CN114689738A

CN114689738A - Method for simultaneously detecting multiple nucleoside components in antler

Info

Publication number: CN114689738A
Application number: CN202210064335.4A
Authority: CN
Inventors: 彭成; 谭玉柱; 谢晓芳; 高继海; 闫洪玲; 郭依田
Original assignee: Chengdu Xinfuyuan Traditional Chinese Medicine Decoction Pieces Co ltd; Chengdu University of Traditional Chinese Medicine
Current assignee: Chengdu Xinfuyuan Traditional Chinese Medicine Decoction Pieces Co ltd; Chengdu University of Traditional Chinese Medicine
Priority date: 2022-01-20
Filing date: 2022-01-20
Publication date: 2022-07-01

Abstract

The invention discloses a method for simultaneously detecting multiple nucleoside components in antler, which comprises the following steps: preparing a test solution: adding water into cornu Cervi powder, shaking, mixing, refrigerating, standing, ultrasonic extracting, centrifuging, collecting supernatant, and filtering; preparation of mixed control solution: accurately weighing proper amounts of cytidine, uracil, hypoxanthine, uridine, thymine, 2 ' -deoxyuridine, inosine, guanosine, 2 ' -deoxyinosine, 2 ' -deoxyguanosine, thymidine, guanine and xanthine respectively, dissolving and diluting to prepare a mixed reference solution; detecting the content of UPLC: and carrying out ultra-high performance liquid chromatography detection on the test solution and the mixed reference solution. The detection method disclosed by the invention is simple and convenient to operate, high in sensitivity, good in stability, repeatability and precision, can be used for comprehensively analyzing nucleoside components in the antler, and provides a basis for developing and utilizing commercial antler instead of medicinal antler.

Description

Method for simultaneously detecting multiple nucleoside components in antler

Technical Field

The invention belongs to the technical field of traditional Chinese medicine quality detection, and particularly relates to a method for simultaneously detecting multiple nucleoside components in deer horns.

Background

Antler is one of the traditional rare Chinese medicines in China, has a long history of medication, and is recorded in Shen nong's herbal Jing. Antler recorded in ' pharmacopoeia of the people's republic of China ' in 2020 edition is ossified horn of Cervus elaphus Linnaeus or Cervus Nippon Temminck of Cervidae or horn base dropped in the spring of the second year after antler sawing, and has the effects of warming kidney yang, strengthening tendons and bones, and relieving swelling and promoting blood circulation. Modern medicine considers that the antler contains rich nutrient substances including amino acids, polypeptides, nucleosides, polysaccharides, lipids, sex hormones, mineral elements and the like, and has wide pharmacological actions of resisting fatigue, resisting osteoporosis, inhibiting hyperplasia of mammary glands, resisting viruses, protecting gastric mucosa, resisting inflammation, easing pain and the like.

Since wild red deer and sika deer are rare, artificially bred deer is mostly matted, commercial deer antler is often replaced by horn of other deer family animals, including reindeer horn, camel horn, roe deer horn, buffalo horn, etc. The antler has similar pharmacological action and chemical components with the medicinal antler, has feasibility of replacing resources with the medicinal antler, but has no scientific evaluation index in the aspect of medicine. Nucleosides are one of the main chemical components of deer antler, not only constitute the basic components of DNA and RNA, but also have various biological activities, such as anti-inflammatory, antiviral, neuroprotective, hepatoprotective, etc. Therefore, the research on the nucleoside components in different deer horns has important significance for developing medicines or health care products taking the deer horns as raw materials.

In view of the above, the invention adopts UPLC-DAD technology to measure the content of 13 nucleosides in 30 deer antler samples of 2 medicinal deer antlers and common counterfeits thereof (moose, reindeer and roe deer), and carries out quality evaluation on the deer antlers by multivariate statistical methods such as principal component analysis, hierarchical clustering analysis and the like so as to provide basis for development and utilization of medicinal deer antler substitute resources, relieve the current situation of resource shortage in the current deer antler market, guide and promote the healthy development of deer antler resource utilization and protection, and provide demonstration for research of animal medicaments.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provides a method for simultaneously detecting multiple nucleoside components in antler, a device and application thereof.

