CN115308317A

CN115308317A - High performance liquid chromatography detection method for index components in diabetic wound healing extract

Info

Publication number: CN115308317A
Application number: CN202210722249.8A
Authority: CN
Inventors: 李福伦; 朱建勇; 年华; 张婧婷; 吴闽枫; 邓禹; 郭冬婕; 郭婉军; 王怡; 陆涵之
Original assignee: Yueyang Hospital of Integrated Traditional Chinese and Western Medicine Shanghai University of TCM
Current assignee: Yueyang Hospital of Integrated Traditional Chinese and Western Medicine Shanghai University of TCM
Priority date: 2022-06-24
Filing date: 2022-06-24
Publication date: 2022-11-08
Anticipated expiration: 2042-06-24
Also published as: CN115308317B

Abstract

The invention discloses a high performance liquid chromatography detection method for index components in a diabetic wound healing extract, wherein the index components in the diabetic wound healing extract are selected from dracorhodin perchlorate, aloe emodin, rhein, imperatorin, emodin, isoimperatorin, chrysophanol, physcion and tanshinone IIA. The high performance liquid chromatography detection method for the index components in the diabetic wound healing extract provided by the invention is accurate, reliable, simple, convenient and feasible, has strong specificity, establishes the quality standard of the extract, and can be used for quality control of the diabetic wound healing extract.

Description

High performance liquid chromatography detection method for index components in diabetic wound healing extract

Technical Field

The invention belongs to the technical field of detection of traditional Chinese medicine components, and particularly relates to a high performance liquid chromatography detection method for index components in a diabetic wound healing extract.

Background

The extract for healing the diabetic wound is an effective component obtained by reflux extraction of 95 percent of a wound healing formula, removal of impurities by petroleum ether and extraction of chloroform, and the component has the effect of promoting the healing of the diabetic wound. In order to effectively control the quality of the production process and ensure the curative effect of the product, a detection method capable of accurately detecting the active ingredients in the extract for healing the diabetic wound needs to be researched and designed.

Disclosure of Invention

The invention aims to provide a high performance liquid chromatography detection method for index components in a diabetic wound healing extract.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the first aspect of the invention provides a high performance liquid chromatography detection method for index components in a diabetic wound healing extract, wherein the index components in the diabetic wound healing extract are selected from dracorhodin perchlorate, aloe-emodin, rhein, imperatorin, emodin, isoimperatorin, chrysophanol, physcion and tanshinone IIA;

the preparation method of the extract for healing the diabetic wound comprises the following steps:

extracting the astragalus, the red sage root, the angelica dahurica, the lithospermum, the raw rhubarb, the calamine, the pearl powder and the dragon's blood twice by reflux according to the mass ratio of (2.5-3.5) to (2.3-2) to (2.5-3.5) to 1; adding 95% ethanol 5-8 times (preferably 6 times) of the total weight of the medicinal materials (including radix astragali, radix Salviae Miltiorrhizae, radix Angelicae Dahuricae, radix Arnebiae, radix Et rhizoma Rhei, galamina, margarita powder, and sanguis Draxonis) for reflux extraction for 0.5-2 h (preferably 1 h) for the first time, adding 95% ethanol 5-8 times (preferably 6 times) of the total weight of the medicinal materials for reflux extraction for 0.5-2 h (preferably 1 h) for the second time, mixing extractive solutions, concentrating under reduced pressure to dry, dissolving in water, and concentrating continuously until no alcohol smell exists to obtain suspension; adding petroleum ether for extraction for at least three times, wherein the mass ratio of the total amount of the petroleum ether to the suspension is 1:1, combining the petroleum ether extraction liquid and concentrating; continuously adding chloroform into the concentrated solution for extraction for at least three times, wherein the mass ratio of the total amount of the chloroform to the concentrated solution is 1:1, combining chloroform extraction liquid and concentrating; continuously adding ethyl acetate into the concentrated solution for extraction for at least three times, wherein the mass ratio of the total amount of the ethyl acetate to the concentrated solution is 1:1, combining the ethyl acetate extraction liquid and concentrating; continuously adding an aqueous solution of saturated n-butyl alcohol into the concentrated solution for extraction for at least three times, wherein the mass ratio of the total using amount of the aqueous solution of the saturated n-butyl alcohol to the concentrated solution is 1:1, and mixing n-butyl alcohol extraction solutions and concentrating (until no solvent comes out) to obtain an extract; dissolving 1mL of the extract in 10mL of methanol, shaking uniformly, filtering with a filter membrane (0.45 mu m), taking the subsequent filtrate as a test solution, and separating and purifying by high performance liquid chromatography to obtain the index components in the diabetic wound healing extract.

