CN114778728B - Method for measuring content of curcumin compounds - Google Patents

Method for measuring content of curcumin compounds Download PDF

Info

Publication number
CN114778728B
CN114778728B CN202210419645.3A CN202210419645A CN114778728B CN 114778728 B CN114778728 B CN 114778728B CN 202210419645 A CN202210419645 A CN 202210419645A CN 114778728 B CN114778728 B CN 114778728B
Authority
CN
China
Prior art keywords
turmeric
curcumin
content
solution
methanol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210419645.3A
Other languages
Chinese (zh)
Other versions
CN114778728A (en
Inventor
张丽霞
罗祖良
郭芳
马小军
唐德英
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Medicinal Plant Development of CAMS and PUMC
Original Assignee
Institute of Medicinal Plant Development of CAMS and PUMC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Medicinal Plant Development of CAMS and PUMC filed Critical Institute of Medicinal Plant Development of CAMS and PUMC
Priority to CN202210419645.3A priority Critical patent/CN114778728B/en
Publication of CN114778728A publication Critical patent/CN114778728A/en
Application granted granted Critical
Publication of CN114778728B publication Critical patent/CN114778728B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers

Landscapes

  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

The invention discloses a method for measuring the content of curcumin compounds, which specifically comprises the following steps: (1) preparation of a standard solution: dissolving a standard substance in methanol to prepare a single stock solution or a mixed standard stock solution; (2) preparation of sample solution: cutting the rootstock of the sample into slices, drying, crushing and sieving, then adding aqueous methanol solution, shaking, ultrasonically extracting, centrifuging, separating, repeatedly extracting residues with the aqueous methanol solution and methanol for 1 time respectively, mixing the supernatant and the two extracts, fixing the volume, and filtering; (3) establishing a regression equation and measuring the content: and (3) measuring and establishing a regression equation of a standard substance by adopting a liquid chromatography-tandem mass spectrometry method, and then measuring and calculating the content of curcumin compounds in a sample solution. According to the invention, the LC-MS/MS is adopted to measure the content of curcumin compounds in turmeric varieties, so that the quality of turmeric medicinal materials and the application prospect thereof are evaluated, and a reference is provided for the quality control of turmeric medicinal materials.

