CN112240914A - Method for detecting flavone components in anoectochilus formosanus with different appearance phenotypes - Google Patents
Method for detecting flavone components in anoectochilus formosanus with different appearance phenotypes Download PDFInfo
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Abstract
The invention provides a method for detecting flavone components in anoectochilus formosanus with different appearance phenotypes, which is to detect by using a high performance liquid chromatography and specifically comprises the following operation steps: 1) preparing a reference substance solution; 2) preparing a test solution; 3) respectively sucking a reference substance solution and a test substance solution, injecting the reference substance solution and the test substance solution into a chromatograph, carrying out chromatographic column: a C18 chromatography column; the mobile phase is gradient eluted with 0.1-0.3% acetonitrile formate as mobile phase A and 0.1-0.3% formic acid as mobile phase B. The detection method can simultaneously detect rutin, narcissin, quercetin, kaempferol and isorhamnetin in anoectochilus roxburghii with different appearance phenotypes, and a one-test-multiple-evaluation method for simultaneously analyzing the rutin, the quercetin, the kaempferol and the isorhamnetin in the anoectochilus roxburghii by taking the narcissin as an internal reference material is simple and reliable, so that a new technology can be provided for quality evaluation of the anoectochilus roxburghii. The one-test-multiple-evaluation method and the external standard method are mutually verified, have no obvious difference and have reliable results.
Description
Technical Field
The invention particularly relates to a method for detecting flavone components in anoectochilus formosanus with different appearance phenotypes.
Background
Anoectochilus roxburghii is a dry whole plant of Lindl, namely Anoectochilus roxburghii, belonging to the genus Anoectochilus of the family Orchidaceae, is called Anoectochilus roxburghii, pinus sylvestris, Anoectochilus roxburghii and the like, is neutral in nature and sweet in taste, enters lung, liver, kidney and bladder channels, has the effects of clearing heat and cooling blood, dispelling wind and removing dampness, is used for treating nephritis, cystitis, diabetes, bronchitis, rheumatic arthritis, acute infantile convulsion and the like, enjoys the name of 'drug king', and can be clinically used for treating type II diabetes, senile hyperuricemia, pediatric diseases and the like. Modern pharmacological research shows that anoectochilus formosanus has pharmacological activities of resisting oxidation, reducing blood sugar, protecting liver, resisting inflammation, resisting virus, resisting melanin and the like. However, because the natural reproduction rate of anoectochilus roxburghii is low, the requirements on ecological environment are strict, the adaptability is poor, and the wild resources are sharply reduced by manual excessive mining, the international trade convention on endangered wild animal and plant species (CITES) is listed as the protection species in appendix II.
In recent years, with research on germplasm resource protection, seedling breeding and artificial cultivation techniques of anoectochilus formosanus by researchers, the planting scale is rapidly enlarged, and a plurality of anoectochilus formosanus with different appearance traits, including tip leaves, big round leaves, small round leaves and cancrinis Carbonisatus, are formed, as shown in fig. 1. However, the research on the quality standard system of the anoectochilus formosanus is relatively lagged, most of the existing literatures consider that flavone is an important pharmacodynamic substance of the anoectochilus formosanus and can be used as an important index for evaluating the quality of the anoectochilus formosanus, but the publicly reported method only measures 2-3 or several flavonoid components respectively, a multi-evaluation method for measuring the flavonoid multi-index components in the anoectochilus formosanus with different phenotypes is not available, and the literature for evaluating the anoectochilus formosanus with different appearance phenotypes by adopting the flavone multi-index is rarely reported, so that the sustainable development of the anoectochil.
Disclosure of Invention
In order to solve the problems, the invention establishes a method for detecting flavone components in anoectochilus formosanus, which is used for detecting by using a high performance liquid chromatography and specifically comprises the following operation steps:
1) preparation of control solutions: dissolving flavone control product in methanol;
2) preparation of a test solution: sieving herba Clematidis Japonicae powder, extracting with methanol, centrifuging, and collecting supernatant;
3) respectively sucking the reference solution and the test solution to be injected into a chromatograph, wherein the chromatographic conditions are as follows: a chromatographic column: c18A chromatographic column; the mobile phase is a mobile phase A which is 0.1 to 0.3 percent of formic acid acetonitrile and a mobile phase B which is 0.1 to 0.3 percent of formic acid water; the gradient elution procedure was as follows:
further, the flavonoid reference substance in the step 1) is rutin, narcissus, quercetin, kaempferol and/or isorhamnetin.
Further, the reference substance solution in the step 1) contains 45-50 mug of rutin, 50-70 mug of narcissus, 25-30 mug of quercetin, 10-15 mug of kaempferol and/or 5-10 mug of isorhamnetin per 1 mL; preferably, each 1mL of the composition contains 49.44 μ g of rutin, 60.06 μ g of narcissus, 28.5 μ g of quercetin, 12.35 μ g of kaempferol and/or 7.884 μ g of isorhamnetin; the methanol is 50-100% methanol, preferably 50% methanol.