The conditions of the high performance liquid chromatography are as follows: a chromatographic column: agilent Zorbax eclipse XDB-C18,4.6 mm. Times.250mm, 5 μm, column number: 990967-902, mobile phase: phase a was acetonitrile, phase B was 0.2% aqueous formic acid, gradient elution procedure, flow rate: 1mL/min, detection wavelength: 254nm, 270nm, 440nm, column temperature: 30 ℃, sample size: 10 mu L, and balancing for 20min by taking the gradient of the mobile phase as an initial condition before sample injection.

The gradient elution procedure was as follows: 0 min-10min, 30% A +70% of B; 11-50min, 45% A +55% by weight B;51 min-60min, 50% A +50% B; 61-75min, 65% A +35% by weight B;76 min-90min, 65% of A +35% of B; 91-95min, 95% A +5% B;96 min-110min, 395% A +5% by weight B; phase A is acetonitrile and phase B is 0.2% formic acid solution in water.

The weight ratio of the astragalus root, the salvia miltiorrhiza, the angelica dahurica, the lithospermum, the raw rhubarb, the calamine, the pearl powder and the dragon's blood is (2.8-3.1), (1.4-1.8), (2.8-3.1) and (2.8-3.1) to (1).

Due to the adoption of the technical scheme, the invention has the following advantages and beneficial effects:

the high performance liquid chromatography detection method for the index components in the diabetic wound healing extract provided by the invention is accurate, reliable, simple, convenient and feasible, has strong specificity, establishes the quality standard of the extract, and can be used for quality control of the diabetic wound healing extract. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.

Drawings

FIG. 1 is a schematic diagram of an HPLC chromatogram of a diabetic wound healing formulation extract, wherein 1 is a mixed reference solution, 2 is dracorhodin perchlorate, 3 is aloe-emodin, 4 is rhein, 5 is imperatorin, 6 is emodin, 7 is isoimperatorin, 8 is chrysophanol, 9 is physcion, and 10 is tanshinone IIA.

FIG. 2 is a schematic diagram of an HPLC chromatogram of a test solution in a diabetic wound healing formulation extract.

FIG. 3 is a schematic diagram of an HPLC chromatogram of a solution of a negative test sample in a diabetic wound healing formula extract.

FIG. 4 is a graph showing the standard curve of dracorhodin perchlorate.

FIG. 5 is a schematic diagram of a standard curve for aloe-emodin.

Figure 6 is a schematic representation of a rhein standard curve.

Fig. 7 is a schematic diagram of a imperatorin standard curve.

FIG. 8 is a schematic diagram of an emodin standard curve.

Fig. 9 is a schematic diagram of the iso-imperatorin standard curve.

Fig. 10 is a schematic diagram of a chrysophanol standard curve.

FIG. 11 is a schematic diagram of a physcion standard curve.

Fig. 12 is a schematic diagram of a tanshinone IIA standard curve.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

Example 1

1 laboratory apparatus

An Agilent 1100 series high performance liquid chromatograph (comprising a DAD detector, an autosampler, an online degasser, a quaternary pump and a column oven); one-tenth-ten-thousandth electronic balance (priceless international trade (shanghai) ltd ES225 SM-DR); the ultrasonic instrument (Shanghai jumping into medical optical instrument factory CQ-250-DST type).