Description

Method for measuring content of curcumin compounds
Technical Field
The invention relates to the technical field of medicine analysis, in particular to a method for measuring the content of curcumin compounds.
Background
The Curcuma rhizome is the dried rhizome of Curcuma longa Curcuma Longa L in Curcuma of Zingiberaceae, and has effects of removing blood stasis, promoting qi circulation, dredging channels and relieving pain. Can be used for treating chest and hypochondrium pain, chest pain, dysmenorrhea, amenorrhea, rheumatism, shoulder and arm pain, and traumatic injury. The active ingredients of turmeric mainly comprise curcumin compounds and volatile oil. Up to now, researchers have separated more than 30 natural curcumin compounds from turmeric, mainly curcumin, demethoxycurcumin, bisdemethoxycurcumin, dihydrocurcumin, tetrahydrocurcumin and the like, wherein the highest curcumin content is the main active ingredient. Pharmacological studies show that curcumin has the functions of resisting tumor, resisting inflammation, resisting oxidation, protecting nerves and the like. Turmeric is an important raw material for both traditional Chinese medicine and curry, and is also a popular dietary supplement for east and west. In recent decades, there has been increasing interest in the research of functional foods and dietary supplements for different diseases, with turmeric being one of the most studied functional foods. Turmeric has a long history as a spice in cooking in asian countries and other regions around the world, and is widely used in pasta, beverages, fruit wines, candies, pastries, cans, juices, cooked dishes, and the like.
At present, curcumin, demethoxycurcumin and bisdemethoxycurcumin are the most interesting in terms of turmeric quality evaluation. But dihydrocurcumin and tetrahydrocurcumin are hydrometabolites in turmeric and have similar pharmacological actions as curcumin. Researches report that the dihydrocurcumin and tetrahydrocurcumin have pharmacological effects of reducing blood sugar, reducing blood fat, resisting metastasis, resisting cancer, resisting depression, protecting nerves and the like, and the pharmacological activities of the dihydrocurcumin and tetrahydrocurcumin in the aspects of resisting oxidization, reducing blood sugar, reducing blood fat and the like are superior to those of the curcumin.
But the turmeric production areas are widely distributed, and the quality difference of turmeric cultivars in different areas is large.
Therefore, how to provide a method for determining the content of curcumin compounds to evaluate the quality of turmeric varieties with different sources is a problem that needs to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for determining the content of curcumin compounds, which is based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to establish a method for simultaneously determining the content of curcumin, demethoxycurcumin, bisdemethoxycurcumin, dihydrocurcumin and tetrahydrocurcumin in turmeric, and to perform content determination on three representative turmeric varieties (high-content turmeric, chuanghuang turmeric and Yun Jianghuang), and provides a reference for quality control of turmeric medicinal materials.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the method for measuring the content of the curcumin compounds specifically comprises the following steps:
(1) Preparation of standard solutions
Dissolving curcumin compound standard substance in methanol to prepare a single stock solution or mixed standard stock solution, and diluting to obtain a standard solution for later use;
(2) Preparation of sample solutions
Cutting rhizome of turmeric sample into slices, oven drying, pulverizing, and sieving to obtain turmeric powder; adding 80% methanol water solution into Curcuma rhizome powder, shaking, ultrasonic extracting, centrifuging, and separating to obtain supernatant and residue; repeatedly extracting the residue with 50% methanol water solution and 100% methanol for 1 time to obtain two extractive solutions; mixing the supernatant with the two extractive solutions, fixing volume, filtering to obtain sample solution, and storing for use;
(3) Establishment of regression equation and content measurement
And determining and establishing a regression equation of the curcumin compound standard substance by adopting a liquid chromatography-tandem mass spectrometry method, and then determining and calculating the curcumin compound content in the sample solution.
Further, in the step (1), the curcumin compound is at least one of curcumin, demethoxycurcumin, bisdeoxycurcumin, dihydrocurcumin and tetrahydrocurcumin.
The curcumin compound has the beneficial effects of resisting oxidation, resisting inflammation, scavenging free radicals, resisting cancer and the like.
Further, in the above step (1), the concentration of the single stock solution was 10. Mu.g.mL -1; the concentration of the mixed standard stock solution is 1 mug.mL -1; the standard solutions were at concentrations of 2ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL and 500ng/mL, respectively.
In the step (2), the turmeric sample is high-content turmeric collected from Yunnan part of the national academy of medical science, china, yunnan, and the Sichuan is at least one of turmeric and Yunnan turmeric.
The further technical scheme has the beneficial effect that the curcumin compound in the turmeric sample selected by the invention is higher in content.
Further, in the step (2), the temperature of the drying is 35 ℃; the number of the screen meshes of the screen is 60 meshes.
The further technical scheme has the beneficial effects that moisture in the rhizome slices of the turmeric sample can be removed rapidly through drying; the curcumin compounds are extracted by crushing and sieving.
Further, in the step (2), the mass-volume ratio of turmeric powder, 80% aqueous methanol solution, 50% aqueous methanol solution and 100% methanol is 0.2g:10mL:10mL:10mL.
The further technical scheme has the beneficial effects that the curcumin compounds in the turmeric powder can be quickly dissolved into the solution through methanol extraction, and the turmeric powder has the advantages of high efficiency, low cost and the like.
Further, in the step (2), the shaking time is 1min; the ultrasonic extraction time is 10min; the rotational speed of the centrifugation was 12000rpm and the time was 5min.
