A method for simultaneously determining 7 active components in northern atractylodes rhizome

Abstract

The application belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for determining seven active components in northern atractylodes medicinal materials. The method adjusts the concentrations of the control substances atractylone, atractylodin, beta-eudesmol, corynol, atractylodes lactone I, atractylodes lactone III and 5-hydroxymethyl furfural to 0.12 mg / mL, 0.21 mg / mL, 0.09 mg / mL, 0.13 mg / mL, 0.09 mg / mL, 0.06 mg / mL and 0.04 mg / mL respectively; and a single detection wavelength 203 nm is adopted. The method is simple, fast and quantitative, and can comprehensively, intuitively and more completely evaluate the efficacy of northern atractylodes.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for determining seven active ingredients in a medicinal material Atractylodes lancea. Background Art

[0002] Atractylodes lancea is the dried rhizome of Atractylodes lancea (Thunb) DC or Atractylodes chinensis (DC) Koidz, a plant of the genus Atractylodes in the Asteraceae family. It is included in the 2020 edition of the Chinese Pharmacopoeia as one of the major medicinal materials. Atractylodes chinensis, as one of the source plants of Atractylodes lancea, is a local medicinal material in Liaoning Province. Atractylodes chinensis has the effects of strengthening the spleen and dampness, dispelling wind and cold, and improving eyesight. It has multiple pharmacological effects such as antibacterial, anti-inflammatory, sedative, diuretic, liver protection, regulating gastrointestinal motility, cardiovascular protection and nervous system effects. Atractylodes chinensis is included in the pharmacopoeia, and the standard component of Atractylodes chinensis in the pharmacopoeia standard is atractylodesin, but atractylodesin alone may not fully represent the three effects of Atractylodes chinensis (strengthening the spleen, drying dampness, and dispelling wind and cold).

[0003] To integrate the active ingredients and efficacy of Atractylodes macrocephala, identify the effective active ingredients corresponding to its efficacy, and more comprehensively evaluate its medicinal quality, we reviewed its active ingredients. We found that the ingredients associated with its "drying dampness" efficacy include β-eudesmol, atractylodesone, atractylodes lactone I, atractylodes lactone III, and atractylol; the ingredients associated with its "spleen-tonifying" efficacy include atractylodesin and 5-hydroxymethylfurfural (5-HMF); and the ingredients associated with its "wind-dispelling and cold-relieving" efficacy include atractylodesin, atractylodes lactone, β-eudesmol, and atractylol. Our previous research suggests that these ingredients may be its active ingredients.

[0004] Currently, there are few quantitative analysis methods for the various active ingredients in Atractylodes lancea. The HPLC method is more commonly used to detect the active ingredients of Atractylodes lancea. Literature reports and national standards list 3-5 quality control ingredients for Atractylodes lancea, including atractylodesin. There is no literature on the determination of the 7 active ingredients related to the efficacy of Atractylodes lancea.

[0005] This study developed a method for simultaneously determining the contents of seven active ingredients in Atractylodes lancea, including atractylodisinone, atractylodin, β-cineole, atractylodisinol, atractylolide I, atractylolide III, and 5-hydroxymethylfurfural (5-HMF). Summary of the Invention

[0006] To address the above issues, the present invention provides a method for determining seven active ingredients in Atractylodes lancea. This method provides a simple and rapid quantitative analysis, and can provide a comprehensive, intuitive, and more complete evaluation of the efficacy of Atractylodes lancea.

[0007] A method for determining seven active ingredients in a medicinal material of Atractylodes macrocephala, the specific steps are as follows:

[0008] Step 1: Preparation of mixed reference solution: Accurately weigh appropriate amounts of atractylone, atractylodesin, β-eudesmol, atractylol, atractylodes lactone I, atractylodes lactone III, and 5-hydroxymethylfurfural (5-HMF) reference substances, add methanol, and prepare a mixed reference solution;

[0009] Step 2: Preparation of the test solution: Accurately weigh 0.5 g of Atractylodes lancea powder passed through an 80-mesh sieve and place it in a stoppered conical flask. Quickly add 20 mL of methanol and seal the conical flask with a sealing film. Wrap it tightly with tin foil and place it in an ultrasonic cleaner with constant water temperature. After ultrasonic extraction for 30 minutes, cool it to room temperature, weigh it again, make up the loss with methanol, shake it well, and filter it. Take the filtrate and filter it with a filter membrane to obtain the test solution.