2 Experimental reagent

TABLE 1 reagent information

3 preparation of the solution

3.1 preparation of control solutions

(1) Dracorhodin perchlorate: precisely weighing and placing 0.02576g of dracorhodin perchlorate in a 10mL volumetric flask, adding methanol for ultrasonic dissolution, and diluting to scale to obtain dracorhodin perchlorate reference solution (2.58 mg/mL).

(2) Aloe-emodin: accurately weighing 5363 g of aloe-emodin 0.01857g, placing in a 10mL volumetric flask, adding methanol, ultrasonically dissolving, and diluting to scale to obtain aloe-emodin reference solution (1.857 mg/mL).

(3) Rhein: weighing 0.02258g rhein accurately, placing in a 10mL volumetric flask, adding methanol, performing ultrasonic treatment to dissolve, and diluting to scale to obtain rhein reference solution (2.26 mg/mL).

(4) Imperatorin: accurately weighing 0.01309g of imperatorin, placing in a 10mL volumetric flask, adding methanol, ultrasonically dissolving, and diluting to scale to obtain imperatorin reference solution (1.31 mg/mL).

(5) Emodin: weighing precisely 0.02042g of emodin in a 10mL volumetric flask, adding methanol, ultrasonically dissolving, and diluting to scale to obtain emodin reference solution (2.042 mg/mL).

(6) Isoimperatorin: precisely weighing 0.04418g of isoimperatorin, placing into a 10mL volumetric flask, adding methanol, ultrasonically dissolving, and diluting to scale to obtain isoimperatorin reference solution (4.42 mg/mL).

(7) Chrysophanol: weighing chrysophanol 0.02757mg precisely, placing into a 10mL volumetric flask, adding methanol, performing ultrasonic treatment to dissolve, and diluting to scale to obtain chrysophanol reference solution (2.76 mg/mL).

(8) Physcion: physcion 0.01592g is precisely weighed and placed in a 10mL volumetric flask, methanol is added for ultrasonic dissolution, and the physcion is diluted to scale, namely physcion reference solution (1.592 mg/mL).

(9) Tanshinone IIA: accurately weighing tanshinone IIA0.01628g, placing in a 10mL volumetric flask, adding methanol, ultrasonically treating to dissolve, and diluting to scale to obtain tanshinone IIA reference solution (1.628 mg/mL).

Mother liquor of a reference product: precisely measuring 1mL of the reference solution (wherein the emodin and the chrysophanol are precisely measured to be 1.5 mL) respectively, placing into a 10mL volumetric flask to scale, and using as reference mother liquor (respectively containing 0.258mg/mL of dracorhodin perchlorate, 0.1857mg/mL of aloe emodin, 0.226mg/mL of rhein, 0.131mg/mL of imperatorin, 0.306mg/mL of emodin, 0.221mg/mL of isoimperatorin, 0.414mg/mL of chrysophanol, 0.1592mg/mL of physcion, and 0.1628mg/mL of tanshinone IIA).

Control solution: gradually diluting the solution in sequence respectively, and fixing the volume to 10mL to obtain 7 concentration gradient mixed reference substance solutions, wherein the concentration ranges of the mixed reference substance solutions are dracorhodin perchlorate 2.58-258 mu g/mL, aloe-emodin 1.86-185.71 mu g/mL, rhein 2.26-226 mu g/mL, imperatorin 1.31-131 mu g/mL, emodin 3.06-306.43 mu g/mL, isoimperatorin 4.42-442 mu g/mL, chrysophanol 4.14-414 mu g/mL, physcion 1.59-159.29 mu g/mL and tanshinone IIA 1.63-162.86 mu g/mL respectively.

