% methanol, weigh it, treat it with ultrasound (power 250 W, frequency 25 kHz) for 1 hour, cool it, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0012] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0013] The current method for determining ellagic acid in the Chinese Pharmacopoeia is as follows: (Pomegranate peel)

[0014] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel is used as filler; acetonitrile-0.2% formic acid solution (21:79) is used as mobile phase; detection wavelength is 254nm. The theoretical plate number calculated based on the ellagic acid peak should be no less than 5000.

[0015] Preparation of reference solution: Take an appropriate amount of ellagic acid reference, weigh accurately, and add methanol to make a solution containing 20 μg per 1 ml.

[0016] Preparation of test solution: Take about 0.2 g of the powder of the product (passed through No. 3 sieve), weigh accurately, place in a stoppered conical flask, add 50 ml of methanol accurately, stopper tightly, weigh, ultrasonically treat (power 150 W, frequency 40 kHz) for 40 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain.

[0017] Determination method: Accurately aspirate 5μl and 10μl of the reference solution and 5-10μl of the test solution, inject into liquid chromatograph, determine, and calculate using the external standard two-point method to obtain the result.

[0018] The existing technology only has a method for determining the content of privet glycoside, wild sumac glycoside or ellagic acid separately. At most, privet glycoside and wild sumac glycoside can be determined simultaneously, but the contents of privet glycoside, wild sumac glycoside and ellagic acid cannot be determined simultaneously. Summary of the invention

[0019] In order to overcome the shortcomings of the prior art, the present invention provides a high precision, high accuracy, more convenient and quick determination method, which can simultaneously detect the content of privet glycosides, wild sumac glycosides and ellagic acid in the medicinal materials, intermediates and related preparations of Scutellaria baicalensis.

[0020] The present invention provides a method for simultaneously detecting the contents of ligusticum glycoside, scutellaria baicalensis glycoside and ellagic acid in leaves of Rhizoma Cyperi, comprising the following steps:

[0021] (1) Preparation of reference solution: Accurately weigh appropriate amounts of privet glycoside reference, wild lacquercoside reference and ellagic acid reference, and prepare them with a solvent to a fixed volume;

[0022] (2) Preparation of test solution: Accurately weigh an appropriate amount of Scutellaria baicalensis leaf powder, add solvent, ultrasonicate, add solvent to make up for the weight loss, filter, and take the filtrate;

[0023] (3) HPLC conditions: C18 chromatographic column; using methanol as mobile phase A and 0.1-1% (V / V) formic acid aqueous solution as mobile phase B, with gradient elution; setting a specific flow rate, injection volume, column temperature and detection wavelength;

[0024] (4) Detection: Take a quantity of the test solution and the reference solution and inject them into a high performance liquid chromatograph respectively, perform the measurement according to the above chromatographic conditions, and record the chromatogram;

[0025] (5) Calculation.

[0026] Preferably, the solvent is a methanol solution, preferably a 30-60% (V / V) methanol solution, and more preferably a 50% (V / V) methanol solution.

[0027] Preferably, the high performance liquid chromatograph is selected from: Agilent 1260, Waters e2695-2998, LC-20AT.

[0028] Preferably, in step (1), in the prepared reference solution, the concentration ratio (ug / ml) of privet glycoside, scutellaria baicalensis glycoside and ellagic acid is 10-15:4-6:1, more preferably 12.5:5:1.

[0029] Preferably, in step (3), the chromatographic column is an ACE Excel 3C18 column, with preferred specifications of: length × inner diameter = 150 mm × 4.6 mm, and a particle size of 3 μm.

[0030] Preferably, in step (3), the flow rate of the mobile phase is 0.4 to 1.0 ml / min, preferably 0.7 to 0.9 ml / min.

[0031] Preferably, in step (3), the mobile phase B is a 0.45-0.55 (V / V) formic acid aqueous solution.

[0032] Preferably, in step (3), the injection volume is 5 to 20 μl, preferably 10 μl.

[0033] Preferably, in step (3), the operation time of the high performance liquid chromatograph is 2 min to 10 min, preferably 5 min.

[0034] Preferably, in step (3), the column temperature is 30-50°C, preferably 38-42°C; and / or the detection wavelength is 300-350nm, preferably 336nm.