Further, step 3) of C18The chromatographic column is
UHPLC AQ-C18 chromatographic column with specification of 2.1mm × 100mm, 1.8 μm.
Further, the amount of the reference solution and the test solution sucked in the step 3) is 5 mu L respectively; the column temperature of the chromatographic conditions is 38 ℃, the wavelength is 368nm, and the flow rate is 0.3 mL/min-1。
Further, the anoectochilus formosanus powder in the step 2) passes through a No. 4 sieve, and the mass volume ratio of the anoectochilus formosanus powder to methanol is 1: (40-60) g/ml, preferably 1: 50 g/ml; the methanol is 80-100% methanol, preferably 80% methanol.
Further, the extraction in the step 2) is ultrasonic extraction, the extraction power is 350W, the frequency is 50Hz, and the time is 30 min.
The invention also provides a method for detecting the content of the flavone component in anoectochilus formosanus with different appearance phenotypes based on a one-test-and-multiple-evaluation method, which takes any one of rutin, narcissus, quercetin, kaempferol and isorhamnetin as an internal reference substance, firstly detects the content of the internal reference substance in a sample to be detected according to the method, and then calculates the content of the other components by using relative correction factors. Further, the relative correction factor is obtained according to the following method:
taking rutin, narcissus, quercetin, kaempferol and isorhamnetin reference substances, detecting according to the detection method of the flavone component in anoectochilus formosanus, and calculating according to the following formula:
fs/i＝fs/fi＝As×Ci/(Ai×Cs) (ii) a In the formula AsIs the area of the peak of the internal reference substance s, CsIs the concentration of internal reference substance s, AiArea of component i peak, CiComponent i concentration.
Furthermore, narcissus is taken as an internal reference, the relative correction factor of narcissus and rutin is 1.016, the relative correction factor of narcissus and quercetin is 0.4650, the relative correction factor of narcissus and kaempferol is 0.4228, and the relative correction factor of narcissus and isorhamnetin is 0.4539.
The detection method can simultaneously detect rutin, narcissin, quercetin, kaempferol and isorhamnetin in anoectochilus roxburghii with different appearance phenotypes, and a one-test-multiple-evaluation method for simultaneously analyzing the rutin, the quercetin, the kaempferol and the isorhamnetin in the anoectochilus roxburghii by taking the narcissin as an internal reference material is simple and reliable, so that a new technology can be provided for quality evaluation of the anoectochilus roxburghii. The one-test-multiple-evaluation method and the external standard method are mutually verified, have no obvious difference and have reliable results.
Compared with the clematis HPLC detection method in the prior art, the invention has the following advantages:
1. the method has short detection time, and can finish the determination of the flavonoid component within 11 min.
2. When the method is applied, only one reference substance is needed to be used, and the determination of other flavonoid components can be completed.
3. The method is established by investigating the influence of different laboratories, different instruments, different chromatographic columns, flow phase change and wavelength change on the durability of the method, and the result reproducibility is good.
4. The method can be used for detecting the content of flavonoid components in anoectochilus formosanus samples with different phenotypes, and overcomes the defect of single variety of the detected samples in the prior art.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1A of Anoectochilus roxburghii (A big round leaf, B small round leaf, C tip leaf, D rosy clouds)
FIG. 2 UPLC chromatogram of mixed control and 36 lots of anoectochilus formosanus samples with different phenotypes (A mixed control, B36 lots of anoectochilus formosanus samples with different phenotypes)
FIG. 336 batch sample Cluster analysis dendrogram (I. Sharp leaf; II. Small round leaf; III. Large round leaf; IV. Hongxia)
FIG. 4 shows a comparison of the contents of Anoectochilus roxburghii (1. round leaf; 2. sauvignon blumea; 3. sharp leaf; 4. round leaf)
Detailed Description
Example 1 test method of the invention (one test for more comments)
(1) Preparation of control solutions
Taking appropriate amount of narcissus reference substance, precisely weighing, adding methanol, and ultrasonic dissolving to obtain narcissus with mass concentration of 567.9 μ g/mL-1Taking a proper amount of the single reference substance stock solution, adding 50% methanol to scale marks to prepare a reference substance solution containing 60.06 mu g of narcissus per 1mL, and further diluting with 50% methanol to prepare the reference substance solution with gradient concentration.
(2) Preparation of test solution
Taking about 0.5g of sample powder to be detected (passing through a No. 4 sieve), precisely weighing, placing in a triangular flask with a plug, precisely adding 25mL of 80% methanol, sealing the plug, weighing, ultrasonically treating for 20min (power 350W and frequency 50Hz), cooling, weighing again, complementing weight loss, shaking up, centrifuging at 10000 r/min for 5min, filtering a supernatant with a 0.22 mu m filter membrane, and taking a subsequent filtrate to obtain the product.
