CN112684059A - Method for detecting chemical components of rhododendron pseudobulb based on UPLC-ESI-MS - Google Patents
Method for detecting chemical components of rhododendron pseudobulb based on UPLC-ESI-MS Download PDFInfo
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- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 title claims abstract description 12
- 241000245165 Rhododendron ponticum Species 0.000 title 1
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Abstract
The invention provides a method for detecting chemical components in a rhododendron pseudobulb extract based on UPLC-ESI-MS, which comprises the following steps: the method comprises the following steps of artificial cultivation conditions of rhododendron, extraction of chemical components of pseudo bulbs, preparation of a standard substance, preparation of a sample to be detected, liquid phase detection conditions, mass spectrum detection conditions, ion source setting and chemical component qualitative analysis. The method for detecting the chemical components of the rhododendron can quickly, accurately and sensitively detect 9 chemical components of colchicine, quercetin, adenosine, rutin, emodin, rhein, physcion, myristic acid and oleanolic acid in the pseudobulb extract of the rhododendron, overcomes the defects of few detection methods of the chemical components of the rhododendron, undefined active ingredients, difficult evaluation of the internal quality of medicinal materials and the like, and provides guidance for the internal quality evaluation of the rhododendron medicinal materials.
Description
Technical Field
The invention relates to a method for detecting chemical components of a Dulan pseudobulb based on UPLC-ESI-MS, belonging to the field of medicinal plant analysis and detection.
Background
Rhododendron (D.Don) Makino, a perennial rare medicinal plant of Rhododendron in Orchidaceae, is used as a medicine with its dry pseudobulb, and was originally recorded in Bencao Shiyi (Yi Xue Yi). Chinese pharmacopoeia records: the Pseudobulbus Cremastrae Seu pleiones is dry pseudobulb of Rhododendron simsii, Pleione bulbocodioides and Pleione yunnanensis, and has effects of clearing heat and toxic substances, moistening lung, relieving cough, promoting blood circulation, removing blood stasis, relieving swelling, and resolving hard mass, and can be used for treating gastric cancer, hepatocarcinoma, breast cancer, metrocarcinoma, etc. by oral administration, and for treating skin scald, snake and insect bite, etc. by external application. The best quality of the rhododendron is selected from the three, and the research is the most.
The detection method related to the chemical components of the rhododendron is few, the phenanthrene compounds and the bibenzyl compounds are more in number, lower in content and unstable in structure in the iphigenia indica medicinal materials, are easy to change under the conditions of illumination and the like, are not suitable for being used as index components, and the inherent quality of the rhododendron medicinal materials is difficult to evaluate comprehensively due to the fact that effective components are not clear enough. The Dactylohin A and the mileanine in the Indian iphigenia medicinal material are simultaneously measured by RP-HPLC (reverse phase high performance liquid chromatography) of the lapacho and the like, and are detected in the Pleione and the Yunnan Pleione, but are hardly detected in the Rhododendron; the chemical components of the edible tulip bulb produced in different areas are analyzed by a high performance liquid chromatography-diode array detector (HPLC-DAD), but three different sources of the edible tulip bulb cannot be distinguished, and the compounds corresponding to chromatographic peaks are not clear.
The effective components and detection method of the edible tulip medicinal material are not specified under the item of the edible tulip medicinal material in newly published pharmacopoeia of the people's republic of China (2020 edition, one part). Therefore, there is a need to further study the pharmacodynamic material basis and chemical composition of rhododendron.
Colchicine is reported to have the activities of resisting tumors, treating gout and the like; through research on an anti-tumor molecular mechanism, Zhongying and the like, the main component playing a role in treating tumors in a cancer-eliminating and detoxifying compound preparation (comprising oldenlandia diffusa, Indian iphigenia bulb and other traditional Chinese medicines) is quercetin, so that the quercetin is presumed to have a certain anti-tumor effect. At present, no report about a method for detecting chemical components such as colchicine and quercetin in pseudobulb of rhododendron is found. Because the pharmacodynamic substance basis of the rhododendron is not clear, the quality control means of the medicinal materials mainly depends on the traditional biopharmaceutical research. Therefore, the method for quickly, efficiently and accurately measuring the main chemical components in the rhododendron simsii medicinal material is established, the comprehensive control of the substance group is realized, a more sufficient basis is provided for the quality evaluation of the rhododendron simsii, and the method has important significance.