[0035] Preferably, in step (3), the gradient elution adopts the following procedure:

[0036]

[0037] Preferably, the calculation formula is:

[0038]

[0039] In the formula, A U is the peak area of ​​ligusticum glycoside / scutellaria baicalensis glycoside / ellagic acid in the test solution;

[0040] A S is the peak area of ​​ligusticum glycoside / scutellaria baicalensis glycoside / ellagic acid in the reference solution;

[0041] C S is the concentration of ligusticum glycoside / scutellaria baicalensis glycoside / ellagic acid in the reference solution (μg / ml);

[0042] C U is the sample concentration in the test solution (μg / ml).

[0043] The beneficial technical effects of the key points of the present invention are:

[0044] 1. The present invention establishes a method for simultaneously detecting the contents of privet glycoside, wild sumac glycoside and ellagic acid.

[0045] 2. The method of the present invention has been verified to have good linearity, precision, accuracy, durability, etc.

[0046] 3. The method of the present invention can be used to accurately detect the content of privet glycoside, wild sumac glycoside and ellagic acid in products related to the safflower oil (such as safflower oil leaf medicinal materials, intermediates, related preparations, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 : Liquid chromatogram of the test sample;

[0048] Figure 2:Liquid phase chromatogram of reference substance;

[0049] Figure 3 : Linear relationship diagram of Ligustrum lucidum concentration-peak area;

[0050] Figure 4 : Linear relationship diagram of wild sumac glycoside concentration-peak area;

[0051] Figure 5 : Linear relationship diagram of ellagic acid concentration-peak area. DETAILED DESCRIPTION

[0052] The technical method of the present invention is described in conjunction with specific implementation methods, but the implementation methods do not limit the scope of the invention. Example 1 Preparation of samples and calculation formula

[0053] 1. Blank solution (diluent): 50% methanol solution.

[0054] 2. Preparation of reference solution: Take 10 mg each of privet glycoside reference substance, wild sumac glycoside and ellagic acid reference substance, accurately weigh, place in 100 ml volumetric flasks respectively, add 50% (V / V) methanol, dissolve in methanol and dilute to the scale, shake well, and obtain the reference stock solution; accurately measure 25 ml of privet glycoside reference stock solution, 10 ml of wild sumac glycoside reference stock solution, and 2 ml of ellagic acid reference stock solution, place in the same 50 ml volumetric flask, add 50% methanol to dilute to the scale, shake well, and obtain (each 1 ml contains 50 μg of privet glycoside, 20 μg of wild sumac glycoside, and 4 μg of ellagic acid).

[0055] 3. Preparation of reference substance positioning solution: Accurately measure 25 ml of Ligustrum lucidum reference substance stock solution, 10 ml of Rhus glycosides reference substance stock solution, and 2 ml of ellagic acid reference substance reserve solution, respectively, and place them in 50 ml volumetric flasks, add 50% methanol to dilute to the scale, shake well, and the reference substance positioning solution is obtained.

[0056] 4. Preparation of test solution: Take about 0.3 g of Scutellaria baicalensis leaf powder (passed through No. 3 sieve), weigh accurately, put it in a stoppered conical flask, accurately add 50 ml of 50% (V / V) methanol, weigh the weight, ultrasonically treat (power 250 W, frequency 25 kHz) for 1 hour, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate.

[0057] 5. Calculation formula:

[0058]

[0059] In the formula, A U is the peak area of ​​ligusticum glycoside / scutellaria baicalensis glycoside / ellagic acid in the test solution;

[0060] A Sis the peak area of ​​ligusticum glycoside / scutellaria baicalensis glycoside / ellagic acid in the reference solution;

[0061] C S is the concentration of ligusticum glycoside / scutellaria baicalensis glycoside / ellagic acid in the reference solution (μg / ml);

[0062] C U is the sample concentration in the test solution (μg / ml).