(3) Respectively sucking the gradient concentration reference substance solution and the test substance solution to be injected into a chromatograph, wherein the chromatographic conditions are as follows: a chromatographic column:
UHPLC AQ-C18 column (2.1 mm. times.100 mm, 1.8 μm); mobile phase, acetonitrile (containing 0.1% formic acid) is used as mobile phase A, and 0.3% formic acid water is used as mobile phase B; flow rate 0.3 mL/min-1Detecting the wavelength of 368 nm; the column temperature is 38 ℃; the sample injection amount is 5 mu L; the separation degree of each peak and adjacent peaks is more than 1.5, and the theoretical plate number is not less than 6000 calculated according to narcissus; the gradient elution procedure was as follows:
(4) drawing a standard curve by taking the narcissus reference quality concentration (X) as a horizontal coordinate and the peak area (Y) as a vertical coordinate, calculating the content of narcissus in the sample according to the standard curve, and calculating the content of rutin, quercetin, kaempferol and isorhamnetin in the sample to be detected by using relative correction factors;
the relative correction factors are:
relative correction factor measurement results
Example 2 detection method of the invention (external standard method)
(1) Preparation of Mixed control
Taking appropriate amount of rutin, narcissus glycoside, quercetin, kaempferol and isorhamnetin reference substance, precisely weighing, adding methanol, and ultrasonic dissolving to obtain rutin, narcissin, quercetin, kaempferol and isorhamnetin with mass concentrations of 494.4, 567.9, 548.0, 514.7 and 109.5 μ g. m L-1Single control stock solution of (a). Taking appropriate amount of reference stock solution, placing into the same volumetric flask, adding 50% methanol to scale mark, making into mixed reference solution containing 49.44, 60.06, 28.5, 12.35, 7.884 μ g above 5 components per 1mL, and further diluting with 50% methanol to obtain mixed reference solution with gradient concentration.
(2) Preparation of test solution
Taking about 0.5g of sample powder to be detected (passing through a No. 4 sieve), precisely weighing, placing in a triangular flask with a plug, precisely adding 25mL of 80% methanol, sealing the plug, weighing, ultrasonically treating for 20min (power 350W and frequency 50Hz), cooling, weighing again, complementing weight loss, shaking up, centrifuging at 10000 r/min for 5min, filtering a supernatant with a 0.22 mu m filter membrane, and taking a subsequent filtrate to obtain the product.
(3) Respectively sucking mixed reference substance solution and test substance solution with series concentrations and injecting into a chromatograph under the following chromatographic conditions: a chromatographic column:
UHPLC AQ-C18 column (2.1 mm. times.100 mm, 1.8 μm); mobile phase, acetonitrile (containing 0.1% formic acid) is used as mobile phase A, and 0.3% formic acid water is used as mobile phase B; flow rate 0.3 mL/min-1Detecting the wavelength of 368 nm; column temperature 38 deg.C(ii) a The sample injection amount is 5 mu L; the separation degree of each peak and adjacent peaks is more than 1.5, and the theoretical plate number is not less than 6000 calculated according to narcissus; the gradient elution procedure was as follows:
(4) and respectively drawing standard curves by taking the reference quality concentration (X) of rutin, narcissus, quercetin, kaempferol and isorhamnetin as a horizontal coordinate and taking the peak area (Y) as a vertical coordinate, and calculating the content of each component in the anoectochilus formosanus according to the standard curves.
The beneficial effects of the invention are illustrated by way of experimental examples below:
1 Material
1.1 Experimental instruments
Agilent 1290 hplc, G4212A 1290DAD detector (Agilent); waters ACQUITY UPLC H-Class ultra high performance liquid chromatograph, 2998 photodiode array detector (WATERS, USA); DF-15 bench continuous feed pulverizer (large mechanical Co., Ltd., forest, Wenling City); AR224CN electronic balance (aohaus instruments ltd); EX225DZH electronic balance (aohaus instruments ltd); KQ-500DE type hospital digital control ultrasonic cleaner (Kunshan ultrasonic instruments Co., Ltd.); MILLI-Q Direct16 integrated ultra pure water machine (Millipore, USA).
1.2 materials of the experiment
Rutin (batch number: 100080-; narcissus (batch No. 111997-201501, China institute for food and drug testing); quercetin (batch number: 100081-201509, China institute for food and drug testing); kaempferol (batch No. 110861-201611, China institute for testing food and drug); isorhamnetin (batch No. 110860-201410, China institute for food and drug testing); chromatographic methanol, chromatographic acetonitrile (merck chemical technology limited); chromatographic formic acid (Shanghai Michelin Biochemical technology, Inc.); chromatographic methanol (national pharmaceutical group chemical agents limited); the experimental water is secondary distilled water which is self-made by a laboratory; anoectochilus roxburghii medicinal materials 36 batches (identified by auxiliary professor of Huang ze Hao of medical college of Fujian Chinese medicinal university, all are anoectochilus roxburghii planting samples), detailed information is shown in Table 1
TABLE 1 sample information
2 methods and results
2.1 chromatographic conditions and System Adaptation
2.2 preparation of control solutions
Taking appropriate amount of rutin, narcissus glycoside, quercetin, kaempferol and isorhamnetin reference substance, precisely weighing, adding methanol, and ultrasonic dissolving to obtain rutin, narcissin, quercetin, kaempferol and isorhamnetin with mass concentrations of 494.4, 567.9, 548.0, 514.7 and 109.5 μ g/mL respectively-1Single control stock solution of (a). Respectively taking appropriate amount of reference stock solution, placing in the same volumetric flask, adding 50% methanol to scale mark to obtain mixed reference solution containing 49.44, 60.06, 28.5, 12.35, 7.884 μ g above 5 components per 1mL, and placing in refrigerator at 4 deg.C for use.