The further technical scheme has the beneficial effects that turmeric powder can be rapidly and uniformly dispersed in 80% methanol aqueous solution by shaking; the ultrasonic extraction can effectively shorten the extraction time on the one hand, and can protect the curcumin active ingredients from being damaged on the other hand, and the ultrasonic extraction equipment is convenient to maintain, simple to operate, low in running cost and obvious in economic benefit; by centrifugation, the supernatant and the residue can be rapidly separated.
Further, in the above step (2), the filtration membrane was a 0.22 μm microporous membrane.
The adoption of the further technical scheme has the beneficial effects that residues in the supernatant and the twice extracting solution can be further removed through filtering, and the accuracy and the sensitivity of the measuring method are ensured.
Further, in the step (3), the chromatographic conditions of the liquid chromatography-tandem mass spectrometry are as follows: chromatographic column: waters ACQUITY UPLC HSS T3 (2.1 mm. Times.100 mm,1.8 μm); mobile phase: phase A is 0.1% formic acid aqueous solution, and phase B is acetonitrile; gradient elution ：0.01-10min,45-58％B;10.00-10.50min,58-95％B;10.50-12.00min,95％B;12.00-12.10min,95-45％B;12.10-15.00min,45％B; flow rate 0.3mL/min; column temperature 40 ℃; the sample injection amount was 2. Mu.L.
Further, in the step (3), mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry are as follows: the ESI ion source is in a positive ion mode; the relevant parameters include a curtain gas, an atomizing gas, and an auxiliary gas, 30psi, 50psi, and 50psi, respectively; the spraying voltage is 5500V, and the ion source temperature is 550 ℃; the collision cell outlet voltage was 10V and the residence time was 50ms for quantitative analysis using a multi-reaction monitoring mode.
The technical scheme has the advantages that the liquid chromatography-tandem mass spectrometry adopts liquid chromatography as a separation system and mass spectrometry as a detection system, and the purified sample is separated and ionized in the liquid chromatography and mass spectrometry part and then a mass spectrogram is obtained through a detector; the method has the advantages of reflecting complementation of the advantages of the chromatograph and the mass spectrum, combining the high separation capacity of the chromatograph on the complex sample and the high selectivity of the mass spectrum, high sensitivity and being capable of providing relative molecular weight and structural information.
Compared with the prior art, the invention has the following beneficial effects:
1. The purpose is as follows: and (3) measuring the contents of 5 curcumin compounds (curcumin, demethoxycurcumin, bisdemethoxycurcumin, dihydrocurcumin and tetrahydrocurcumin) in three turmeric varieties (high-content turmeric, chuanjiu and Yun Jianghuang) by adopting a liquid chromatography-tandem mass spectrometry (LC-MS/MS) so as to evaluate the medicinal material quality of the high-content turmeric and the application prospect thereof.
2. The method comprises the following steps: sequentially extracting 5 curcumin compounds from three turmeric varieties by using 80% methanol, 50% methanol and methanol through ultrasonic, determining under a multi-reaction monitoring (MRM) mode through LC-MS/MS, carrying out qualitative and quantitative analysis on each component by two pairs of ions, and carrying out statistical analysis on content change among different varieties.
3. Results: the content and the component proportion of 5 curcumin compounds in the three turmeric varieties are greatly different, the content of the index component curcumin in the high-content turmeric is highest and reaches 3.23 percent, which is 2 times of the content of the Chuanhong turmeric and 15 times of the cloud turmeric.
4. Conclusion: the contents of five types of xanthophyll compounds in the high-content turmeric are higher than those of Sichuan turmeric and cloud turmeric, and the result is that the high-content turmeric is superior to the Sichuan turmeric and cloud turmeric in terms of accumulation of active ingredients of the curcumin.
Drawings
FIG. 1 is a total ion flow diagram of three variety samples and mixed standards (wherein A is the mixed standard, B is the high-content turmeric, C is Sichuan turmeric, D is cloud turmeric, peak 1 is tetrahydrocurcumin, peak 2 is dihydrocurcumin, peak 3 is curcumin, peak 4 is demethoxycurcumin, peak 5 is bisdemethoxycurcumin);
FIG. 2 shows the content of 5 curcuminoids in three turmeric varieties (wherein A is curcumin content, B is demethoxycurcumin content, C is bisdemethoxycurcumin content, D is dihydrocurcumin content, E is tetrahydrocurcumin content, and F is total content).
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the following examples, the concentration of each aqueous reagent solution was the volume concentration.
Example 1
The method for measuring the content of the curcumin compounds specifically comprises the following steps:
(1) Preparation of standard solutions
Precisely weighing curcumin standard substances respectively, dissolving in chromatographic grade methanol to prepare a single stock solution with the concentration of 10 mug.mL -1, diluting to obtain standard solutions with the concentrations of 2ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL and 500ng/mL respectively, and storing in a refrigerator at 4 ℃ for later use;
(2) Preparation of sample solutions
Sampling by five-point sampling method, cutting fresh rhizome of high-content Curcuma rhizome collected by Yunnan of China medical science sciences of Xishuangbanna of Yunnan province into slices, oven drying at 35deg.C, pulverizing, and sieving with 60 mesh sieve to obtain Curcuma rhizome powder; weighing Curcuma rhizome powder 0.2g, precisely weighing, placing in a 10mL centrifuge tube, adding 10mL 80% methanol water solution, shaking for 1min, ultrasonic extracting for 10min, centrifuging at 12000rpm for 5min, and separating to obtain supernatant and residue; repeatedly extracting the residue with 10mL of 50% methanol aqueous solution and 10mL of 100% methanol for 1 time to obtain two extractive solutions; mixing the supernatant with the two extracts, and adding 1mL of methanol to a constant volume of 20mL; finally, filtering through a microporous membrane with the thickness of 0.22 mu m to obtain a sample solution, and storing the sample solution in a sample injection bottle for sample injection analysis;
(3) Establishment of regression equation and content measurement
Determining and establishing a regression equation of a curcumin compound standard substance by adopting a liquid chromatography-tandem mass spectrometry method, and then determining and calculating the content of the curcumin compound in a sample solution;