3.2 sample preparation

Weighing 30.14g of astragalus, 15.54g of salvia miltiorrhiza, 30.05g of angelica dahurica, 30.27g of lithospermum, 15.32g of raw rhubarb, 30.62g of calamine, 30.22g of pearl powder and 10.02g of dragon's blood in a round-bottom flask, and performing reflux extraction twice; adding 1500mL95% ethanol for reflux extraction for 1h for the first time, adding 1000mL95% ethanol for reflux extraction for 1h for the second time, mixing extractive solutions, and concentrating under reduced pressure to 400mL. Concentrating the extractive solution under reduced pressure, adding a large amount of water to dissolve, and concentrating continuously until no alcohol smell exists to obtain suspension. Adding petroleum ether for extraction for four times, wherein the mass ratio of the total amount of the petroleum ether to the suspension is 1:1, combining the petroleum ether extraction liquid and concentrating to 84mL; continuously adding chloroform into the concentrated solution for extraction for four times, wherein the mass ratio of the total amount of the chloroform to the concentrated solution is 1:1, combining chloroform extraction liquid and concentrating to 132mL; continuously adding ethyl acetate into the concentrated solution for extraction for four times, wherein the mass ratio of the total amount of the ethyl acetate to the concentrated solution is 1:1, combining ethyl acetate extraction solutions, and concentrating to 74mL; continuously adding an aqueous solution of saturated n-butyl alcohol into the concentrated solution for extraction for four times, wherein the mass ratio of the total using amount of the aqueous solution of the saturated n-butyl alcohol to the concentrated solution is 1:1, mixing n-butyl alcohol extract, and concentrating to 75mL (until no solvent exists) to obtain an extract; dissolving 1mL of the extract in a 10mL volumetric flask, adding methanol to a constant volume to a scale mark, shaking up, filtering with a 0.45 mu m filter membrane, and taking the subsequent filtrate as a test solution.

4 chromatographic conditions

A chromatographic column: agilent Zorbax eclipse XDB-C18 (4.6 mm. Times.250mm, 5 μm), column number: 990967-902, mobile phase: phase a was acetonitrile and phase B was 0.2% aqueous formic acid, the gradient elution procedure is shown in table 2, flow rates: 1mL/min, detection wavelength: 254nm, 270nm, 440nm, column temperature: 30 ℃, sample introduction: 10 mu L, and balancing for 20min by taking the gradient of the mobile phase as an initial condition before sample injection.

TABLE 2 gradient elution schedule

5 methodological investigation

5.1 System Adaptation Studies

Under the experimental conditions, the reference solution is injected to obtain a chromatogram shown in figures 1-3, and the adaptability of the system is calculated according to the chromatographic parameters.

As shown in fig. 1-3, fig. 1 is a schematic diagram of HPLC spectra of extracts of diabetic wound healing formula, wherein 1 is mixed reference solution, 2 is dracorhodin perchlorate, 3 is aloe-emodin, 4 is rhein, 5 is imperatorin, 6 is emodin, 7 is isoimperatorin, 8 is chrysophanol, 9 is physcion, and 10 is tanshinone IIA. FIG. 2 is a schematic diagram of an HPLC chromatogram of a test solution in a diabetic wound healing formulation extract. FIG. 3 is a schematic representation of the HPLC profile of the negative test solution in the diabetic wound healing formulation extract.

The high performance liquid chromatogram of the control solution is shown in figure 1, the high performance liquid chromatogram of the test solution is shown in figure 2, and the high performance liquid chromatogram of the negative control solution is shown in figure 3. As can be seen from the graphs 1-3, the separation coefficients of the 9 index components and the adjacent chromatographic peaks are all larger than 1.5, the components are not interfered with each other, the theoretical plate number meets the requirement, the extract is negative and free of interference, the specificity is good, and the method can be used for establishing the quality control method of the diabetic wound healing formula extract.

TABLE 3 results of system adaptability examination

5.2 Linear relationship examination

And (3) sequentially and continuously feeding mixed reference substance solutions with different concentration gradients, feeding samples according to the chromatographic conditions for determination, drawing a standard curve by taking the concentration of the reference substance as a horizontal coordinate (x) and the peak area value as a vertical coordinate (Y), and calculating a regression equation.

TABLE 4 Linear relationship between the peak area and concentration of dracorhodin perchlorate

The dracorhodin perchlorate linear regression equation is Y =33.1196x +72.5400 (r = 0.9996) and the concentration range is 12.90-258.00 mug/mL, as shown in FIG. 4, and FIG. 4 is a dracorhodin perchlorate standard curve diagram. The results show that: the linear relation between the peak area of the dracorhodin perchlorate and the concentration thereof is good.