2.3 preparation of test solutions
Taking about 0.5g of the powder (passing through a No. 4 sieve), precisely weighing, placing in a triangular flask with a plug, precisely adding 25mL of 80% methanol, sealing the plug, weighing, ultrasonically treating for 20min (power 350W, frequency 50Hz), cooling, weighing again, complementing weight loss, shaking uniformly, centrifuging at 10000 r/min for 5min, filtering the supernatant with a 0.22 mu m filter membrane, and taking the subsequent filtrate to obtain the product.
2.4 methodological considerations
2.4.1 examination of the Linear relationship it was appropriate to precisely extract the stock solution of the mixed control prepared in item 2.2 and dilute it with 50% methanol solution to prepare a mixed control solution of gradient concentration. Precisely absorbing 5 mu L of each mixed reference substance solution, respectively, injecting into a liquid chromatograph, measuring according to the chromatographic condition under the item of 2.1, respectively drawing standard curves by taking the mass concentrations (X) of rutin, narcissus glycoside, quercetin, kaempferol and isorhamnetin as abscissa and taking the peak area integral value (Y) as ordinate, obtaining a regression equation, and the result is shown in a table 2, wherein each component has a good linear relation in a study range.
TABLE 25 regression equation, correlation coefficient (r) and Linear Range of the components
2.4.2 precision test 5 μ L of the mixed control solution under item 2.2 was precisely extracted, peak areas of the components were measured under the chromatographic conditions under item 2.1, sample introduction was continued 6 times within 1d, and the peak areas were recorded. As a result: the RSD of the peak areas of rutin, narcissus, quercetin, kaempferol and isorhamnetin are respectively 0.56%, 0.57%, 0.58%, 0.68% and 0.80%, which indicates that the precision of the instrument is good.
2.4.3 repeatability test the same sample of Anoectochilus roxburghii (batch number: S31) was precisely weighed, 6 parts were paralleled, the test solution was prepared according to the method under item "2.3", the peak areas of the components were measured according to the chromatographic conditions under item "2.1", and the standard curve was substituted to calculate the contents of rutin, narcissus glycoside, quercetin, kaempferol and isorhamnetin. As a result, the average contents of the components in the sample were 0.0332%, 0.1215%, 0.00481%, 0.0025% and 0.0058%, respectively, and the contents of RSD were 1.29%, 1.54%, 2.15%, 3.75% and 3.85%, respectively, indicating that the method was excellent in reproducibility.
2.4.4 stability test 0.5g of the same test solution (batch number: S31) was weighed precisely, and the test solution was prepared according to the method under item "2.3" and was subjected to sample injection measurement under the chromatographic conditions under item "2.1" at 0, 2, 4, 6, 8, 10, 12, 24 and 48 hours, respectively. The results show that the peak areas RSD of the rutin, the narcissus glycoside, the quercetin, the kaempferol and the isorhamnetin are respectively 1.15%, 1.48%, 2.28%, 2.63% and 2.42%, which indicates that the stability of the test solution is good within 48 h.
2.4.5 sample application recovery test about 0.25g of sample powder (lot: S31) with known content is precisely weighed, a certain amount of reference substance is added according to the ratio of the reference substance to the content of the medicinal material (1:1), 6 parts are prepared in parallel, sample application recovery and RSD of each component are calculated according to sample injection measurement under the chromatographic condition of '2.1'. The results show that the sample recovery rates of rutin, narcissus, quercetin, kaempferol and isorhamnetin are 106.17%, 98.04%, 97.06%, 101.30% and 99.63% respectively, and the RSD are 2.41%, 2.35%, 2.17%, 3.05% and 3.24% respectively, which indicates that the method has good accuracy.
2.5 establishment of relative correction factor and evaluation of durability thereof
2.5.1 establishment of relative correction factor precisely absorbs the mixed reference solution under the term of "2.2", 1, 2, 3, 4, 5, 7, 10 and 15 μ L are respectively taken and injected into a liquid chromatograph, peak areas are recorded, narcissin is taken As an internal reference, a formula fs/i is fs/fi As x Ci/(Ai x Cs) is calculated according to a relative correction factor (fs/i) which is evaluated on one side more (Ci in the formula is the concentration of an internal standard substance, Ai is the peak area of the internal standard substance, Cs is the concentration of a component s, and As is the peak area of the component s), relative correction factors of rutin, quercetin, kaempferol and isorhamnetin are respectively calculated, the respective average values are taken As the relative correction factors, and the result is shown in table 3.