[0063] Example 2 High performance liquid chromatography detection

[0064] 1. HPLC conditions: instrument Agilent 1260; ACE Excel 3C18 column, specification: length × inner diameter = 150 mm × 4.6 mm, particle size 3 μm; injection volume 10 μl; chromatographic operation 5 min, column temperature 40°C, detection wavelength 336 nm; 50% (V / V) methanol solution as mobile phase A, 0.5% (V / V) formic acid aqueous solution as mobile phase B, mobile phase flow rate 0.8 ml / min, gradient elution, the program is as follows:

[0065]

[0066] 2. Detection: Take the test solution and reference solution of Example 1 and inject them into the high performance liquid chromatograph respectively, and measure them according to the above chromatographic conditions, and record each chromatogram (see Figure 1 , Figure 2 );

[0067] 3. Calculation results: Ligustrum lucidum content 0.72%; Rhus glycoside content 0.40%; Ellagic acid content 0.06%.

[0068] Example 3 Methodology Verification

[0069] 1. Specificity test: Take blank solution, reference solution, reference solution, and test solution, and inject them respectively according to the conditions of Example 2, and record the chromatogram. The blank solvent should have no interference, and the main peak retention time of the reference solution and the test solution should be consistent.

[0070] 2. Linearity test: Take 25 mg of Ligustrum lucidum glycoside, accurately weigh it, place it in a 50 ml volumetric flask, add an appropriate amount of diluent to dissolve and dilute to the mark, shake well, and use it as the Ligustrum lucidum glycoside reference stock solution; take 10 mg of Rhus sutchuenensis glycoside, accurately weigh it, place it in a 100 ml volumetric flask, add an appropriate amount of diluent to dissolve and dilute to the mark, shake well, and use it as the Rhus sutchuenensis glycoside reference stock solution. Take 10 mg of ellagic acid, accurately weigh it, place it in a 100 ml volumetric flask, add an appropriate amount of diluent to dissolve and dilute to the mark, shake well, and use it as the ellagic acid reference stock solution. Accurately measure 2.5ml, 4.0ml, 5ml, 6.0ml, 7.5ml of Ligustrum lucidum reference substance stock solution, 5ml, 8ml, 10ml, 12ml, 15ml of Rhus cinnamomea reference substance stock solution and 1ml, 1.6ml, 2ml, 2.4ml, 3ml of Ellagic acid reference substance stock solution respectively in the same 50ml volumetric bottle, dilute to scale with diluent and shake well, as a series of linear solutions. Accurately measure 10μl respectively and inject the liquid chromatograph (double needle) under the conditions of Example 2, record the chromatogram, perform linear regression on the concentration with peak area, and obtain the linear equation and correlation coefficient. The test results are shown in Table 1.

[0071] Table 1 Linearity test results of ligustrin, scutellaria baicalensis glycosides and ellagic acid

[0072]

[0073] Conclusion: In the concentration range of 23.6000~70.8000μg / ml, the linear equation of Ligustrum lucidum is y=0.9975x+4.5493, the correlation coefficient is r=0.9999, and the peak area has a good linear relationship with the concentration (see Figure 3 ); In the concentration range of 10.0753-30.2258 μg / ml, the linear equation was y=1.002x-0.8979, the correlation coefficient was r=1.0000, and the peak area had a good linear relationship with the concentration (see Figure 4 ); Ellagic acid has a good linear relationship with concentration in the range of 2.1148 to 6.3445 μg / ml, with a linear equation of y = 0.9988x + 0.9817 and a correlation coefficient of r = 1.0000 (see Figure 5 ).

[0074] 3. Repeatability test: Prepare 2 parts of reference solution and 6 parts of test solution according to the method of the present invention.

[0075] 10 μl of the reference solution and the test solution were accurately drawn respectively, and injected into the chromatograph according to the conditions of Example 2, and the chromatogram was recorded, and the contents of privet glycoside and wild sumac glycoside were calculated respectively. The test results are shown in Table 2.

[0076] Table 2 Repeatability test results

[0077] serial number Ligustrum lucidum (%) Rhusin(%) Ellagic acid(%) 1 0.717 0.401 0.058 2 0.727 0.404 0.058 3 0.713 0.401 0.058 4 0.716 0.404 0.059 5 0.717 0.399 0.058 6 0.722 0.402 0.058 Average content (%) 0.72 0.40 0.06 RSD(%) 0.8 0.5 0.9

[0078] Conclusion: The RSD of the 6 test sample content results is less than 1.5%, and the repeatability is good.