The invention is implemented by the following technical scheme:

a method for simultaneously detecting multiple nucleoside components in antler comprises the following steps:

preparation of a test solution: cutting cornu Cervi into small pieces, pulverizing, and sieving to obtain cornu Cervi powder; adding distilled water into cornu Cervi powder, shaking, mixing, refrigerating, standing, ultrasonic extracting, centrifuging, collecting supernatant, and filtering to obtain test solution;

preparation of mixed control solution: accurately weighing proper amounts of cytidine, uracil, guanine, hypoxanthine, xanthine, uridine, thymine, 2 ' -deoxyuridine, inosine, guanosine, 2 ' -deoxyinosine, 2 ' -deoxyguanosine and thymidine respectively, and adding distilled water to dissolve to prepare a single reference mother solution, wherein 2mmoL/L NaOH solution is required to be added for assisting in dissolving the guanine and the xanthine; respectively taking a proper amount of each reference mother liquor, mixing, and diluting with distilled water to prepare a mixed reference solution;

detecting the content of the nucleoside by UPLC: respectively and precisely sucking 5 mu L of each prepared test solution and mixed reference solution, injecting the solutions into an ultra-high performance liquid chromatograph, and measuring to obtain the content of cytidine, uracil, hypoxanthine, uridine, thymine, 2 ' -deoxyuridine, inosine, guanosine, 2 ' -deoxyinosine, 2 ' -deoxyguanosine and thymidine.

Preferably, said deer horn is taken from elk, sika deer, roe deer, moose deer or reindeer.

Preferably, the specific preparation method of the test solution is as follows: taking 0.1g of antler powder sieved by a 40-mesh sieve, placing the antler powder into a 2mL centrifuge tube, precisely weighing, accurately adding 1mL of distilled water, uniformly mixing by oscillation, refrigerating and standing for 10-16h, and performing ultrasonic extraction at room temperature for 60min, wherein the ultrasonic frequency is 50 KHz; and (4) filtering the supernatant with a 0.45-micron microporous filter membrane to obtain a test solution.

Preferably, the addition amount of the NaOH solution is 0-200ul in the preparation process of the mixed control solution.

Preferably, the mixed control solution contains cytidine, uracil, guanine, hypoxanthine, xanthine, uridine, thymine, 2 ' -deoxyuridine, inosine, guanosine, 2 ' -deoxyinosine, 2 ' -deoxyguanosine, and thymidine in the following mass concentrations: 10.24. mu.g/mL, 10.05. mu.g/mL, 10.54. mu.g/mL, 10.07. mu.g/mL, 10.14. mu.g/mL, 10.07. mu.g/mL, 10.17. mu.g/mL, 10.11. mu.g/mL, 10.03. mu.g/mL, 10.02. mu.g/mL, 10.51. mu.g/mL, 10.01. mu.g/mL, 10.05. mu.g/mL.

Preferably, the chromatographic conditions in the step of detecting the content of the nucleosides by UPLC are: adopting an ACQUITY HSS T3100X 2.1mm, 1.7 μm chromatographic column; the column temperature is 30 ℃; the flow rate is 0.3 mL/min; the detection wavelength is 260 nm; gradient elution was performed with 0.06% formic acid aqueous solution as mobile phase a and acetonitrile as mobile phase B.

Preferably, the procedure of the gradient elution is: 0-3min, the mobile phase A is 100%, and the mobile phase B is 0%; 3-7min, changing the mobile phase A from 100% to 95% and the mobile phase B from 0% to 5%; 7-12min, changing the uniform speed of the mobile phase A from 95% to 85%, and changing the uniform speed of the mobile phase B from 5% to 15%; 12-13min, the mobile phase A is 85%, and the mobile phase B is 15%.