2.5.2 ultra high performance liquid chromatography and column investigation experiments were performed on Agilent 1290 and Waters ACQUITY UPLC H-Class ultra high performance liquid chromatography systems, respectively, examining 3 different brands of columns, such as Yuehao, Waters and Thermo Fisher Scientific,
UHPLC AQ-C18 chromatographic column (100mm × 2.1mm, 1.8 μm), ACQUITY
HSS T3C 18 chromatographic column (100mm x 2.1mm, 1.8 μm) and Syncronis aQ chromatographic column (100mm x 2.1mm, 1.7 μm)3 kinds of relative correction factors of the chromatographic columns, and the results are shown in Table 4, and the reproducibility of the relative correction factors of the components is good.
TABLE 3 determination of relative correction factors
TABLE 4 relative correction factors measured by different instruments and chromatographic columns
2.5.3 investigation of different laboratories in three different laboratories, the same root was used
The influence of 3 different laboratories on relative correction factors was examined respectively by using a UHPLC AQ-C18(100 mm. times.2.1 mm, 1.8 μm) chromatographic column and two Agilent 1290 and a Waters ACQUITY UPLC H-Class high performance liquid chromatography system, and the results are shown in Table 5.
TABLE 5 relative correction factors measured in different laboratories
2.5.4 investigation of different column temperatures in laboratory 1, Agilent 1290 high Performance liquid chromatography SystemThe system is provided with a plurality of communication interfaces,
UHPLC AQ-C18(100 mm. times.2.1 mm, 1.8 μm) column was examined for relative correction factors for 5 components at different column temperatures (35, 38, 40 ℃ C.). The results show that the components are very reproducible with respect to the correction factor, see Table 6.
TABLE 6 relative correction factors measured at different column temperatures
2.5.5 investigation of the flow rates of the various mobile phases in laboratory 1, using an Agilent 1290 high performance liquid chromatography system,
UHPLC AQ-C18(100 mm. times.2.1 mm, 1.8 μm) column was examined for different mobile phase flow rates (0.25, 0.30, 0.35 mL. min.)-1) Relative correction factors for the next 5 components. The results show that the components are very reproducible with respect to the correction factor, see Table 7.
TABLE 7 relative correction factors measured at different flow rates
2.5.6 investigation of different detection wavelengths in laboratory 1, using an Agilent 1290 high performance liquid chromatography system,
UHPLC AQ-C18(100mm × 2.1mm, 1.8 μm) column was examined for relative correction factors for 5 components under small changes in wavelength (367nm, 368nm, 369 nm). The results show that the components are very reproducible with respect to the correction factor, see Table 8.
TABLE 8 relative correction factors measured at different wavelengths
2.5.7 examination of the proportions of the various mobile phases in laboratory 1, using an Agilent 1290 high performance liquid chromatography system,
UHPLC AQ-C18(100 mm. times.2.1 mm, 1.8 μm) column was examined for relative correction factors for 5 components under small changes in all flow phases (ratio-1, original ratio, ratio +1) in the liquid phase method. The results show that the components are highly reproducible with respect to the correction factor, as shown in Table 9.
TABLE 9 relative correction factors measured for different flow phase ratios
2.5.8 examination of the different acid water concentrations in laboratory 1, using an Agilent 1290 high performance liquid chromatography system,
UHPLC AQ-C18(100 mm. times.2.1 mm, 1.8 μm) column was examined for relative correction factors for 5 components at different acid water concentrations (0.2% acid water, 0.3% acid water, 0.4% acid water). The results show that the components are highly reproducible with respect to the correction factor, as shown in Table 10.
TABLE 10 relative correction factors measured for different acid water concentrations
2.6 location of chromatographic Peak of component to be measured
The reproducibility of the relative retention time and retention time difference between 5 components of Anoectochilus roxburghii in different brands of instruments and different brands of chromatographic columns is respectively examined, and the results are shown in Table 11. The relative retention value refers to the ratio of retention time between each component to be measured and the internal reference, and the calculation formula is that ras is tRa/tRAnd s. The result shows that the relative retention time value fluctuation among the components to be detected is small, and the RSDLess than 5 percent, therefore, under the condition of lacking a reference substance, the relative retention value is utilized to position the chromatographic peaks of the 5 detected components of the anoectochilus formosanus, and the positions of the target components can be determined more accurately and reasonably by combining the ultraviolet absorption characteristics of the chromatographic peaks according to the retention time of the internal reference substance.
TABLE 11 relative retention times measured under different instruments and chromatography columns
2.7 comparison of results between one-test-multiple-evaluation method and external standard method
About 0.5g of a goldthread root sample is respectively taken, precisely weighed, a test solution is prepared according to a method under the item '2.3', the determination is carried out according to chromatographic conditions under the item '2.1', the content of narcissus, rutin, quercetin, kaempferol and isorhamnetin is determined by adopting an external standard method and a one-test-multiple evaluation method, and the RSD values of the narcissus, rutin, quercetin, kaempferol and isorhamnetin are calculated, the result is shown in table 12, the result can be seen from the table, the RSD values calculated by an internal standard method and the one-test-multiple evaluation method of the content of each component of 36 batches of samples are less than 5 percent, the result shows that the two have no obvious difference, and the established one-test-multiple evaluation.