Wherein the chromatographic conditions of the liquid chromatography-tandem mass spectrometry are as follows: chromatographic column: waters ACQUITY UPLC HSS T3 (2.1 mm. Times.100 mm,1.8 μm); mobile phase: phase A is 0.1% formic acid aqueous solution, and phase B is acetonitrile; gradient elution ：0.01-10min,45-58％B;10.00-10.50min,58-95％B;10.50-12.00min,95％B;12.00-12.10min,95-45％B;12.10-15.00min,45％B; flow rate 0.3mL/min; column temperature 40 ℃; the sample injection amount is 2 mu L;
the mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry were: the ESI ion source is in a positive ion mode; the relevant parameters include a curtain gas, an atomizing gas, and an auxiliary gas, 30psi, 50psi, and 50psi, respectively; the spraying voltage is 5500V, and the ion source temperature is 550 ℃; the collision cell outlet voltage was 10V and the residence time was 50ms for quantitative analysis using a multi-reaction monitoring mode.
Example 2
The method for measuring the content of the curcumin compounds specifically comprises the following steps:
(1) Preparation of standard solutions
Precisely weighing curcumin, demethoxycurcumin, bisdeoxycurcumin, dihydrocurcumin and tetrahydrocurcumin standard substances respectively, dissolving in chromatographic grade methanol to prepare a single stock solution with the concentration of 10 mug.mL -1, then mixing the stock solution to prepare a mixed standard stock solution with the concentration of 1 mug.mL -1, diluting to obtain standard solutions with the concentrations of 2ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL and 500ng/mL respectively, and storing in a refrigerator at 4 ℃ for later use;
(2) Preparation of sample solutions
Sampling by five-point sampling method, cutting rhizoma Curcumae Longae and Sichuan-bond which are high content and are collected by Yunnan part of the medical plant institute of China, xishuangbangna, yunnan, into slices, oven drying at 35deg.C, pulverizing, and sieving with 60 mesh sieve to obtain rhizoma Curcumae Longae powder; weighing Curcuma rhizome powder 0.2g, precisely weighing, placing in a 10mL centrifuge tube, adding 10mL 80% methanol water solution, shaking for 1min, ultrasonic extracting for 10min, centrifuging at 12000rpm for 5min, and separating to obtain supernatant and residue; repeatedly extracting the residue with 10mL of 50% methanol aqueous solution and 10mL of 100% methanol for 1 time to obtain two extractive solutions; mixing the supernatant with the two extracts, and adding 1mL of methanol to a constant volume of 20mL; finally, filtering through a microporous membrane with the thickness of 0.22 mu m to obtain a sample solution, and storing the sample solution in a sample injection bottle for sample injection analysis;
(3) Establishment of regression equation and content measurement
Determining and establishing a regression equation of a curcumin compound standard substance by adopting a liquid chromatography-tandem mass spectrometry method, and then determining and calculating the content of the curcumin compound in a sample solution;
Wherein the chromatographic conditions of the liquid chromatography-tandem mass spectrometry are as follows: chromatographic column: waters ACQUITY UPLC HSS T3 (2.1 mm. Times.100 mm,1.8 μm); mobile phase: phase A is 0.1% formic acid aqueous solution, and phase B is acetonitrile; gradient elution ：0.01-10min,45-58％B;10.00-10.50min,58-95％B;10.50-12.00min,95％B;12.00-12.10min,95-45％B;12.10-15.00min,45％B; flow rate 0.3mL/min; column temperature 40 ℃; the sample injection amount is 2 mu L;
the mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry were: the ESI ion source is in a positive ion mode; the relevant parameters include a curtain gas, an atomizing gas, and an auxiliary gas, 30psi, 50psi, and 50psi, respectively; the spraying voltage is 5500V, and the ion source temperature is 550 ℃; the collision cell outlet voltage was 10V and the residence time was 50ms for quantitative analysis using a multi-reaction monitoring mode.
Performance testing
1 Experimental materials
1.1 Sample for sample feeding
In 2016, the inventor collects a turmeric cultivar with orange-red rhizome in Guizhou Tongren in the investigation of ginger yellow germplasm, the curcumin content of the turmeric cultivar is obviously higher than other germplasm, the turmeric cultivar is temporarily named as 'high-content turmeric', and the turmeric cultivar is subjected to continuous third generation cultivation in Yunnan part of medicine orchard of medical plant institute of China academy of medical sciences, so that the turmeric cultivar is stable in agronomic property and quality property. The invention uses high-content turmeric collected by Yunnan part of medical plant institute of China medical science sciences, xishuangbangna, yunnan, szechuan, turmeric as a sample and Yunnan turmeric as a test material, and three turmeric varieties are identified as turmeric Curcuma longa L by Yunnan part Zhang Lixia of medical plant institute, china medical science sciences, unless otherwise specified, and are collectively called high-content turmeric (GJH), sichuan turmeric (CJH) and Yun Jianghuang (YJH).
1.2 Reagents
Curcumin, demethoxycurcumin, bisdeoxycurcumin, dihydrocurcumin and tetrahydrocurcumin standards were purchased from adzuki biotechnology limited and Shanghai source leaf biotechnology limited (HPLC purity greater than 98%). Methanol and acetonitrile (chromatographic grade) were purchased from FISHER SCIENTIFIC (Fair down, NJ, USA); formic acid was purchased from guangdong chemical industry, china; purified water (baby haha, hangzhou, china).
1.3 Instruments
The LC-MS/MS analysis system comprises Shimadzu LC-20A high performance liquid chromatography system (Shimadzu corporation, including LC-20A infusion pump, SIL-20AC autosampler, CTO-20A column incubator and DGU-20A3 degasser) and AB SCIEX5500 System (applied biosystems, usa, including Turbo V ion source and triple quadrupole linear ion hydrazine tandem mass analyzer; Software version 1.6 for synchronous control, data acquisition and analysis of LC-MS/MS system); AB-135-S electronic analytical balance, mettler Toledo group, switzerland; KQ-300 ultrasonic cleaner, kunshan ultrasonic instruments Inc.; LG16-B type Lei Boer high speed centrifuge, beijing Lei Boer centrifuge Co., ltd; targinVX-02 type multitube vortex oscillator, beijing pedal brocade technology Co., ltd; DHG-9030A type electrothermal blowing drying oven, shanghai-Heng science instruments Co., ltd.
2 Methods and results
2.1 Preparation of standard solutions