[0010] Step 3: Separately take the mixed reference solution and the test solution and determine them by HPLC, wherein the detection wavelength is 203 nm, the run time is 10 minutes after injection, and the mobile phase is acetonitrile (A)-water (B) for gradient elution.

[0011] Furthermore, in step 1, the concentrations of atractylone, atractylodesin, β-eudesmol, atractylodesinol, atractylodes lactone I, atractylodes lactone III and 5-hydroxymethylfurfural (5-HMF) reference substances were 0.12 mg / mL, 0.21 mg / mL, 0.09 mg / mL, 0.13 mg / mL, 0.09 mg / mL, 0.06 mg / mL and 0.04 mg / mL, respectively.

[0012] Furthermore, in step 2, the water temperature is constant at 25±1°C, the ultrasonic wave is 240W, 40kHz, and the filter membrane is 0.45μm.

[0013] Furthermore, in step 3, the chromatographic conditions are: chromatographic column Dikma Diamonsil Plus C18 (250 mm × 4.6 mm, 5 μm); mobile phase gradient elution program: 0-10 min, 10%-15% A; 10-15 min, 15%-45% A; 15-45 min, 45%-90% A; 45-50 min, 90%-100% A; 50-55 min, 100%-10% A; flow rate 0.8 mL / min; column temperature 30°C; injection volume 10 μL.

[0014] Compared with the closest prior art, the present invention has the following advantages.

[0015] The method provided by the present invention can simultaneously determine multiple active ingredients of Atractylodes lancea, and can simultaneously determine seven ingredients including atractylodesone, atractylodesin, atractylodesinol, atractylodes lactone I, atractylodes lactone III, β-eudesmol, and 5-hydroxymethylfurfural (5-HMF), thereby improving the integrity of the quality evaluation of Atractylodes lancea.

[0016] Compared with multi-wavelength measurement methods, this method selects a single wavelength of 203 nm for measurement, which is simple and quick. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The chromatograms are of 7 mixed reference substances.

[0018] Figure 2 Examine the chromatogram for linearity.

[0019] Figure 3 This is the chromatogram of the precision experiment.

[0020] Figure 4 Chromatogram of the repeatability experiment.

[0021] Figure 5 This is the chromatogram of the stability experiment.

[0022] Figure 6 This is the chromatogram of the sample recovery experiment.

[0023] Figure 7 It is the chromatogram of the mixed reference substance at wavelengths of 203nm, 220nm, and 280nm.

[0024] Figure 8 It is the chromatogram of the mixed reference substance at wavelengths of 203nm, 210nm, and 215nm.

[0025] Figure 9 This is the chromatogram of the sample matching experiment.

[0026] Figure 10 Chromatogram of the sample extraction time experiment.

[0027] Figure 11 The chromatogram of the sample.

[0028] Among them, 1 atractylodesone; 2 atractylodesin; 3 β-eudesmol; 4 atractylodes alcohol; 5 atractylodes lactone 1; 6 atractylodes lactone 3; 7 5-hydroxymethylfurfural. DETAILED DESCRIPTION

[0029] To facilitate understanding of the present invention, the present invention is described more fully below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present invention. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.

[0030] Example

[0031] 1. Prepare test solution and reference solution

[0032] (1) Preparation of test solution:

[0033] Accurately weigh 0.5 g of Atractylodes lancea powder (pass through an 80-mesh sieve) and place it in a stoppered conical flask. Quickly add 20 ml of methanol and seal the conical flask with sealing film. Wrap tightly with tin foil and place in an ultrasonic cleaner (240 W, 40 kHz) at a constant water temperature of 25 ± 1 ° C. After ultrasonic extraction for 30 minutes, cool to room temperature, weigh again, make up the loss with methanol, shake well, and filter. Take the filtrate and filter it through a 0.45 μm filter membrane to obtain the test solution.