TABLE 5 Linear relationship between Aloe-emodin peak area and concentration

The aloe-emodin linear regression equation is Y =72.7708x-192.4914 (r = 0.9998), the concentration range is 9.29-185.71 mug/mL, as shown in FIG. 5, and FIG. 5 is a graph of aloe-emodin standard curve. The results show that: the linear relation between the peak area of the aloe-emodin and the concentration thereof is good.

TABLE 6 Linear relationship between peak area and concentration of rhein

The rhein linear regression equation is Y =61.1810x-45.5310 (r = 0.9999), the concentration range is 11.30-226.00 mug/mL, as shown in FIG. 6, and FIG. 6 is a rhein standard curve diagram. The results show that: the linear relation between the peak area of rhein and the concentration of rhein is good.

TABLE 7 Linear relationship of imperatorin peak area to concentration

The imperatorin linear regression equation is Y =46.0345x +65.9926 (r = 0.9998), the concentration range is 6.55-131.00 mug/mL, as shown in FIG. 7, and FIG. 7 is a schematic diagram of the imperatorin standard curve. The results show that: the linear relation between the imperatorin peak area and the concentration thereof is good.

TABLE 8 Linear relationship between emodin peak area and concentration

The emodin linear regression equation is Y =37.0220x +45.3357 (r = 0.9996), the concentration range is 15.32-306.43 mug/mL, as shown in FIG. 8, and FIG. 8 is a diagram of an emodin standard curve. The results show that: the linear relation between the peak area of the emodin and the concentration of the emodin is good.

TABLE 9 Linear relationship between Isoimperatorin peak area and concentration

The isoimperatorin linear regression equation is Y =42.1108x-4.6907 (r = 0.9999), the concentration range is 4.42-221.00 mug/mL, as shown in FIG. 9, and FIG. 9 is a schematic diagram of the isoimperatorin standard curve. The results show that: the linear relation between the isoimperatorin peak area and the concentration thereof is good.

TABLE 10 Linear relationship of chrysophanol peak area to concentration

The chrysophanol linear regression equation is Y =61.0807x +245.7570 (r = 0.9997), the concentration range is 20.7-414.00 mug/mL, as shown in FIG. 10, and FIG. 10 is a chrysophanol standard curve diagram. The results show that: the peak area of chrysophanol has good linear relation with the concentration thereof.

TABLE 11 Linear relationship between physcion peak area and concentration

The linear regression equation of physcion is Y =35.5278x +107.9925 (r = 0.9997), the concentration range is 7.96-159.29 mug/mL, as shown in FIG. 11, and FIG. 11 is a graph of physcion standard curve. The results show that: the linear relation between the peak area of physcion and the concentration of physcion is good.

TABLE 12 Linear relationship between peak area and concentration of tanshinone IIA

The tanshinone IIA linear regression equation is Y =56.8428x +9.1982 (r = 0.9999), the concentration range is 8.14-162.86 μ g/mL, as shown in fig. 12, and fig. 12 is a schematic diagram of a tanshinone IIA standard curve. The results show that: the linear relation between the peak area of tanshinone IIA and its concentration is good.

5.3 precision test

And respectively taking the reference substance solution with each concentration in the standard curve, continuously injecting samples for 3 times within one day, and continuously injecting samples for 3 days with three concentrations of low, medium and high respectively, calculating the RSD of the peak area, and performing intra-day precision and inter-day precision investigation.

TABLE 13 results of precision within and between days

In the table: 1 is dracorhodin perchlorate, 2 is aloe-emodin, 3 is rhein, 4 is imperatorin, 5 is emodin, 6 is isoimperatorin, 7 is chrysophanol, 8 is physcion, and 9 is tanshinone IIA.

In summary, the following steps: the daily and daytime precision RSD of the nine reference substance solutions with different concentrations of low, medium and high components are less than 5 percent, which shows that the precision of the method is good.