[0079] 4. Intermediate precision test: According to the "Repeatability" item, the test was carried out on different dates, by different analysts, and with different instruments. The results of the repeatability test were compared with the results of this test, and the RSD of the 12 results was calculated. The test results are shown in Table 3.

[0080] Table 3 Precision test results

[0081]

[0082] Conclusion: The RSDs of the results of ligusticum glycosides, scutellaria baicalensis glycosides and ellagic acid were all less than 3.0%, and the intermediate precision was good.

[0083] 5. Accuracy test: Take 0.15g of the herbal material of Rhizoma Cibotii, weigh it accurately, weigh 9 portions, place them in stoppered conical bottles, and divide them into 3 groups; weigh 22mg of Ligustrum lucidum glycosides, weigh it accurately, place them in a 100ml volumetric flask, add diluent to dissolve and dilute to the scale, then accurately measure 2.5ml, 5ml, and 7.5ml (n=3) and place them in the above conical flasks; weigh 12mg of Rhus sutchuenensis glycosides, weigh it accurately, place them in a 100ml volumetric flask, and add diluent to dissolve and dilute to the scale; Bottle, add methanol solvent and dilute to the scale, then accurately measure 2.5ml, 5ml, 7.5ml (n=3) respectively and place them in the above-mentioned conical flask; then weigh 11mg of ellagic acid, accurately weigh it, place it in a 250ml volumetric flask, add methanol solvent and dilute to the scale, then accurately measure 1ml, 2ml, 3ml (n=3) respectively and place them in the above-mentioned conical flask, add diluent to make up to 50ml, shake well, and get the spiked recovery solution.

[0084] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the high performance liquid chromatograph according to the conditions of Example 2, record the chromatogram, and calculate the recovery rate.

[0085] Recovery rate % = (C-A) ÷ B × 100%

[0086] Where A is the amount of the component to be measured contained in the test sample;

[0087] B is the amount of reference substance added;

[0088] C is the measured amount.

[0089] The test results are shown in Table 4, as follows:

[0090] Table 4 Accuracy test results

[0091]

[0092]

[0093] Conclusion: The average recoveries at each concentration level were all in the range of 95% to 102%, and the RSDs of the recoveries were all less than 2.0%, indicating good accuracy.

[0094] 6. Range: refers to the range of upper and lower limits of concentration or quantity when the analytical method can achieve certain precision, accuracy and linearity requirements. It is determined by combining the concentrations used to verify linearity and accuracy, and the intersection of the two is the range.

[0095] Concentrations for verification of linearity: Ligustrum lucidum glycoside concentration 23.6000~70.8000μg / ml, Rhus chinensis glycoside concentration 10.0753~30.2258μg / ml, ellagic acid concentration 2.1148~6.3445μg / ml.

[0096] Concentrations for verification of accuracy: Ligustrum lucidum concentration 32.8549 ~ 55.6064μg / ml, Rhus chinensis glycoside concentration 18.1695 ~ 30.3584μg / ml, ellagic acid concentration 2.6254 ~ 4.4537μg / ml.

[0097] The determined ranges are: ligusticum glycoside concentration 32.8549~55.6064μg / ml, asteridoxine concentration 18.1695~30.2258μg / ml, and ellagic acid concentration 2.62548~4.4537μg / ml.

[0098] 7. Durability test: By changing the chromatographic conditions, the degree to which the measurement results can be tolerated without being affected by small changes in the measurement conditions is evaluated, and whether the system suitability test meets the requirements.

[0099] The changes in the instrument's chromatographic behavior were investigated under the following conditions, with each condition tested twice. The changes in chromatographic conditions are summarized in Table 5.