Compared with the prior art, the invention has the following beneficial effects: the ultrahigh performance liquid chromatography detection method can simultaneously detect the content of 13 nucleosides in deer horns, including cytidine, uracil, guanine, hypoxanthine, xanthine, uridine, thymine, 2 ' -deoxyuridine, inosine, guanosine, 2 ' -deoxyinosine, 2 ' -deoxyguanosine, and thymidine; the method is simple and convenient to operate, high in sensitivity and good in stability; the obtained chromatogram has stable baseline, good peak shape and high separation degree, can well reflect the real content of the 13 nucleosides in the sample so as to comprehensively analyze the nucleoside components of the antler, provide scientific basis for the quality control of the antler and provide reference for replacing medicinal antler with commercial antler to expand medicinal antler resources.

Drawings

FIG. 1 is a UPLC profile of 13 nucleoside mixed controls and five antler samples of the invention.

In the figure: 1. cytidine; 2. uracil; 3. guanine; 4. hypoxanthine; 5. xanthine; 6. uridine; 7. thymine; 8.2' -deoxyuridine; 9. inosine; 10. guanosine; 11.2' -deoxyinosine; 12.2' -deoxyguanosine; thymidine; A. a nucleoside control; B. a deer antler sample; C. a spotted deer horn sample; D. reindeer horn sample; E. a camel antler sample; F. roe deer horn sample; G. a blank solution.

Detailed Description

The invention is further illustrated by the following figures and examples, without however restricting the scope of the invention to these examples.

Example 1

1.1 optimization of the extraction Process

Preliminary experiments were carried out on the extraction solvent (pure water, 10% methanol, 20% methanol) and extraction time (60min, 120min) of the nucleoside components in deer antlers. The test solutions obtained by using different extraction methods were analyzed under the same chromatographic conditions, and the results showed that: the method comprises the following steps of (1) extracting a sample solution by pure water to obtain a sample solution, wherein the sample solution contains most nucleoside components, and has a better peak shape and a larger peak area; the results of the measurement of the extraction time of 60min and the measurement of 120min are basically the same, and even the content of the extraction time of 120min is lower; the acetonitrile precipitation method is adopted to remove protein in the aqueous extract of antler, and the result shows that the content of nucleoside ingredients is reduced probably due to the protein removal operation. Therefore, the extraction efficiency is comprehensively considered, the final extraction process is determined to be pure water extraction, the extraction time is 60min, and protein is not removed.

1.2 examination of chromatographic conditions

When the content of nucleoside components in antler is measured, an acquisition UPLC HSS T3 chromatographic column, an acquisition UPLC BEH Amide chromatographic column and an acquisition UPLC BEH C18 chromatographic column are selected from the chromatographic columns for preliminary experiments. The experimental results show that: the obtained chromatogram column has poor peak shape, and the obtained chromatogram column HSS T3 has better water phase resistance and better separation effect on nucleoside components than the obtained chromatogram column BEH C18. The mobile phase selects aqueous formic acid solution-acetonitrile, the proportion of formic acid and water is continuously adjusted, the separation conditions of 13 nucleosides under different proportions are compared, and finally aqueous formic acid solution-acetonitrile with the concentration of 0.06 percent is selected as the mobile phase. As the nucleoside components have ultraviolet absorption, the ultraviolet detection of the 13 nucleoside reference substances shows that the maximum absorption wavelength of the 13 components is near 260nm, so the detection wavelength is selected to be 260 nm.

1.3 methodological investigation of the detection of Deerhorn nucleoside Components by UPLC

1.3.1 instruments and reagents

A Ultimate 3000RSLC ultra high performance liquid chromatograph equipped with a DAD detector (seimer feishell science and technology ltd); electronic balances of BSA224S type (d 0.1mg) and BP211D type (d 0.01mg) were both manufactured by sedolis scientific instruments ltd, beijing; KQ5200E ultrasonic cleaner (kunshan ultrasonic instruments ltd); TG16-WS model desk-top high-speed centrifuge (Hunan instruments laboratory development, Hunan).