5.4 stability test

Preparing a sample solution to be detected according to the method of the item 3.2; the peak areas and retention times of the 9 main chemical components were measured at 0h,2h,4h,8h,12h, and 24h, respectively, and the average peak area values (A) and RSD% thereof were calculated for stability examination.

TABLE 14 Peak area stability results for the major chemical Components

In summary, the following steps: the peak areas RSD of the nine components in 24h are all less than 3%, which shows that the nine components have good stability in 24 h.

5.5 repeatability test

Preparing 6 parts of sample solution to be detected according to the method of the item 3.2; the content and retention time of the 9 main chemical components were determined. And calculating the peak area average value (A) and RSD% of the sample, and performing repeatability inspection.

TABLE 15 Peak area repeatability results for major chemical Components

In summary, the following steps: the peak areas RSD of the nine components are all less than 4%, which shows that the nine components have good repeatability under the measurement of the method.

5.6 inspection line and quantitative Limit investigation

The control solution of dracorhodin perchlorate, aloe emodin, rhein, imperatorin, emodin, isoimperatorin, chrysophanol, physcion and tanshinone IIA is diluted, the lowest detection limit is determined by a signal-to-noise ratio of 3:1, and the lowest quantitative limit is determined by a signal-to-noise ratio of 10.

TABLE 16 detection limit and quantitation limit results

5.7 sample recovery test

Weighing 15.07g of astragalus, 7.77g of salvia miltiorrhiza, 15.12g of angelica dahurica, 15.03g of lithospermum, 7.56g of raw rhubarb, 15.38g of calamine, 15.26g of pearl powder and 5.21g of dragon's blood, precisely weighing and adding 6 parts of mixed reference solution with the same concentration gradient, preparing a test solution according to the method under the item 3.2, measuring the content of 9 components in the test solution, and calculating the recovery rate and RSD of the test solution.

TABLE 17 sample recovery of dracorhodin perchlorate results

TABLE 18 results of sample recovery of aloe-emodin

TABLE 19 results of sample recovery of rhein

TABLE 20 results of recovery of imperatorin from sample application

TABLE 21 results of recovery of emodin from sample application

TABLE 22 results of sample recovery of isoimperatorin

TABLE 23 results of sample recovery of chrysophanol

TABLE 24 results of sample recovery of physcion

TABLE 25 sample recovery results for tanshinone IIA

6 sample assay

3 parts of raw medicinal materials in the same batch are taken to prepare a test solution according to the method under the item 3.2, the contents of 9 chemical components in the test solution are calculated according to a regression equation (wherein the dracorhodin content = dracorhodin perchlorate content/1.377), and the results are shown in a table 26.

TABLE 26 measurement of granulation promoting and blood stasis removing formula (mg/g)

7 analysis and discussion

7.1 selection of the Mobile phase

In the experiment, the elution capability, column pressure and other aspects of the flowing phase of four components of methanol-water, acetonitrile-0.1% phosphoric acid, acetonitrile-0.1% formic acid and acetonitrile-0.2% formic acid relative to a sample are examined. Compared with methanol, the acetonitrile and water phase mixture has lower column pressure, stronger elution capacity and better peak shape, and the acetonitrile is used as an organic phase for separating and analyzing chemical components in a sample more reasonably. 0.1% phosphoric acid, 0.1% formic acid, 0.2% formic acid are added into the aqueous phase respectively, and compared with phosphoric acid, the formic acid has stronger acidity, so that the separation degree between chemical components is larger and better, and in addition, the 0.2% formic acid has stronger separation capability and lower tailing degree than the 0.1% formic acid, so that the peak shape can be better improved. Since the column can tolerate a pH in the range of 2.0 to 9.0, the formic acid fraction is 0.2%.