Discussion of 3
3.1 chromatographic Condition optimization
The experiment establishes an ultra-high performance liquid chromatography method for simultaneously measuring 5 flavonoid components in anoectochilus formosanus, and compares the separation effects of different mobile phases (methanol-water, acetonitrile-formic acid water and acetonitrile (containing formic acid) -formic acid water) on target components in the experiment, so that the separation degree of the acetonitrile (containing formic acid) -formic acid water system is better; and the influence of different formic acid concentrations on chromatographic peak tailing factors is investigated, and as a result, the acetonitrile (0.1% formic acid) -0.3% formic acid water gradient elution method is short in time and good in separation degree.
3.2 selection of measurement wavelength
The content measurement is carried out by adopting one-measurement multi-evaluation correction factor, and because the deviation is larger under different wavelengths, how to select proper wavelength is critical to reduce the measurement error. Through the analysis of a diode array detector, the maximum absorption wavelengths of rutin, narcissus, quercetin, kaempferol and isorhamnetin are 355nm, 354nm, 371nm, 368nm and 371nm respectively, 5 components are considered comprehensively, and the result shows that at the position of 368nm, the 5 components have large absorption, the chromatographic peak separation degree is more than 1.5, the chromatographic analysis requirement is met, and therefore 368nm is selected as the detection wavelength.
3.3 selection of internal reference
Narcissus is a flavonoid component with the highest content in anoectochilus formosanus, the total ratio of narcissus components in 5 flavones in samples of big round leaf, red clouds, tip leaf, small round leaf and wild anoectochilus formosanus is 68.45-78.27%, 67.52-81.24%, 73.68-85.84%, 55.81-77.28% and 57.08-86.55%, and the retention time is moderate, so that narcissus with relatively high content is selected as an internal reference substance, narcissus, rutin, quercetin, kaempferol and isorhamnetin are in a linear range, relative correction factors are 1.016, 0.4650, 0.4228 and 0.4539 respectively, the durability is good, the content measurement result of 36 batches of anoectochilus formosanus shows that the calculated values of 4 components have no significant difference from the measured values, and the content of four different anoectochilus formosanus is different. The research also considers the influence of micro-changes of different ultra-high performance chromatographs, chromatographic columns of different brands, different column temperatures and flow rates, different laboratories, detection wavelengths, mobile phase proportions and acid water proportions on relative correction factors, and the result shows that the reproducibility of the relative correction factors of rutin, quercetin, kaempferol, isorhamnetin and the reference substance narcissus is good. Therefore, the one-test-multiple-evaluation method which is established by the experiment and takes the narcissus as an internal reference meets the analysis requirement.
3.4 analysis of content
The external standard method and the one-test-multiple evaluation method are respectively adopted to compare the content of 5 flavonoid components in 36 batches of anoectochilus formosanus samples, and the RSD values of 2 methods are calculated, so that the calculated values of rutin, narcissus, quercetin, kaempferol and isorhamnetin in 36 batches of anoectochilus formosanus in the content range by the 2 methods have no significant difference, and the established one-test-multiple evaluation method can be used for determining the content of 5 flavonoid components in the anoectochilus formosanus.
The content measurement result shows that the content of 5 flavonoid components in anoectochilus formosanus with different phenotypes has difference. Firstly, applying DPS14.50 software to perform systematic clustering analysis on 5 main flavone components in 36 batches of anoectochilus roxburghii planted with different phenotypes, and performing data conversion: principal component conversion and clustering distance: chi-square distance and clustering method: the results of the Lijian square sum analysis are shown in figure 3, when the threshold value is more than 27 and less than 37, the four samples with different phenotypes, namely the tip leaf, the small round leaf, the big round leaf and the rosy pinus can be independently gathered into one, so that the qualitative distinction can be carried out on the samples of the anoectochilus formosanus planted with the four samples with different phenotypes, namely the tip leaf, the small round leaf, the big round leaf and the rosy pinus (except that the samples with the large round leaf and the rosy pinus of the batches S1 and S4 are gathered into one type, and the samples with the rosy pinus of the number S9 are gathered; the wild samples measured at this time are scattered in different phenotype samples and cannot be independently gathered into one type, and the reason may be related to the harvesting time of the wild samples, so that the content distribution is not uniform. Further, GraphPad Prism 6.01 software was used to statistically analyze the content of 5 flavonoids and the total amount of five flavonoids in four phenotypic samples, namely, round leaf, nepheline, sharp leaf and small leaf, and the results are shown in fig. 4.