Uracil (BG16a045), guanine (BK19a006), hypoxanthine (BE30a110), xanthine (CJ08B108), uridine (G23a0097), thymine (CD16B125), inosine (CJ09B206), guanosine (CJ08B112), thymidine (BG16a097) are produced by kypt biotechnology limited, cytidine (DDYJ1014-32), 2 ' -deoxyguanosine (DDYJ1014-34), 2 ' -deoxyuridine (DDYJ1014-35), 2 ' -deoxyinosine (DDYJ1014-36) are provided by medall-while-era medical technology limited, and all controls have a purity greater than 98%. Acetonitrile was chromatographically pure, produced by Sigma-aldrich. Formic acid is chromatographically pure and is produced by metropolis chemical company Limited. Ultrapure water was prepared by a Milli-Q system (Millipore, USA).

30 deer horn samples are collected in places such as Jilin Changchun, Heilongjiang peony river and the like, and are derived from five varieties of ML (red deer), MHL (spotted deer), PZ (roe deer), TL (camel deer) and XL (reindeer) according to character identification of professor of Longfei assistant and graduator of Chengjiang university of Chinese medicine and university of Chengdu, wherein the 30 deer horn samples are respectively numbered as ML1-6, MHL1-6, PZ1-6, TL1-6 and XL 1-6.

1.3.2 methods and results

(1) Preparation of Mixed control solutions

Precisely weighing appropriate amount of each reference substance, placing in a 25mL volumetric flask, and dissolving with distilled water to obtain reference substance mother liquor, wherein appropriate amount of 2mmoL/L NaOH solution is added for guanine and xanthine. Then, an appropriate amount of each control solution is diluted with distilled water to obtain a mixed control solution containing 10.24 μ g/mL of cytidine, 10.05 μ g/mL of uracil, 10.54 μ g/mL of guanine, 10.07 μ g/mL of hypoxanthine, 10.14 μ g/mL of xanthine, 10.07 μ g/mL of uridine, 10.17 μ g/mL of thymine, 10.11 μ g/mL of 2 ' -deoxyuridine, 10.03 μ g/mL of inosine, 10.02 μ g/mL of guanosine, 10.51 μ g/mL of 2 ' -deoxyinosine, 10.01 μ g/mL of 2 ' -deoxyguanosine, and 10.05 μ g/mL of thymidine.

(2) Preparation of test solution

All antler samples are cut into small pieces by an electric saw, crushed and sieved by a 40-mesh sieve for later use. Weighing cornu Cervi powder about 0.10g, precisely weighing, adding 1mL distilled water into 2mL centrifuge tube, shaking, mixing, refrigerating, standing overnight, ultrasonic extracting at room temperature (50KHz) for 60min, centrifuging, and filtering the supernatant with 0.45 μm microporous membrane.

(3) Chromatographic conditions

The column was ACQUITYHSST3 (100X 2.1mm, 1.7 μm, Waters); column temperature: 30 ℃; flow rate: 0.3 mL/min; sample injection amount: 5 μ L. Detection wavelength of 260nm, mobile phase: aqueous formic acid (a) -acetonitrile (B) at a concentration of 0.06%, gradient elution: 0-3min, 0% B; 3-7min, 0% -5% B; 7-12min, 5% -15% B; 12-13min, 15% B. Under this chromatographic condition, chromatographic peaks of 13 nucleosides were well separated. The results are shown in FIG. 1.

(4) Investigation of Linear relationships

Sucking up the mixed control solution under the item (1),adding water to dilute into a series of control solutions with different concentrations, and injecting sample for analysis according to the chromatographic conditions under the item (3). And (3) performing linear regression on the concentration (X, mu g/mL) by using the peak area (Y), calculating a regression equation and a correlation coefficient, and determining the detection limit and the quantification limit when the reference solution is diluted until the signal-to-noise ratio obtained by detection is about 3: 1 and 10: 1. The results of the linear relationship and the detection and quantification limits, see table 1, show that: the 13 nucleoside components show better linear relation (r) in the measurement range²>0.9990) with detection and quantitation limits both within the feasible range.

TABLE 113 regression equation, detection limits, quantitation limits for nucleoside components

(5) Precision survey

A control solution (0.5. mu.g/mL) was sampled continuously for 6 needles, and the peak area of each component was measured and the RSD value was calculated to evaluate the precision. The RSD of the peak area of each nucleoside was 0.38% (cytidine), 0.22% (uracil), 0.77% (guanine), 1.15% (hypoxanthine), 1.07% (xanthine), 0.45% (uridine), 2.49% (thymine), 0.74% (2 ' -deoxyuridine), 0.48% (inosine), 0.42% (guanosine), 4.23% (2 ' -deoxyinosine), 0.68% (2 ' -deoxyguanosine), 0.66% (thymidine), respectively. Indicating that the precision of the instrument is good.