[0034] (2) Preparation of reference solution:

[0035] Atractylodesin, atractylodesin, β-eudesmol, atractylodesinol, atractylodesinol, atractylodesinol, atractylodesinol I, atractylodesinol III, and 5-hydroxymethylfurfural (5-HMF) were accurately weighed into a 1 mL volumetric flask, dissolved with methanol, and diluted to the mark. The concentrations of atractylodesin, β-eudesmol, atractylodesinol, atractylodesinol, atractylodesinol, atractylodesinol I, atractylodesinol III, and 5-hydroxymethylfurfural (5-HMF) were obtained, respectively. The concentrations were 2.38 mg / mL, 2.11 mg / mL, 0.94 mg / mL, 1.26 mg / mL, 0.93 mg / mL, 0.58 mg / mL, and 2.00 mg / mL. Accurately measure the above 7 reference substance stock solutions respectively, and use a pipette to draw 200 μL of atractylodesin, 100 μL each of atractylodesin, β-eudesin, atractylodes lactone I, and atractylodes lactone III, 50 μL of atractylodesone, and 20 μL of 5-hydroxymethylfurfural (5-HMF) into a 1 mL volumetric flask, make up to the scale, and shake well to obtain a mixed reference substance solution, in which the concentration of atractylodesone is 0.12 mg / mL, the concentration of atractylodesin is 0.21 mg / mL, the concentration of β-eudesin is 0.09 mg / mL, the concentration of atractylodes lactone is 0.13 mg / mL, the concentration of atractylodes lactone I is 0.09 mg / mL, the concentration of atractylodes lactone III is 0.06 mg / mL, and the concentration of 5-hydroxymethylfurfural (5-HMF) is 0.04 mg / mL.

[0036] 2. Measurement process

[0037] Take the mixed reference solution and the test solution respectively, and use HPLC to determine the mixed reference solution. Figure 1 The detection wavelength was 203 nm, and the chromatographic conditions were as follows: the chromatographic column was Dikma Diamonsil Plus C18 (250 mm × 4.6 mm, 5 μm); the column temperature was 30°C, the flow rate was 0.8 mL / min, the injection volume was 10 μL, the post-run was 10 min, and the mobile phase was acetonitrile (A)-water (B) for gradient elution. The elution program is shown in Table 1.

[0038] Table 1 Elution schedule

[0039]

[0040] 3. Methodological Investigation

[0041] (1) Linear relationship investigation

[0042] Prepare the aforementioned mixed reference solution, set the injection volume to 1, 2, 4, 6, 8, and 10 μL, and determine according to the above chromatographic conditions. Use the reference solution mass (mg) as the horizontal coordinate and the reference peak area (Y) as the vertical coordinate to draw a standard curve. The results show that the correlation coefficient r values ​​of the 7 reference substances, atractylodesone, atractylodesin, β-eudesmol, atractylodesinol, atractylodes lactone I, atractylodes lactone III, and 5-HMF, are all greater than 0.999, indicating a good linear relationship. The results are shown in Table 2 and the chromatogram is shown in Figure 2 .

[0043] Table 2 Regression equations and linear ranges of seven components of Atractylodes lancea

[0044]

[0045]

[0046] (2) Precision experiment

[0047] Accurately weigh 0.5 g of Atractylodes lancea powder, prepare the test solution according to the above test solution preparation method, inject 6 times continuously, record the peak areas of atractylodesone, atractylodesin, β-eudesmol, atractylodes alcohol, atractylodes lactone I, atractylodes lactone III, and 5-hydroxymethylfurfural, and calculate the contents of the 7 components and RSD. The measured RSD is between 1.3% and 2.9%. The results show that the instrument has good precision. The results are shown in Table 3 and the chromatogram is shown in Figure 3 .