5 Curcumin compound standards were precisely weighed and dissolved in chromatographic grade methanol to prepare a single stock solution with a concentration of 10. Mu.g.mL -1. Then, the stock solution was mixed to prepare a mixed standard stock solution of 1. Mu.g.mL -1, and the standard solution was stored in a refrigerator at 4℃for use.
2.2 Preparation of sample solutions
Sampling by five-point sampling method, taking more than 6 strains of high-content Curcuma rhizome, curcuma rhizome and Curcuma xanthorrhiza, respectively, mixing fresh rhizome, cutting into uniform slices, oven drying at 35deg.C, pulverizing, and sieving with 60 mesh sieve. Weighing Curcuma rhizome powder 0.2g, precisely weighing, placing in a 10mL centrifuge tube, adding 10mL 80% methanol water solution, shaking for 1min, ultrasonic extracting for 10min, centrifuging at 12000rpm for 5min, and separating to obtain supernatant and residue; repeatedly extracting the residues with 10mL of 50% methanol aqueous solution and 10mL of 100% methanol for 1 time to obtain two extractive solutions; the supernatant and the two extracts are mixed, 1mL of methanol is added to fix the volume to 20mL. Finally, filtering through a microporous membrane with the thickness of 0.22 mu m to obtain a sample solution, and storing the sample solution in a sample injection bottle for sample injection analysis. To avoid photodegradation of curcumin, all manipulations were protected from light.
2.3 LC-MS/MS analysis conditions
Chromatographic conditions: chromatographic column: waters ACQUITY UPLC HSS T3 (2.1 mm. Times.100 mm,1.8 μm); mobile phase: phase A is 0.1% formic acid aqueous solution, and phase B is acetonitrile; gradient elution ：0.01-10min,45-58％B;10.00-10.50min,58-95％B;10.50-12.00min,95％B;12.00-12.10min,95-45％B;12.10-15.00min,45％B; flow rate 0.3mL/min; column temperature 40 ℃; the sample injection amount was 2. Mu.L.
Mass spectrometry conditions: the ESI ion source, the scanning mode is positive ion mode. Relevant parameters include a curtain gas (CUR), an atomizing gas (GS 1) and an auxiliary gas (GS 2) of 30psi, 50psi and 50psi, respectively. The spray voltage (IS) was 5500V and the ion source Temperature (TEM) was 550 ℃. The collision cell outlet voltage (CXP) was 10V and the residence time was 50ms for quantitative analysis using the Multiple Reaction Monitoring (MRM) mode. The spectral parameters of each target compound are shown in Table 1.
Table 1 mass spectrum acquisition parameters for each target compound
Note that: * Indicating quantitative ion pairs.
2.4 Methodological verification
2.4.1 Linearity and detection Limit
The mixed standard stock solution was diluted to give 2ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL and 500ng/mL standard solutions, respectively, and the samples were analyzed. Calibration curves were established with the peak area as a dependent variable (Y-axis) and the analyte concentrations as independent variables (X-axis), and linearity was evaluated by the correlation coefficient (r 2) of each calibration curve. The mixed standard solutions were diluted stepwise, and the amounts of each standard were defined as the detection Limit (LOD) and the quantification Limit (LOQ) at signal to noise ratios S/n=3 and S/n=10, respectively. Regression equations, linear ranges, detection limits and quantification limits for the five target compounds are shown in Table 2.
TABLE 2 regression equation, linear range, detection limit and quantitative limit for each target compound
As shown in Table 2, the target compounds have good linear relationship in the mass concentration range of 2.0-500 ng.mL -1, and the correlation coefficient (r 2) is more than 0.999, so that the quantitative analysis requirement is met.
2.4.2 Precision test
The same mixed control solution was continuously sampled 6 times, peak areas of curcumin, demethoxycurcumin, bisdeoxycurcumin, dihydrocurcumin and tetrahydrocurcumin were recorded and RSD (relative standard deviation) values of precision (precision in day and precision in daytime) were calculated, respectively. The results are shown in Table 3.
TABLE 3 results of precision experiments
As shown in Table 3, the RSD value of the daily precision is between 1.31% and 3.04%, and the RSD value of the daily precision is between 0.58% and 4.10%, which indicates that the instrument precision is good.
2.4.3 Recovery test
9 Parts of turmeric samples with known content in the same batch are taken, each part is about 0.1g, each part is precisely weighed, each three parts are respectively added into 80 percent (low concentration), 100 percent (medium concentration) and 120 percent (high concentration) standard substance solutions with the content of each component in the samples, a sample solution is prepared by adding a standard sample according to a method under 2.2, target analytes are measured according to chromatographic conditions under 2.3, and the sample adding recovery rate and RSD value are calculated. The results are shown in Table 4.
TABLE 4 recovery test results
As shown in Table 4, the recovery rate of the sample is between 84.36 and 108.09 percent, the RSD value of the recovery rate is between 1.39 and 5.16 percent, and the quantitative analysis requirement is met.
2.4.4 Repeatability test
6 Parts of turmeric samples of the same batch are taken in parallel, and turmeric sample solution prepared according to the method of 2.2 is prepared according to the following steps
The chromatographic conditions were measured under 2.3 entries, the peak area of each target compound was recorded and RSD values were calculated. The results are shown in Table 5.
TABLE 5 repeatability test results
Compounds of formula (I) Repeatability (RSD,%)
Tetrahydrocurcumin 0.44
Dihydrocurcumin 2.68
Curcumin 6.66
Demethoxycurcumin 5.70
Bisdemethoxycurcumin 2.61
As can be seen from Table 5, the RSD values of the reproducibility were between 0.44% and 6.66%, indicating good reproducibility of the process.
2.4.5 Stability test
One portion of turmeric sample solution prepared by the method of item 2.2 was taken and measured once at time points 0, 2,4, 8, 24, each according to chromatographic conditions of item 2.3, and RSD values were calculated. The results are shown in Table 6.
TABLE 6 stability test results
Compounds of formula (I) Stability (RSD,%)
Tetrahydrocurcumin 5.54
Dihydrocurcumin 3.43
Curcumin 6.10
Demethoxycurcumin 6.26
Bisdemethoxycurcumin 6.56
As can be seen from Table 6, the RSD values for the stability were between 3.43% and 6.56%, indicating that the test solutions were stable within 24 hours.
2.6 Determination results of 5 curcumin compounds in turmeric sample