(6) Repeatability survey

Precisely weighing 6 parts of antler powder of the same batch, preparing a sample solution in parallel according to the method under the item (2), injecting sample under the chromatographic condition under the item (3), and evaluating the repeatability of the method according to the RSD value of each component content in the sample. RSDs for each nucleoside content were 2.08% (cytidine), 1.02% (uracil), 3.05% (guanine), 0.80% (hypoxanthine), 1.38% (xanthine), 1.18% (uridine), 4.73% (thymine), 2.99% (2 ' -deoxyuridine), 2.28% (inosine), 1.87% (guanosine), 1.52% (2 ' -deoxyinosine), 1.28% (2 ' -deoxyguanosine), and 1.09% (thymidine, respectively. The results show that: the method has good repeatability.

(7) Stability survey

Sampling the same sample solution for 0, 3, 6, 9, 12 and 24h respectively according to the chromatographic conditions under the item (3), and calculating the RSD value of the peak area of each component to evaluate the stability of the sample. The RSD of each nucleoside peak area was 3.78% (cytidine), 3.27% (uracil), 2.19% (guanine), 1.55% (hypoxanthine), 0.73% (xanthine), 2.25% (uridine), 4.33% (thymine), 3.52% (2 ' -deoxyuridine), 1.06% (inosine), 0.37% (guanosine), 2.58% (2 ' -deoxyinosine), 2.89% (2 ' -deoxyguanosine), 1.24% (thymidine), respectively. The results show that: the stability of the tested sample is good.

(8) Investigation of sample recovery

The same batch of deer antler samples of known content were taken and added to the control at 80%, 100% and 120%, respectively, in triplicate. The preparation was carried out according to the method under item "(2)", and then the measurement was carried out under the chromatographic conditions under item "(3)", and the sample recovery rate and the RSD value were calculated to verify the accuracy of the method. As a result: the average sample recovery of each nucleoside was 106.49% (cytidine), 100.77% (uracil), 99.48% (guanine), 94.19% (hypoxanthine), 86.25% (xanthine), 101.93% (uridine), 91.17% (thymine), 100.48% (2 ' -deoxyuridine), 101.71% (inosine), 92.05% (guanosine), 90.62% (2 ' -deoxyinosine), 100.43% (2 ' -deoxyguanosine), and 100.99% (thymidine). RSDs are 1.35% (cytidine), 0.79% (uracil), 2.20% (guanine), 0.82% (hypoxanthine), 3.20% (xanthine), 0.56% (uridine), 2.09% (thymine), 0.46% (2 ' -deoxyuridine), 0.96% (inosine), 1.27% (guanosine), 1.03% (2 ' -deoxyinosine), 1.32% (2 ' -deoxyguanosine), 0.52% (thymidine), respectively. Shows that: the method can well reflect the real content of the 13 nucleosides in the sample.

(9) Content measurement of 30 deer horn samples

Taking 30 batches of antler medicinal material samples, preparing a test solution according to the method under the item (2), recording peak areas, and calculating the content of each component. Each batch of medicinal materials is subjected to parallel measurement twice, and the average value is calculated to be used as the content of nucleoside in the medicinal material sample, and the result is shown in the following table 2. The analysis finds that: the reindeer of five deer horn species has the highest total nucleoside content of 87.53-180.50 mug/g, and the red deer and the moose have 44.58-141.18 mug/g and 38.78-156.37 mug/g respectively. Xanthine, cytidine, uracil and hypoxanthine are main nucleoside components in deer horns, the average content of xanthine in five varieties of deer horns is the highest (25.14-44.34 mu g/g), and cytidine, uracil and hypoxanthine are arranged in the second place; the contents of the four nucleosides in the reindeer horn are equivalent to or even superior to those of the medicinal deer horn. The different components in different varieties of deer antler are inosine, guanosine, 2 ' -deoxyguanosine and thymidine, and the average relative content of inosine, guanosine, 2 ' -deoxyinosine, 2 ' -deoxyguanosine and thymidine is lower. Thus, the nucleoside components of the five varieties of deer antlers are not clearly distinguished at the overall nucleoside level.