[0048] Table 3 Precision test results

[0049]

[0050] (3) Repeatability experiment

[0051] Take 6 portions of the same batch of Atractylodes lancea, weigh them accurately, and prepare the test solutions according to the above test solution preparation method, and measure them separately. Record the peak areas of atractylodesone, atractylodesin, β-eudesmol, atractylodesinol, atractylodes lactone I, atractylodes lactone III, and 5-hydroxymethylfurfural, and calculate the contents and RSDs of the 7 components. The RSDs were measured to be between 0.9% and 2.8%, indicating good repeatability. The results are shown in Table 4, and the chromatograms are shown in Figure 4 .

[0052] Table 4 Repeatability test results

[0053]

[0054]

[0055] (4) Stability test

[0056] Accurately weigh 0.5 g of Atractylodes lancea powder, prepare the test solution according to the test solution preparation method, inject samples for analysis at 0, 2, 4, 8, 12, and 24 hours, record the peak areas of atractylodesone, atractylodesin, β-eudesmol, atractylodes alcohol, atractylodes lactone I, atractylodes lactone III, and 5-hydroxymethylfurfural, and calculate the contents of the seven components and RSD. The measured RSD is between 1.2% and 2.9%. The results show that the test solution has good stability for 24 hours. The results are shown in Table 5 and the chromatogram is shown in Figure 5 .

[0057] Table 5 Stability test results

[0058]

[0059] (5) Sample recovery experiment

[0060] Accurately weigh 6 portions of 0.5g of Atractylodes lancea powder, add 6 portions of prepared mixed reference solution, prepare the test solution according to the test solution preparation method, inject the samples separately, record the peak areas of atractylodesone, atractylodesin, β-eudesmol, atractylodes alcohol, atractylodes lactone I, atractylodes lactone III, and 5-hydroxymethylfurfural, and calculate the contents of the 7 components and RSD. The sample recovery was measured to be between 95.51% and 102.64%, and the RSD was between 1.4% and 3.8%. The results show that the method has good accuracy. The results are shown in Table 6 and the chromatogram is shown in Figure 6 .

[0061] Table 6 Sample recovery test results

[0062]

[0063]

[0064] 4. Determination of measurement wavelength

[0065] The prepared mixed reference solution was analyzed at wavelengths of 203 nm, 210 nm, 215 nm, 220 nm, and 280 nm. The results showed that 203 nm was the optimal wavelength, which could simultaneously analyze 7 components. Figure 7 、 8 .

[0066] 5. Sample preparation method screening

[0067] (1) Sample ratio

[0068] Accurately weigh 4 portions of 1.0g and 0.5g of Atractylodes lancea powder respectively, and place them in stoppered conical flasks according to different ratios of 1:20, 1:25, 1:40, 1:50, 0.5:10, 0.5:15, 0.5:20, and 0.5:25. Quickly add different amounts of methanol, seal the conical flask mouth with a sealing film, wrap it tightly with tin foil, and place it in an ultrasonic cleaner (240W, 40kHz) with a constant water temperature of 25±1°C. After ultrasonic extraction for 60 minutes, cool it to room temperature, weigh it again, make up the lost weight with methanol, shake it well, and filter it. Take the filtrate, filter it with a 0.45μm filter membrane, and obtain the test solution. The results are shown in Table 7, and the chromatogram is shown in Figure 9 .

[0069] Table 7 Sample ratio experimental results

[0070]

[0071] Combined with the sample chromatogram, a solid-liquid ratio of 0.5:20 was finally selected, which resulted in a higher content of extracted active ingredients and better separation in the chromatogram.

[0072] (2) Extraction time

[0073] Accurately weigh 4 portions of 0.5g of Atractylodes lancea powder, place them in a stoppered conical flask, quickly add 20mL of methanol, and seal the conical flask mouth with a sealing film. Wrap it tightly with tin foil, and place it in an ultrasonic cleaner (240W, 40kHz) with a constant water temperature of 25±1°C. Set the ultrasonic extraction time to 30min, 40min, 50min, and 60min, respectively, then cool to room temperature, weigh it again, make up the lost weight with methanol, shake well, and filter. Take the filtrate, filter it with a 0.45μm filter membrane, and obtain the test solution. Finally, 30min extraction time was selected, and the extracted active ingredient content was the highest. The results are shown in Table 8, and the chromatogram is shown in Figure 10 .