7.2 selection of chromatography columns

The influence of chromatographic columns with different lengths and different filler types on the separation result of various chemical components in a test sample is investigated in the experiment. The type of the chromatographic column: agilent Zorbax eclipse XDB-C18 (4.6 mm. Times.250mm, 5 μm), agilent Zorbax eclipse XDB-C18 (4.6. Times.150mm, 5 μm), agilent Zorbax SB-C18 (4.6 mm. Times.250mm, 5 μm), and the columns were examined based on the degree of separation of the peaks to be measured, the shape of the peaks, and the like. Agilent Zorbax eclipse XDB-C18 (4.6 mm. Times.250mm, 5 μm) was found to separate the chemical components well and gave more satisfactory absorption peaks and retention times. Other chromatographic columns have the problems of overlapping absorption peaks, poor peak shape, poor separation degree and the like. Agilent Zorbax eclipse XDB-C18 (4.6 mm. Times.250mm, 5 μm) was therefore selected as the column for methodological investigations.

7.3 selection of mobile phase proportion and detection wavelength

The aspects of mobile phase proportion, column temperature, detection wavelength and the like are investigated in the experiment. With respect to the flow phase ratio problem, properly decreasing the organic phase ratio, and extending the retention time can increase the degree of separation between the absorption peaks. According to the reference of Chinese pharmacopoeia 2015 edition, satisfactory peak areas can be obtained at three wavelengths of 254nm, 270nm and 440nm, and full-wavelength scanning patterns at three detection wavelengths of 254nm, 270nm and 440nm are obtained through a DAD detector, so that the detection wavelength is further determined.

7.4 Final determination of HPLC conditions

An Agilent Zorbax eclipse XDB-C18 (4.6 mm. Times.250mm, 5 μm) column was used with acetonitrile and 0.2% aqueous formic acid as mobile phases, acetonitrile as phase A and 0.2% aqueous formic acid as phase B, gradient elution program Table 27, flow rates: 1mL/min, detection wavelength: 254nm, 270nm, 440nm, column temperature: 30 ℃, sample introduction: 10 mu L, and balancing for 20min by taking the gradient of the mobile phase as an initial condition before sample injection. Under the chromatographic condition, each chemical component is well separated, and the investigation result of the content determination methodology shows that the liquid phase condition is suitable for simultaneously determining a plurality of main chemical components in the chloroform part of the tissue regeneration promoting and blood stasis removing prescription.

TABLE 27 gradient elution schedule

8 small knot

On the basis of influence of system adaptability inspection chromatographic conditions on a content determination method of index components in the diabetic wound healing extract, a content determination method for simultaneously determining multiple chemical components in the diabetic wound healing extract by adopting an HPLC method is established, and system methodology verification of the method is carried out; the result shows that the chromatographic condition is suitable for simultaneously measuring a plurality of main chemical components in the diabetic wound healing extract.

The high performance liquid chromatography has wide application range, accurate quantitative analysis result, high separation efficiency, high analysis speed, high sensitivity, repeated use of chromatographic column, automatic operation and easy collection of effluent components, and is widely applied to the fields of traditional Chinese medicine component analysis and the like. The content determination method established in the part can be used for qualitative and quantitative analysis of main chemical components in the diabetic wound healing extract, and provides a certain scientific basis for comprehensively, effectively and accurately evaluating the quality of medicinal materials. Unknown components can be further analyzed, and the properties of the compounds, the molecular structure and the molecular weight are estimated through the chromatography-mass spectrometry, so that scientific basis is provided for analyzing the effective active components in the diabetic wound healing extract.

Comparative example 1

CN113952333A was used as comparative example.

The invention establishes an HPLC method with strong specificity to measure the content of the extract, adopts the HPLC method, and establishes a mixed control of 9 standard substance components through overall methodology investigation (linearity, precision, stability, repeatability, detection line and quantitative limit), and detects 9 components under the same wavelength, thereby controlling the quality of the extract.