(1) And (3) carrying out data acquisition on the high-content turmeric, turmeric and turmeric cloud samples by adopting a multi-reaction detection mode (MRM) of LC-QTRAP-MS/MS, and carrying out mass spectrum data processing by utilizing analysis 1.6.2 software to obtain a total ion flow diagram (TIC) of three variety samples and a mixed standard. The results are shown in FIG. 1.
As can be seen from FIG. 1, the peak separation degree of each spectrum of the target analytes such as tetrahydrocurcumin, dihydrocurcumin, curcumin, demethoxycurcumin, bisdemethoxycurcumin and the like is high, the peak is good, and the peak is correspondingly high.
(2) And (3) calculating the content of 5 curcumin compounds in three turmeric varieties by adopting an external standard method through comparison with the retention time of a standard product and the abundance ratio of parent ions, child ions and ions. The results are shown in FIG. 2 and Table 7.
TABLE 7 content of 5 curcuminoids in different turmeric varieties
Note that: the different lower case letters in the same column indicate significant differences between treatments, P <0.05.
As can be seen from fig. 2 and table 7, the three turmeric varieties have large differences in the contents of five curcumin compounds, namely tetrahydrocurcumin, dihydrocurcumin, curcumin, demethoxycurcumin and bisdemethoxycurcumin, and the 5 components are the highest in content of the high-content turmeric, and the total content of 5 curcumin in the high-content turmeric is about 2.4 times that of the Chuanjia turmeric and 28 times that of the yunnan turmeric. Except for the cloud turmeric, the content of curcumin in the high-content turmeric and the Sichuan turmeric accords with the regulation of not less than 1% of the content of curcumin in the Chinese pharmacopoeia, and the content of curcumin in the high-content turmeric reaches 3.23%, which is 2 times of that of the Sichuan turmeric and 15 times of that of the cloud turmeric. Except for the difference of the content, the content distribution ratio of the five compounds in three turmeric varieties is also inconsistent, and the content ratio of 5 curcumin compounds in high-content turmeric is tetrahydrocurcumin (1.76%), dihydrocurcumin (20.15%), curcumin (35.57%), demethoxycurcumin (17.73%), bisdemethoxycurcumin (24.78%); the five curcumin compounds in the turmeric comprise 2.05%, 39.77%, 28.18%, 7.50% and 8.64% respectively; the content ratio of 5 curcumin compounds in Curcuma xanthorrhiza is 6.25%, 21.87%, 56.25%, 9.38% and 6.25%, respectively. The curcumin in the cloud turmeric accounts for 56.25% of the total content of curcumin, while the content of the dihydrocurcumin in the Sichuan turmeric exceeds the content of curcumin, and the ratio is 39.77%. In a word, compared with Chuanjia turmeric and Yun Jianghuang, the curcumin compounds in the high-content turmeric have the advantage of larger content, and the proportion difference of 5 components in different varieties is larger.
Discussion 3
3.1 Optimization of extraction conditions
In order to obtain the optimal extraction conditions of the curcumin components, the extraction solvent (ethanol, methanol, 80% aqueous methanol solution and 50% aqueous methanol solution), the extraction solvent volume (10 mL, 20mL and 30 mL) and the extraction time (10 min, 20min and 30 min) were optimized, respectively. Comparing chromatographic peaks of each component under different extraction conditions, and finally determining the optimal extraction conditions of the turmeric effective components as follows: each sample was 0.2g, 10mL of 80% methanol was added, the ultrasonic extraction time was 10min, and the residue was repeatedly extracted 1 time with 10mL of 50% methanol and 10mL of pure methanol, respectively. Under these conditions, the standard recovery of 5 target compounds was within an acceptable range (84.36% -108.09%).
3.2 Mass Spectrometry Condition optimization
In order to obtain optimal mass spectrum conditions for curcumin component analysis, 100 ng.mL -1 standard solution is taken, and ionization monitoring is carried out under ESI + and ESI － modes respectively by peristaltic pump injection sample injection mode. The comparison result shows that 5 curcumin components have better response under the ESI + mode. And then, each substance is subjected to parent ion scanning in an ESI + mode, excimer ion [ M+H ] + is obtained in a primary full-scanning mass spectrogram, instrument parameters are adjusted to maximize the abundance of the parent ion, secondary mass spectrum scanning is further carried out on the aimed molecule ion, fragment ion information is obtained, two pairs of ions with stronger abundance and smaller interference are selected as qualitative and quantitative ions, parameter conditions such as declustering voltage (DP) and collision voltage (CE) are optimized to reach optimal sensitivity, finally, parameters such as atomizing gas, drying gas, ion source temperature and electrospray voltage are optimized, the detection limit of a target compound in the condition of optimizing mass spectrum is 0.2 ng-mL -1, and the established method has good sensitivity.
3.2 Analysis of measurement results
The sample measurement results show that the content of five curcumin compounds in three turmeric varieties is greatly different, and the proportions of the components are different. The content of curcumin compounds and the ratio of components can influence the appearance and the property of turmeric materials and the change of the drug effect. In the aspect of variety evaluation, the contents of five types of xanthophyll compounds in the high-content turmeric are higher than those of Sichuan turmeric and cloud turmeric, and the quality of the high-content turmeric can be considered to be better than those of Sichuan turmeric and cloud turmeric from the aspect of accumulation of active ingredients of the curcumin. Pharmacological activity researches on natural curcumin including curcumin, demethoxycurcumin, bisdemethoxycurcumin and the like show that the bisdemethoxycurcumin has higher activity than demethoxycurcumin and curcumin in the aspects of anti-tumor, anti-inflammatory, anti-oxidation and the like, and is the curcumin with the largest activity; the pharmacological activity of tetrahydrocurcumin in the aspects of antioxidation, blood sugar reduction, blood fat reduction and the like is superior to that of curcumin. Although all five curcumin compounds have similar pharmacological actions, the activities are inconsistent. This shows that the curcumin compounds in turmeric have different proportions, and the drug effects of the curcumin compounds are different, so that the curcumin compounds can be further verified through pharmacological experiments.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (2)