The invention aims to provide a method for qualitatively analyzing chemical components in the rhododendron dry pseudo bulb.
Disclosure of Invention
In view of the above, the invention aims to provide a method for detecting chemical components in a rhododendron pseudobulb extract based on UPLC-ESI-MS, which overcomes the problems of difficulty in detecting chemical components, uncertain effective components and the like of the rhododendron in the prior art.
In order to achieve the above purpose, the invention provides an analytical method for detecting the chemical components of the pseudobulb of azalea based on UPLC-ESI-MS, which comprises the following steps: the method comprises the following steps of artificial cultivation conditions of azalea, extraction of chemical components of pseudo bulbs of azalea, preparation of a standard substance, preparation of a sample to be detected, liquid phase detection conditions, mass spectrum detection conditions, ion source setting and chemical component qualitative analysis. The method comprises the following specific steps:
(1) artificial cultivation condition for rhododendron
Cremastra appendiculata (D.Don) Makino, collected from the high slope of the Huaxi region of Guiyang city of Guizhou province, planted in the sunshade greenhouse of the Guizhou university test farm according to the plant spacing of 7cm, the culture medium is loose humus soil on the surface layer collected by broad leaf forest, the illumination intensity of the greenhouse is 180--2·s-1The relative humidity is 70-85%.
(2) Ericaria chemical component extraction
Taking healthy and strong rhododendron pseudobulb which has been cultivated for 3 years as an experimental material, cleaning, cutting into small pieces, drying at 60 ℃, crushing, and sieving with a 40-mesh sieve. Extracting with 1.0g pseudobulb powder with 70% and 55% ethanol under ultrasound for three times (each time for 20 min), mixing extractive solutions, and recovering ethanol until no alcohol smell is produced.
(3) Preparation of standards
Precisely weighing 9 standard substances of colchicine, physcion, rhein, emodin, quercetin, rutin, oleanolic acid, myristic acid and adenosine 0.01g respectively, adding methanol to constant volume to 50mL volumetric flask, and filtering with 0.22 μm microporous membrane.
(4) Preparation of samples to be tested
Adding the Rhododendron pseudobulb extract into chromatographic grade methanol, performing ultrasonic treatment to dissolve completely, diluting to 50mL, and introducing sample through 0.22 μm microporous membrane.
(5) Chromatographic detection conditions
And (3) taking 5 mu L of 9 standard substance mixed liquid in the step (3) and 50 mu L of the sample to be detected in the step (4), and detecting the chemical components of the rhododendron pseudobulb extract by respectively adopting a liquid chromatography-mass spectrometry system (Thermo Fisher Scientific in America) and a HESI-II ion source.
Liquid system is Dionex Ultimate 3000RSLC (HPG) (thermo Fisher scientific) and chromatographic column is thermo Fisher Hypersil GOLD aQ (100 x 2.1mm x 9 μm). The gradient elution solvent is acetonitrile solution containing 0.1% formic acid (organic phase A) and 0.1% methanol water (water phase B), and the elution time and the organic phase ratio are respectively as follows: 0-2min (20% A + 80% B); 3-6min (40% A + 60% B); 12-20min (90% A + 10% B); 20.1-25min (20% A + 80% B), flow rate of 0.2mL/min, column temperature of 40 ℃.
(6) Mass spectrometric detection conditions
The mass spectrum system is Thermo Scientific Q active Focus (Thermo Fisher Scientific), the scanning range of the whole mass spectrum is 100-200m/z, the resolution is 17500-70000, the maximum is 50-100ms, and the separation width is 1.5 m/z. Setting AGC and threshold to 1e6And 2e5And the dynamic emission reduction is 5 s.