Compared with the literature, the literature uses macroporous resin separation, forward silica gel column elution, LH-20 semi-preparative liquid column separation and purification and the like, the invention only adopts high performance liquid chromatography for content determination, and the specific high performance liquid chromatography conditions are as follows: a chromatographic column: agilent Zorbax eclipse XDB-C18 (4.6 mm'250mm,5 μm), column number: 990967-902, mobile phase: phase A is acetonitrile, phase B is 0.2% formic acid aqueous solution, and gradient elution is carried out at the flow rate: 1mL/min, detection wavelength: 254nm, 270nm, 440nm, column temperature: 30 ℃, sample introduction: 10 μ L. The invention adopts simple high performance liquid chromatography conditions to perform chromatographic separation on 9 substances, and establishes a simple, reliable and good-repeatability determination method.

Comparative example 2

CN114099495A was used as comparative example.

Although the present invention has been described with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the present invention.

Claims

1. A high performance liquid chromatography detection method for index components in a diabetic wound healing extract is characterized in that the index components in the diabetic wound healing extract are selected from dracorhodin perchlorate, aloe emodin, rhein, imperatorin, emodin, isoimperatorin, chrysophanol, physcion and tanshinone IIA;

extracting the astragalus, the red sage root, the angelica dahurica, the lithospermum, the raw rhubarb, the calamine, the pearl powder and the dragon's blood resin twice in a mass ratio of (2.5-3.5) to (1); adding 95% ethanol which is 5-8 times of the total mass of the medicinal materials for reflux extraction for 0.5-2 h for the first time, adding 95% ethanol which is 5-8 times of the total mass of the medicinal materials for reflux extraction for 0.5-2 h for the second time, combining the extracting solutions, concentrating under reduced pressure until the extracting solutions are dried, adding water for dissolution, and continuously concentrating until no alcohol smell exists to obtain a suspension; adding petroleum ether for extraction for at least three times, wherein the mass ratio of the total amount of the petroleum ether to the suspension is 1:1, combining the petroleum ether extraction liquid and concentrating; continuously adding chloroform into the concentrated solution for extraction for at least three times, wherein the mass ratio of the total amount of the chloroform to the concentrated solution is 1:1, combining chloroform extraction liquid and concentrating; continuously adding ethyl acetate into the concentrated solution for extraction for at least three times, wherein the mass ratio of the total amount of the ethyl acetate to the concentrated solution is 1:1, combining the ethyl acetate extraction liquid and concentrating; continuously adding an aqueous solution of saturated n-butyl alcohol into the concentrated solution for extraction for at least three times, wherein the mass ratio of the total using amount of the aqueous solution of saturated n-butyl alcohol to the concentrated solution is 1:1, mixing n-butyl alcohol extract, and concentrating to obtain an extract; dissolving 1mL of the extract in 10mL of methanol, shaking up, filtering with a filter membrane, taking a subsequent filtrate as a test solution, and separating and purifying by high performance liquid chromatography to obtain an index component in the diabetic wound healing extract;

the conditions of the high performance liquid chromatography are as follows: a chromatographic column: agilent Zorbax Eclipe XDB-C18,4.6 mm. Times.250mm, 5 μm, column number: 990967-902, mobile phase: phase a was acetonitrile, phase B was 0.2% aqueous formic acid, gradient elution procedure, flow rate: 1mL/min, detection wavelength: 254nm, 270nm, 440nm, column temperature: 30 ℃, sample introduction: 10 mu L, and balancing for 20min by taking the gradient of the mobile phase as an initial condition before sample injection.

2. The HPLC detection method for detecting an index component in a diabetic wound healing extract according to claim 1, wherein the gradient elution procedure is as follows: 0 min-10min, 30% A +70% of B; 11-50min, 45% A +55% B;51 min-60min, 50% A +50% B; 61-75min, 65% A +35% by weight B;76 min-90min, 65% of A +35% of B; 91-95min, 95% A +5% B;96 min-110min, 395% A +5% B; phase A is acetonitrile, phase B is 0.2% formic acid water solution.

3. The HPLC detection method for detecting the index components in the extract for diabetic wound healing according to claim 1, wherein the mass ratio of Astragalus membranaceus, salvia miltiorrhiza, angelica dahurica, lithospermum erythrorhizon, raw rhubarb, galamina, pearl powder and sanguis Draxonis is (2.8-3.1), (1.4-1.8), (2.8-3.1) and (2.8-3.1).