1. The method for measuring the content of the curcumin compounds is characterized by comprising the following steps of:
(1) Preparation of standard solutions
Dissolving curcumin compound standard substance in methanol to prepare a single stock solution or mixed standard stock solution, and diluting to obtain a standard solution for later use;
The curcumin compound is at least one of curcumin, demethoxycurcumin, bisdeoxycurcumin, dihydrocurcumin and tetrahydrocurcumin;
(2) Preparation of sample solutions
Cutting rhizome of turmeric sample into slices, oven drying, pulverizing, and sieving to obtain turmeric powder; adding 80% methanol water solution into Curcuma rhizome powder, shaking, ultrasonic extracting, centrifuging, and separating to obtain supernatant and residue; repeatedly extracting the residue with 50% methanol water solution and 100% methanol for 1 time to obtain two extractive solutions; mixing the supernatant with the two extractive solutions, fixing volume, filtering to obtain sample solution, and storing for use;
the temperature of the drying is 35 ℃; the number of the screened screen meshes is 60 meshes;
The mass volume ratio of the turmeric powder, the 80% methanol aqueous solution, the 50% methanol aqueous solution and the 100% methanol is 0.2g:10mL:10mL:10mL;
the shaking time is 1min; the ultrasonic extraction time is 10min; the rotational speed of the centrifugation is 12000rpm, and the time is 5min;
The filtering membrane is a 0.22 mu m microporous membrane;
(3) Establishment of regression equation and content measurement
Determining and establishing a regression equation of a curcumin compound standard substance by adopting a liquid chromatography-tandem mass spectrometry method, and then determining and calculating the content of the curcumin compound in a sample solution;
The chromatographic conditions of the liquid chromatography-tandem mass spectrometry are as follows: chromatographic column: waters ACQUITY UPLC HSS T3,2.1 mm. Times.100 mm,1.8 μm; mobile phase: phase A is 0.1% formic acid aqueous solution, and phase B is acetonitrile; gradient elution ：0.01-10min,45-58%B;10.00-10.50min,58-95%B;10.50-12.00min,95%B;12.00-12.10min,95-45%B;12.10-15.00min,45%B; flow rate 0.3mL/min; column temperature 40 ℃; the sample injection amount is 2 mu L;
The mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry are as follows: the ESI ion source is in a positive ion mode; the relevant parameters include a curtain gas, an atomizing gas, and an auxiliary gas, 30psi, 50psi, and 50psi, respectively; the spraying voltage is 5500V, and the ion source temperature is 550 ℃; the collision cell outlet voltage was 10V and the residence time was 50ms for quantitative analysis using a multi-reaction monitoring mode.
2. The method for measuring the content of curcumin compounds according to claim 1, wherein in the step (1), the concentration of the single stock solution is 10 μg/mL -1; the concentration of the mixed standard stock solution is 1 mug.mL -1; the standard solutions were at concentrations of 2ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL and 500ng/mL, respectively.
CN202210419645.3A 2022-04-20 2022-04-20 Method for measuring content of curcumin compounds Active CN114778728B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210419645.3A CN114778728B (en) 2022-04-20 2022-04-20 Method for measuring content of curcumin compounds