(7) Ion source setup
The HESI-II ionization mode is electrospray ionization (ESI), and detection is performed in a positive ion mode and a negative ion mode respectively, and the parameters are set as follows: the atomization voltage is 3.0kV (+)/2.5kV (-), the capillary temperature is 320 ℃, the probe heating temperature is 350 ℃, the protective gas is 35arb, the AUX gas is 10arb, and the tail gas is 0 arb.
(8) Qualitative analysis of chemical composition
The comparison of the chemical components of the pseudobulb of azalea with the retention times and molecular weights of the 9 standards shows that the pseudobulb of three-year old azalea contains the following 9 components as shown in fig. 3: colchicine (1), quercetin (2), adenosine (3), rutin (4), emodin (5), rhein (6), physcion (7), myristic acid (8) and oleanolic acid (9). Wherein adenosine and colchicine are detected in a cation mode, and the rest 7 components are detected in an anion mode.
The invention has the beneficial effects that:
(1) overcomes the defects of few methods for detecting the chemical components of the rhododendron, undefined active ingredients, difficult evaluation of the internal quality of medicinal materials and the like.
(2) The invention adopts a liquid chromatography-mass spectrometry combined system (Thermo Fisher Scientific in America) and a HESI-II ion source, uses an acetonitrile solution containing 0.1 percent of formic acid (an organic phase A) and 0.1 percent of methanol (a water phase B) as a gradient elution solvent, and the elution time and the organic phase ratio are respectively as follows: 0-2min (20% A + 80% B); 3-6min (40% A + 60% B); 12-20min (90% A + 10% B); 20.1-25min (20% A + 80% B), flow rate of 0.2mL/min, column temperature of 40 ℃. The specific parameters of the above experiment are obtained by searching for two or more months, and if the experimental parameters such as the proportion of the mobile phase and the like change, the chemical components in the rhododendron pseudobulb cannot be detected.
(3) The method for detecting the chemical components of the rhododendron can quickly, accurately and sensitively detect 9 chemical components of colchicine, quercetin, adenosine, rutin, emodin, rhein, physcion, myristic acid and oleanolic acid of the pseudobulb extract of the rhododendron, and provides guidance for the internal quality evaluation of the rhododendron.
Drawings
FIG. 1 is an ESI ion flow chromatogram of 9 standards, ESI + mode: adenosine (1), colchicine (2); ESI-mode: rutin (3), quercetin (4), emodin (5), rhein (6), physcion (7), myristic acid (8) and oleanolic acid (9);
FIG. 2 is a graph showing the ion pattern of the chemical components ESI in the pseudobulb extract of Rhododendron simsii;
FIG. 3 is a liquid chromatogram of chemical components of a pseudobulb extract of Rhododendron simsii;
FIG. 4 is a mass spectrum of chemical components in the Ericatum pseudobulb extract.
Detailed Description
The present invention will be further described with reference to the following embodiments, which are fully understood and implemented by the following embodiments, for how to apply technical means to solve the technical problems and achieve the technical effects.
Example 1:
(1) artificial cultivation condition for rhododendron
Cremastra appendiculata (D.Don) Makino, collected from the high slope of the Huaxi region of Guiyang city of Guizhou province, planted in the sunshade greenhouse of the Guizhou university test farm according to the plant spacing of 7cm, the culture medium is loose humus soil on the surface layer collected by broad leaf forest, the illumination intensity of the greenhouse is 180--2·s-1The relative humidity is 70-85%.
(2) Ericaria chemical component extraction
Taking healthy and strong rhododendron pseudobulb which has been cultivated for 3 years as an experimental material, cleaning, cutting into small pieces, drying at 60 ℃, crushing, and sieving with a 40-mesh sieve. Extracting with 1.0g pseudobulb powder with 70% and 55% ethanol under ultrasound for three times (each time for 20 min), mixing extractive solutions, and recovering ethanol until no alcohol smell is produced.