Comparing the content of 5 main flavones in 4 phenotypes of Anoectochilus roxburghii, firstly, the content range of rutin in Anoectochilus roxburghii samples planted in different phenotypes is 66.25-623.7 mug-1Wherein the mean value of the contents of the small round leaves is the highest, and the second is the tip leaf which is larger than the first is the red clouds and the big round leaves; secondly, the content of narcissus components in 4 strains ranges from 924.1 to 1931 mu g-1The component with the highest mean value of 15 in 5 components, wherein the content of the tip leaf sample is the highest, the minor lobe and the major lobe are the lowest, and the red clouds are the lowest(ii) a The content of quercetin is slightly lower than that of the first two components, and the content range is 22.06-230.8 mug-1The tip leaves were relatively slightly lower than the other three phenotypic samples; the content of kaempferol and isorhamnetin in the 5 components is the lowest, and the content ranges are 10.11-156.9 mug.g-1And 16.98 to 152.5. mu.g.g-1The kaempferol content in the big round leaf sample is highest, the first time of the rosy clouds sample is the lowest, and the tip leaf sample and the small round leaf sample are the lowest, but the isorhamnetin content in the tip leaf sample is highest, the second time of the rosy clouds sample is the lowest, and the rosy clouds sample and the small round leaf are lower; finally, the total content of the flavone is 1207-3179 mug-1The total content of spirifera is highest, the minor lobe and the major lobe are inferior, and the rosy clouds are lowest. For wild samples, the growth time cannot be determined, and the growth environments are different, which may be important reasons that the content ranges of the components fluctuate greatly and the systematic clustering cannot become unique. In conclusion, the narcissus content in the planted anoectochilus formosanus is the highest, the content of quercetin, kaempferol and isorhamnetin is relatively low, the narcissus content and the total flavone content in the tip leaf sample are higher than those of other phenotypes, the tip leaf sample may have stronger expression capability on secondary metabolism of flavone components, and certain advantages may be achieved. Experimental results show that the method can effectively detect the effective components in the anoectochilus formosanus.
The one-test-multiple-evaluation method for simultaneously analyzing rutin, quercetin, kaempferol and isorhamnetin in the anoectochilus formosanus by taking narcissus glycoside as an internal reference substance is simple, convenient and reliable, and can provide a new technical means for quality evaluation of the anoectochilus formosanus.
In conclusion, the detection method can simultaneously detect different appearance phenotypes, and a one-test-multiple-evaluation method for simultaneously analyzing rutin, quercetin, kaempferol and isorhamnetin in the anoectochilus roxburghii by taking narcissus glycoside as an internal reference is simple, convenient and reliable, and can provide a new technology for quality evaluation of the anoectochilus roxburghii. The one-test-multiple-evaluation method and the external standard method are mutually verified, have no obvious difference and have reliable results.
Claims (10)
1. A method for detecting flavone components in anoectochilus formosanus is characterized by comprising the following steps: the method adopts high performance liquid chromatography for detection, and specifically comprises the following operation steps:
1) preparation of control solutions: dissolving flavone control product in methanol;
2) preparation of a test solution: sieving herba Clematidis Japonicae powder, extracting with methanol, centrifuging, and collecting supernatant;
3) respectively sucking the reference solution and the test solution to be injected into a chromatograph, wherein the chromatographic conditions are as follows: a chromatographic column: c18A chromatographic column; the mobile phase is a mobile phase A which is 0.1 to 0.3 percent of formic acid acetonitrile and a mobile phase B which is 0.1 to 0.3 percent of formic acid water; the gradient elution procedure was as follows:
2. the method of claim 1, further comprising: the flavonoid reference substance in the step 1) is rutin, narcissus, quercetin, kaempferol and/or isorhamnetin.
3. The method of claim 1, further comprising: step 1) the reference substance solution contains 45-50 mug of rutin, 50-70 mug of narcissus, 25-30 mug of quercetin, 10-15 mug of kaempferol and/or 5-10 mug of isorhamnetin per 1 mL; preferably, each 1mL of the composition contains 49.44 μ g of rutin, 60.06 μ g of narcissus, 28.5 μ g of quercetin, 12.35 μ g of kaempferol and/or 7.884 μ g of isorhamnetin; the methanol is 50-100% methanol, preferably 50% methanol.
4. The method of claim 1, further comprising: step 3) said C18The chromatographic column is
UHPLC AQ-C18 chromatographic column with specification of 2.1mm × 100mm, 1.8 μm.
5. The method of claim 1, further comprising: the column temperature of the chromatographic conditions in the step 3) is 38 ℃ and the waveA length of 368nm, a flow rate of 0.3 mL/min-1The amount of the sample was 5. mu.L.
6. The method of claim 1, further comprising: step 2), sieving the anoectochilus formosanus powder by a No. 4 sieve, wherein the mass volume ratio of the anoectochilus formosanus powder to methanol is 1: (40-60) g/ml, preferably 1: 50 g/ml; the methanol is 80-100% methanol, preferably 80% methanol.