Further, the main component analysis, the hierarchical clustering analysis and the orthogonal partial least square discriminant analysis find that: taking nucleoside components as evaluation indexes, the domesticated antler and the medicinal antler have chemical equivalence and can be used as potential medicinal antler resources.

TABLE 230 nucleoside content in batch samples (. mu.g/g) (n ═ 2)

Measurement result below limit of quantitation

Claims

1. A method for simultaneously detecting multiple nucleoside components in antler is characterized in that: the method comprises the following steps:

detecting the content of the nucleoside by UPLC: precisely absorbing 5 mu L of each of the test solution and the mixed reference solution, injecting the solution into an ultra-high performance liquid chromatograph, and measuring to obtain the content of cytidine, uracil, hypoxanthine, uridine, thymine, 2 ' -deoxyuridine, inosine, guanosine, 2 ' -deoxyinosine, 2 ' -deoxyguanosine and thymidine.

2. The method for simultaneously detecting multiple nucleoside ingredients in antler according to claim 1, wherein: the cornu Cervi is selected from Cervus elaphus Linnaeus, Cervus nippon Temminck, roe deer, moose deer or reindeer.

3. The method for simultaneously detecting multiple nucleoside components in antler according to claim 1, characterized in that: the specific preparation method of the test solution comprises the following steps: taking 0.1g of antler powder sieved by a 40-mesh sieve, placing in a 2mL centrifuge tube, precisely weighing, accurately adding 1mL of distilled water, oscillating, mixing uniformly, refrigerating and standing for 10-16h, performing ultrasonic extraction at room temperature for 60min, wherein the ultrasonic frequency is 50 KHz; and (4) filtering the supernatant with a 0.45-micron microporous filter membrane to obtain a test solution.

4. The method for simultaneously detecting multiple nucleoside ingredients in antler according to claim 1, wherein: in the preparation process of the mixed reference solution, the addition amount of the NaOH solution is 0-200 ul.

5. The method for simultaneously detecting multiple nucleoside ingredients in antler according to claim 1, wherein: the mixed reference solution contains cytidine, uracil, guanine, hypoxanthine, xanthine, uridine, thymine, 2 ' -deoxyuridine, inosine, guanosine, 2 ' -deoxyinosine, 2 ' -deoxyguanosine, and thymidine in the mass concentrations respectively as follows: 10.24. mu.g/mL, 10.05. mu.g/mL, 10.54. mu.g/mL, 10.07. mu.g/mL, 10.14. mu.g/mL, 10.07. mu.g/mL, 10.17. mu.g/mL, 10.11. mu.g/mL, 10.03. mu.g/mL, 10.02. mu.g/mL, 10.51. mu.g/mL, 10.01. mu.g/mL, 10.05. mu.g/mL.

6. The method for simultaneously detecting multiple nucleoside ingredients in antler according to claim 1, wherein: the chromatographic conditions in the step of detecting the content of the nucleosides by UPLC are as follows: using an ACQUITY HSS T3100 × 2.1mm, 1.7 μm chromatographic column; the column temperature is 30 ℃; the flow rate is 0.3 mL/min; the detection wavelength is 260 nm; gradient elution was carried out using 0.06% formic acid aqueous solution as mobile phase a and acetonitrile as mobile phase B.

7. The method for simultaneously detecting multiple nucleoside ingredients in antler according to claim 5, wherein: the procedure for the gradient elution was: 0-3min, the mobile phase A is 100%, and the mobile phase B is 0%; 3-7min, changing the mobile phase A from 100% to 95% and the mobile phase B from 0% to 5%; 7-12min, changing the uniform speed of the mobile phase A from 95% to 85%, and changing the uniform speed of the mobile phase B from 5% to 15%; 12-13min, the mobile phase A is 85%, and the mobile phase B is 15%.