(3) Preparation of standards
Precisely weighing 9 standard substances of colchicine, physcion, rhein, emodin, quercetin, rutin, oleanolic acid, myristic acid and adenosine 0.01g respectively, adding methanol to constant volume to 50mL volumetric flask, and filtering with 0.22 μm microporous membrane.
(4) Preparation of samples to be tested
Adding the Rhododendron pseudobulb extract into chromatographic grade methanol, performing ultrasonic treatment to dissolve completely, diluting to 50mL, and introducing sample through 0.22 μm microporous membrane.
(5) Chromatographic detection conditions
And (3) taking 5 mu L of 9 standard substance mixed liquid in the step (3) and 50 mu L of the sample to be detected in the step (4), and detecting the chemical components of the rhododendron pseudobulb extract by respectively adopting a liquid chromatography-mass spectrometry system (Thermo Fisher Scientific in America) and a HESI-II ion source.
Liquid system is Dionex Ultimate 3000RSLC (HPG) (thermo Fisher scientific) and chromatographic column is thermo Fisher Hypersil GOLD aQ (100 x 2.1mm x 9 μm). The gradient elution solvent is acetonitrile solution containing 0.1% formic acid (organic phase A) and 0.1% methanol water (water phase B), and the elution time and the organic phase ratio are respectively as follows: 0-2min (20% A + 80% B); 3-6min (40% A + 60% B); 12-20min (90% A + 10% B); 20.1-25min (20% A + 80% B); the flow rate was 0.2mL/min and the column temperature was 40 ℃.
(6) Mass spectrometric detection conditions
The mass spectrum system is Thermo Scientific Q active Focus (Thermo Fisher Scientific), the scanning range of the whole mass spectrum is 100-200m/z, the resolution is 17500-70000, the maximum is 50-100ms, and the separation width is 1.5 m/z. Setting AGC and threshold to 1e6And 2e5And the dynamic emission reduction is 5 s.
(7) Ion source setup
The HESI-II ionization mode is electrospray ionization (ESI), and detection is performed in a positive ion mode and a negative ion mode respectively, and the parameters are set as follows: the atomization voltage is 3.0kV (+)/2.5kV (-), the capillary temperature is 320 ℃, the probe heating temperature is 350 ℃, the protective gas is 35arb, the AUX gas is 10arb, and the tail gas is 0 arb.
(8) Qualitative analysis of chemical composition
The comparison of the chemical components of the pseudobulb of azalea with the retention times and molecular weights of the 9 standards shows that the pseudobulb of three-year old azalea contains the following 9 components as shown in fig. 3: colchicine (1), quercetin (2), adenosine (3), rutin (4), emodin (5), rhein (6), physcion (7), myristic acid (8) and oleanolic acid (9).
TABLE 1 analysis of 9 chemical components of Cremastra pseudobulb
| Compound (I) | Retention time (min) | ESI ionization mode | Pioneer ion (m/z) | Molecular formula | Name of |
| 1 | 5.69 | + | 400 | C22H25NO6 | Colchicine |
| 2 | 6.12 | - | 301 | C15H10O7 | Quercetin |
| 3 | 1.05 | + | 268 | C10H13N5O4 | Adenosine (I) |
| 4 | 2.15 | - | 609 | C14H28O2 | Rutin |
| 5 | 11.75 | - | 269 | C15H10O5 | Emodin |
| 6 | 13.37 | - | 253 | C15H8O6 | Rhein |
| 7 | 14.01 | - | 283 | C16H12O5 | |
| 8 | 15.37 | - | 228 | C14H28O2 | Myristic acid |
| 9 | 15.53 | - | 456 | C30H48O3 | Oleanolic acid |
The above-described embodiments are merely illustrative of the methods and principles of this patent and are not to be construed as limiting the invention. It will be appreciated by those skilled in the art that variations may be made in the methods and principles of the invention described herein without departing from the teachings or the skill or knowledge of the relevant art and yet still be within the scope of the invention as defined in the appended claims.