[0074] Table 8 Extraction time experimental results

[0075]

[0076]

[0077] 6. Determination of seven components in Atractylodes lancea samples

[0078] Octadecylsilane bonded silica gel was used as the filler; mobile phase: acetonitrile (A) - water (B). The mobile phase gradient elution program is shown in Table 1, and the flow rate is 0.8 ml / min. -1 ; Column temperature 30℃; Injection volume 10μl; Post-run 10min.

[0079] Preparation of mixed reference solution: Accurately weigh appropriate amounts of atractylone, atractylodesin, β-eudesmol, atractylodesinol, atractylodes lactone I, atractylodes lactone III and 5-hydroxymethylfurfural (5-HMF) reference substances and dissolve them in methanol to prepare mixed reference solutions with concentrations of 0.12 mg / mL, 0.21 mg / mL, 0.09 mg / mL, 0.13 mg / mL, 0.09 mg / mL, 0.06 mg / mL and 0.04 mg / mL, respectively.

[0080] Preparation of sample solution: Accurately weigh 0.5g of Atractylodes lancea powder that has passed through an 80-mesh sieve, place it in a stoppered conical flask, quickly add 20mL of methanol, and seal the conical flask mouth with a sealing film. Wrap it tightly with tin foil and place it in an ultrasonic cleaner (240W, 40kHz) with a constant water temperature of 25±1°C. After ultrasonic extraction for 30 minutes, cool it to room temperature, weigh it again, make up the lost weight with methanol, shake it well, and filter it. Take the filtrate, filter it with a 0.45μm filter membrane to obtain the sample solution, and determine it by HPLC. The results are shown in Table 9 and the color spectrum is shown in Figure 11 .

[0081] Table 9 Sample measurement results

[0082]

Claims

1. A method for determining 7 active ingredients in a Chinese herbal medicine, characterized in that: The specific steps are as follows: Step 1: Preparation of mixed reference solution: accurately weigh appropriate amounts of atractylone, atractylosin, β-eudesyl alcohol, atractylol, atractylolactone I, atractylolactone III and 5-hydroxymethylfurfural reference substances, add methanol to prepare a mixed reference solution; the concentrations of atractylone, atractylosin, β-eudesyl alcohol, atractylolactone I, atractylolactone III and 5-hydroxymethylfurfural reference substances are 0.12 mg / mL, 0.21 mg / mL, 0.09 mg / mL, 0.13 mg / mL, 0.09 mg / mL, 0.06 mg / mL and 0.04 mg / mL, respectively; Step 2: Preparation of the test solution: Accurately weigh 0.5 g of Atractylodes lancea powder passed through an 80-mesh sieve and place it in a stoppered conical flask. Quickly add 20 mL of methanol and seal the conical flask with a sealing film. Wrap it tightly with tin foil and place it in an ultrasonic cleaner with constant water temperature. After ultrasonic extraction, cool it to room temperature, weigh it again, make up the weight loss with methanol, shake it well, and filter it; take the filtrate and filter it with a filter membrane to obtain the test solution; the water temperature is constant at 25±1°C; the ultrasonic wave is 240W, 40kHz, and the ultrasonic time is 30min; the filter membrane is 0.45μm; Step 3: Separately, the mixed reference solution and the test solution were measured by HPLC, with a detection wavelength of 203 nm and a 10-min run after injection. The mobile phase A was acetonitrile and the mobile phase B was water for gradient elution. Chromatographic conditions: chromatographic column Dikma Diamonsil Plus C18, 250 mm × 4.6 mm, 5 μm; mobile phase gradient elution program: 0-10 min, 10%-15% A. 10~15min, 15%~45%A; 15~45min, 45%~90%A; 45~50min, 90%~100%A; 50~55min, 100%~10%A; flow rate 0.8mL / min; column temperature 30℃; injection volume 10μL.

Images