[0100] Table 5 Summary of changes in chromatographic conditions

[0101] Chromatographic conditions Proposed value Range of variation Flow rate (ml / min) 0.8 0.7、09 Column temperature (℃) 40 38、42 Mobile phase formic acid concentration 0.5% formic acid 0.45% and 0.55% instrument Agilent1260 Waters e2695-2998, LC-20AT

[0102] The results of the investigation of each condition are shown in Table 6, Table 7, Table 8, and Table 9, as follows:

[0103] Table 6 Flow rate investigation results

[0104] Flow rate (ml / min) 0.7 0.8 0.9 RSD(%) Ligustrum lucidum (%) 0.72 0.72 0.72 0.5 Rhusin(%) 0.40 0.40 0.40 0.6 Ellagic acid(%) 0.06 0.06 0.06 2.5

[0105] Table 7 Column temperature investigation results

[0106] Column temperature (℃) 38 40 42 RSD(%) Ligustrum lucidum (%) 0.72 0.72 0.72 0.2 Rhusin(%) 0.40 0.40 0.40 0.4 Ellagic acid(%) 0.06 0.06 0.06 1.7

[0107] Table 8 Mobile phase formic acid concentration results

[0108] Mobile phase formic acid concentration 0.45% Formic acid 0.5% formic acid 0.55% Formic acid RSD(%) Ligustrum lucidum (%) 0.72 0.72 0.72 0.4 Rhusin(%) 0.40 0.40 0.40 0.6 Ellagic acid(%) 0.06 0.06 0.06 2.4

[0109] Table 9 Instrument investigation results

[0110] instrument LC-20AT Agilent 1260 Water e2695 RSD(%) Ligustrum lucidum (%) 0.73 0.72 0.72 0.9 Rhusin(%) 0.40 0.40 0.41 1.3 Ellagic acid(%) 0.06 0.06 0.06 2.6

[0111] Note: The above results are rounded to two decimal places. The actual values ​​are slightly different, so the RSD is not 0 when the results are the same.

[0112] Conclusion: With the same parameters, different flow rates, different column temperatures, different formic acid concentrations and different instrument conditions, the RSDs of the measured contents were all within 4.0%, and the method had good durability.

[0113] The results of methodological validation show that the developed method can meet the needs of determining the contents of privet glycoside, wild tonic glycoside and ellagic acid, and during the methodological validation process, the standard deviation (RSD) of each indicator is small, the method has good specificity, good linearity, good repeatability, high accuracy and good durability, and can be used for the detection of privet glycoside, wild tonic glycoside and ellagic acid in Scutellaria baicalensis leaves and related preparations (such as single-ingredient preparations).

Claims

1. A method for simultaneously detecting the contents of ligusticum glycoside, scutellaria baicalensis glycoside and ellagic acid in the leaves of Rhizoma Cibotii, comprising the following steps: (1) Preparation of reference solution: Accurately weigh appropriate amounts of privet glycoside reference, wild lacquercoside reference and ellagic acid reference. The concentration (μg / mL) ratio of privet glycoside, wild lacquercoside and ellagic acid in the prepared reference solution is 10-15:4-6:1, and the solution is prepared using 50% V / V methanol solution; (2) Preparation of test solution: Accurately weigh an appropriate amount of Scutellaria baicalensis leaf powder, add 50% v / v methanol solution, treat with ultrasound, make up the weight loss with solvent, filter, and take the filtrate; (3) HPLC conditions: The chromatographic column was an ACE Excel 3C18 column, with the specifications of length × inner diameter = 150 mm × 4.6 mm, and a particle size of 3 μm; the injection volume was 10 μL, the column temperature was 40°C, the detection wavelength was 336 nm, methanol was used as mobile phase A, 0.5% V / V formic acid aqueous solution was used as mobile phase B, the flow rate of the mobile phase was 0.8 mL / min, and gradient elution was performed, and the procedure was as follows: (4) Detection: Take a quantity of the test solution and the reference solution and inject them into a high performance liquid chromatograph respectively, perform the measurement according to the above chromatographic conditions, and record the chromatogram; (5) Calculation.

2. The method according to claim 1, characterized in that: The calculation formula is: In the formula, A U is the peak area of ​​ligusticum glycoside / scutellaria baicalensis glycoside / ellagic acid in the test solution; A S is the peak area of ​​ligusticum glycoside / scutellaria baicalensis glycoside / ellagic acid in the reference solution; C S is the concentration of ligusticum glycoside / scutellaria baicalensis glycoside / ellagic acid in the reference solution (μg / mL); C U is the sample concentration in the test solution (μg / mL).

Images