Claims (6)
1. A method for detecting chemical components of pseudobulb of rhododendron based on UPLC-ESI-MS is characterized by comprising the following steps:
(1) ericaria chemical component extraction
Drying pseudobulb of Cremastra Appendiculata, pulverizing, sieving, ultrasonic extracting with ethanol for three times, and mixing extractive solutions;
(2) preparation of standards
Precisely weighing 9 standard substances including colchicine, physcion, rhein, emodin, quercetin, rutin, oleanolic acid, myristic acid and adenosine, adding methanol to desired volume, and filtering with microporous membrane;
(3) preparation of samples to be tested
Adding the Rhododendron pseudobulb extract into chromatographic grade methanol, performing ultrasonic treatment, fixing volume, and introducing sample through microporous filter membrane;
(4) chromatographic detection conditions
Taking the 9 standard substance mixed solutions in the step (2) and the sample to be detected in the step (3), and respectively detecting the chemical components of the rhododendron pseudobulb extract by adopting a liquid chromatography-mass spectrometry combined system and a HESI-II ion source;
(5) qualitative analysis of chemical composition
According to the comparison of the chemical components of the pseudobulb of the azalea orchid with the retention time and the molecular weight of 9 standard substances, the pseudobulb of the three-year-old azalea orchid contains the following 9 components: colchicine, quercetin, adenosine, rutin, emodin, rhein, physcion, myristic acid and oleanolic acid, wherein adenosine and colchicine are detected in a cation mode, and the rest 7 components are detected in an anion mode.
2. The method for determining the chemical composition of pseudobulb of azalea based on UPLC-ESI-MS as claimed in claim 1, wherein the ethanol in step (1) is 70% ethanol and 55% ethanol, and the ultrasonic extraction is performed for 3 times, and each extraction time is 20 min.
3. The method for determining the chemical composition of the pseudobulb of azalea based on UPLC-ESI-MS as claimed in claim 1, wherein the diameter of the microporous filter membrane in step (2) and step (3) is 0.22 μm.
4. The method for determining the chemical composition of the pseudobulb of Rhododendron simsii based on UPLC-ESI-MS as claimed in claim 1, wherein the liquid system in step (4) is Dionex Ultimate 3000RSLC (HPG) (thermo Fisher scientific), and the chromatographic column is ThermoFisher Hypersil GOLD aQ (100 × 2.1mm 9 μm); the gradient elution solvent is acetonitrile solution containing 0.1% formic acid (organic phase A) and 0.1% methanol water (water phase B), and the elution time and the organic phase ratio are respectively as follows: 0-2min (20% A + 80% B); 3-6min (40% A + 60% B); 12-20min (90% A + 10% B); 20.1-25min (20% A + 80% B), flow rate of 0.2mL/min, column temperature of 40 ℃.
5. The method for determining the chemical composition of the pseudobulb of azalea based on UPLC-ESI-MS as claimed in claim 1, wherein the mass spectrometry system in step (4) is Thermo Scientific Q active Focus (Thermo Fisher Scientific), the scanning range of the whole mass spectrum is 100-1200m/z, the resolution is 17500-70000, the maximum is 50-100MS, the separation width is 1.5m/z, the AGC and the threshold are set to 1e6And 2e5And the dynamic emission reduction is 5 s.
6. The method for determining the chemical composition of the pseudobulb of azalea based on UPLC-ESI-MS as claimed in claim 1, wherein the HESI-II ionization mode in step (4) is ESI, which is an electrospray ion source, and is respectively detected in a positive and negative ion mode, and the parameters are set as follows: the atomization voltage is 3.0kV (+)/2.5kV (-), the capillary temperature is 320 ℃, the probe heating temperature is 350 ℃, the protective gas is 35arb, the AUX gas is 10arb, and the tail gas is 0 arb.
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