7. The method of claim 1, further comprising: the extraction in the step 2) is ultrasonic extraction, the extraction power is 350W, the frequency is 50Hz, and the time is 30 min.
8. A method for measuring the content of a flavone component in anoectochilus formosanus is characterized by comprising the following steps: the method is characterized in that a one-test and multi-evaluation method is adopted for detection, any one of rutin, narcissus, quercetin, kaempferol and isorhamnetin is used as an internal reference substance, the content of the internal reference substance in a sample to be detected is detected according to the method of any one of claims 1 to 7, and then the relative correction factors are used for calculating the content of the rest components.
9. The method of claim 8, further comprising: the relative correction factor is obtained according to the following method:
taking rutin, narcissus, quercetin, kaempferol and isorhamnetin as reference substances, detecting according to the method of any one of claims 1-5, and calculating according to the following formula:
fs/i＝fs/fi＝As×Ci/(Ai×Cs) (ii) a In the formula AsIs the area of the peak of the internal reference substance s, CsIs the concentration of internal reference substance s, AiArea of component i peak, CiComponent i concentration.
10. The method according to claim 8 or 9, wherein: narcissus glycoside is used as an internal reference, the relative correction factor of narcissus glycoside and rutin is 1.016, the relative correction factor of narcissus glycoside and quercetin is 0.4650, the relative correction factor of narcissus glycoside and kaempferol is 0.4228, and the relative correction factor of narcissus glycoside and isorhamnetin is 0.4539.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115266976A (en) * | 2022-07-25 | 2022-11-01 | 山东步长制药股份有限公司 | Ultra-high performance liquid phase detection method for Shenxian pulse-rising oral liquid |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2626049A1 (en) * | 2004-10-15 | 2006-04-20 | Biopharmacopae Design International Inc. | Methods and therapeutic compositions comprising plant extracts for the treatment of cancer |
| CN102768254A (en) * | 2012-06-19 | 2012-11-07 | 福建省金草生物集团有限公司 | Method for establishing Anoectochilus roxburghii fingerprint, and Anoectochilus roxburghii fingerprint and Anoectochilus formosanus Hay fingerprint |
| CN106706809A (en) * | 2016-12-26 | 2017-05-24 | 河北神威药业有限公司 | Method for simultaneously determining contents of multiple components in Shuxuening injection |
| CN106770828A (en) * | 2016-12-26 | 2017-05-31 | 河北神威药业有限公司 | It is a kind of while the method for determining multicomponent content in ginkgo biloba p.e and its preparation |
| CN108279278A (en) * | 2017-12-29 | 2018-07-13 | 广州白云山和记黄埔中药有限公司 | A kind of method and its application of separating flavone constituents |
| CN109908266A (en) * | 2019-04-24 | 2019-06-21 | 华侨大学 | A kind of preparation method of roxburgh anoectochilus terminal bud granule |
-
2019
- 2019-07-18 CN CN201910653665.5A patent/CN112240914A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2626049A1 (en) * | 2004-10-15 | 2006-04-20 | Biopharmacopae Design International Inc. | Methods and therapeutic compositions comprising plant extracts for the treatment of cancer |
| CN102768254A (en) * | 2012-06-19 | 2012-11-07 | 福建省金草生物集团有限公司 | Method for establishing Anoectochilus roxburghii fingerprint, and Anoectochilus roxburghii fingerprint and Anoectochilus formosanus Hay fingerprint |
| CN106706809A (en) * | 2016-12-26 | 2017-05-24 | 河北神威药业有限公司 | Method for simultaneously determining contents of multiple components in Shuxuening injection |
| CN106770828A (en) * | 2016-12-26 | 2017-05-31 | 河北神威药业有限公司 | It is a kind of while the method for determining multicomponent content in ginkgo biloba p.e and its preparation |
| CN108279278A (en) * | 2017-12-29 | 2018-07-13 | 广州白云山和记黄埔中药有限公司 | A kind of method and its application of separating flavone constituents |
| CN109908266A (en) * | 2019-04-24 | 2019-06-21 | 华侨大学 | A kind of preparation method of roxburgh anoectochilus terminal bud granule |
Non-Patent Citations (6)
| Title |
|---|
| 王澄林 等: "金线莲总黄酮的最佳超声提取工艺", 《福建中医药》 * |
| 甄汉深 等: "《药物分析化学》", 31 August 2017, 中国中医药出版社 * |
| 陈杨 等: "一测多评法在中药质量控制中的应用及关键问题", 《环球中医药》 * |
| 陈莹 等: "HPLC法同时测定金线莲中6种成分", 《中成药》 * |
| 马迟 等: "金线莲中六种黄酮成分的含量测定及其主成分、聚类分析", 《广州化工》 * |
| 黄庆佐 等: "HPLC法测定不同施肥模式栽培的金线莲中黄酮类成分", 《分析测试技术与仪器》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115266976A (en) * | 2022-07-25 | 2022-11-01 | 山东步长制药股份有限公司 | Ultra-high performance liquid phase detection method for Shenxian pulse-rising oral liquid |
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