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210419645.3A CN114778728B (en) 2022-04-20 2022-04-20 Method for measuring content of curcumin compounds

Publications (2)

Publication Number Publication Date
CN114778728A CN114778728A (en) 2022-07-22
CN114778728B true CN114778728B (en) 2024-07-16

Family

ID=82431842

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210419645.3A Active CN114778728B (en) 2022-04-20 2022-04-20 Method for measuring content of curcumin compounds

Country Status (1)

Country Link
CN (1) CN114778728B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115469044A (en) * 2022-09-26 2022-12-13 中国农业科学院农业质量标准与检测技术研究所 Liquid chromatography-tandem mass spectrometry detection method for gingerol in ginger rhizome, overground stem and leaf
CN117607300A (en) * 2023-11-29 2024-02-27 中国农业科学院农业质量标准与检测技术研究所 Liquid chromatography-tandem mass spectrometry detection method for curcumin compounds in ginger and ginger products

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5068209B2 (en) * 2008-03-27 2012-11-07 学校法人金城学院 Separation and purification of curcumin from turmeric powder (turmeric powder) by high-speed counter-current chromatography
CN103163244B (en) * 2013-03-08 2014-12-10 南京中医药大学 Method for simultaneously detecting sesquiterpene and curcumin components
CN104914191A (en) * 2015-06-29 2015-09-16 四川新绿色药业科技发展股份有限公司 Method for simultaneously determining content of three effective components in turmeric products
CN106841422A (en) * 2016-12-30 2017-06-13 广州中大南沙科技创新产业园有限公司 A kind of UHPLC MS/MS analysis methods for determining rat curcumin PC
CN108459117A (en) * 2017-02-17 2018-08-28 中国中医科学院中药研究所 A method of turmeric secondary metabolite is measured using HPLC-DAD-MS
CN109444283A (en) * 2018-12-12 2019-03-08 广西中医药大学 The detection method of curcumin chemical compounds in a kind of osmanthus Radix Curcumae
US20220178936A1 (en) * 2019-04-14 2022-06-09 The Regents Of The University Of California A sensitive lc-ms assay to measure curcuminoids in complex biological samples

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
姜黄配方颗粒的高效液相指纹图谱研究;毕晓黎;胥爱丽;罗文汇;;时珍国医国药;20120520(第05期);第1104-1105页 *
微波辅助萃取－ 高效液相色谱－串联质谱法测定温郁金中3 个姜黄素类化合物;韩超;《药物分析杂志》;20131231;第33卷(第2期);第185-188页 *
高含型姜黄新品系的质量评价研究;郭芳;《医药卫生科技辑》;20230131;第E057-187页 *

Also Published As

Publication number Publication date
CN114778728A (en) 2022-07-22

Similar Documents

Publication Publication Date Title
CN114778728B (en) Method for measuring content of curcumin compounds
Stachniuk et al. Liquid chromatography-mass spectrometry in the analysis of pesticide residues in food
Rajski et al. Large pesticide multiresidue screening method by liquid chromatography-Orbitrap mass spectrometry in full scan mode applied to fruit and vegetables
Pan et al. Non-targeted metabolomic analysis of orange (Citrus sinensis [L.] Osbeck) wild type and bud mutant fruits by direct analysis in real-time and HPLC-electrospray mass spectrometry
Xu et al. Rapid determination of 1-deoxynojirimycin in Morus alba L. leaves by direct analysis in real time (DART) mass spectrometry
Kecojević et al. Evaluation of LC-MS/MS methodology for determination of 179 multi-class pesticides in cabbage and rice by modified QuEChERS extraction
Bongiorno et al. Bio‐phenols determination in olive oils: Recent mass spectrometry approaches
da Silva Antonio et al. Food pungency: the evolution of methods for capsaicinoid analysis
Avula et al. Characterization of steroidal saponins from Dioscorea villosa and D. cayenensis using ultrahigh performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry
Bae et al. Analysis of prenylflavonoids from aerial parts of Epimedium grandiflorum and dietary supplements using HPTLC, UHPLC-PDA and UHPLC-QToF along with chemometric tools to differentiate Epimedium species
Zhu et al. Comparative quality of the forms of decoction pieces evaluated by multidimensional chemical analysis and chemometrics: Poria cocos, a pilot study
CN109212054B (en) Method for determining tomatidine by high performance liquid chromatography-mass spectrometry
Shin et al. Metabolomic approach for discrimination of four-and six-year-old red ginseng (Panax ginseng) using UPLC-QToF-MS
CN114280206A (en) Method for detecting mycotoxin in spice and product thereof
CN117310032A (en) Method for detecting content of illegally added medicines for tonifying kidney and strengthening yang in traditional Chinese medicine
CN113325103A (en) Analysis method for simultaneously measuring gelsemium, gelsemine and gelsemine in hair
CN105954434B (en) A kind of detection method of phenols fragrance
CN106066367A (en) The method of nine kinds of Amadori compound concentrations in detection pastry food simultaneously
Song et al. Simultaneous quantification of six nonpolar ginsenosides in white ginseng by reverse-phase high-performance liquid chromatography coupled with integrated pulsed amperometric detection
Singh et al. Determination of Curcumin in Rat Plasma by Liquid-liquid Extraction using LC-MS/MS with Electrospray Ionization: Assay Development, Validation and Application to a Pharmacokinetic Study
Pan et al. Development and validation of a UPLC-MS/MS method for the simultaneous determination and detection of four neuritogenic compounds in different parts of Gentiana rigescens Franch using multiple reaction monitoring and precursor ion scanning
Yao et al. HILIC‐UPLC‐MS/MS combined with hierarchical clustering analysis to rapidly analyze and evaluate nucleobases and nucleosides in Ginkgo biloba leaves
CN112255327B (en) Method for detecting content of glufosinate-ammonium in dairy product
CN114563497A (en) Quantitative fingerprint detection method for bupleurum tenue capsule components
Zeng et al. Investigation on the Contents of Acrylamide in Baked and Fried foods

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant