CN114778728A

CN114778728A - Determination method for curcumin compound content

Info

Publication number: CN114778728A
Application number: CN202210419645.3A
Authority: CN
Inventors: 张丽霞; 罗祖良; 郭芳; 马小军; 唐德英
Original assignee: Institute of Medicinal Plant Development of CAMS and PUMC
Current assignee: Institute of Medicinal Plant Development of CAMS and PUMC
Priority date: 2022-04-20
Filing date: 2022-04-20
Publication date: 2022-07-22

Abstract

The invention discloses a determination method of curcumin compound content, which specifically comprises the following steps: (1) preparation of 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 rhizomes of a sample into slices, drying, crushing and sieving, adding a methanol aqueous solution, shaking, ultrasonically extracting, centrifuging, separating, repeatedly extracting residues for 1 time by using the methanol aqueous solution and methanol respectively, mixing a supernatant and the two extracting solutions, fixing the volume, and filtering; (3) establishing a regression equation and measuring the content: and (3) determining and establishing a regression equation of the standard substance by adopting a liquid chromatography-tandem mass spectrometry method, and then determining and calculating the content of the curcumin compound in the sample solution. According to the invention, the content of the curcumin compounds in the turmeric varieties is determined by adopting LC-MS/MS so as to evaluate the quality of the turmeric medicinal materials and the application prospect thereof, and a reference is provided for the quality control of the turmeric medicinal materials.

Description

Determination method for curcumin compound content

Technical Field

The invention relates to the technical field of drug analysis, in particular to a determination method of curcumin compound content.

Background

In 2020 edition, the Chinese pharmacopoeia records that Curcuma rhizome is dried rhizome of Curcuma rhizome Curcuma Longa L of Curcuma of Zingiberaceae, and has effects of removing blood stasis, activating qi-flowing, dredging channels and relieving pain. Can be used for treating chest and hypochondrium pricking pain, thoracic obstruction, cardialgia, dysmenorrhea, amenorrhea, abdominal mass, rheumatic shoulder and arm pain, and traumatic injury with swelling and pain. The active ingredients of the turmeric mainly comprise curcumin compounds and volatile oil. At present, researchers have separated more than 30 natural curcumin compounds from turmeric, mainly including curcumin, demethoxycurcumin, bisdemethoxycurcumin, dihydrocurcumin, tetrahydrocurcumin and the like, wherein the curcumin has the highest content and is the main active component. Pharmacological research shows that curcumin has the effects of resisting tumor, inflammation, oxidation, neuroprotection and the like. Turmeric is not only a traditional Chinese medicine, but also an important raw material of curry, and is also a dietary supplement for the east and west of the wind. Over the last several decades there has been an increasing interest in the study of functional foods and dietary supplements for different diseases, of which turmeric is one of the most studied. Turmeric as a spice has a long history in cooking in asian countries and other regions around the world, and is widely used in wheaten foods, beverages, fruit wines, candies, cakes, cans, juices, cooking dishes and the like.

Currently, curcumin, demethoxycurcumin and bisdemethoxycurcumin are the most interesting in the evaluation of turmeric quality. However, dihydrocurcumin and tetrahydrocurcumin are hydrogenated metabolites in the body of turmeric, and have similar pharmacological effects to curcumin. The research reports that the dihydrocurcumin and the 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 the tetrahydrocurcumin in the aspects of resisting oxidation, reducing blood sugar, reducing blood fat and the like are better than those of the curcumin.

However, the production areas of the curcuma longa are widely distributed, and the quality of the curcuma longa cultivars in different areas is greatly different.

Therefore, the problem to be solved by those skilled in the art is how to provide a method for measuring the content of curcumin compounds to evaluate the quality of turmeric varieties from different sources.

Disclosure of Invention

In view of the above, the invention aims to provide a method for determining the content of curcumin compounds, which is based on a liquid chromatography-tandem mass spectrometry technology (LC-MS/MS), establishes a method for simultaneously determining the content of curcumin, demethoxycurcumin, bisdemethoxycurcumin, dihydrocurcumin and tetrahydrocurcumin in turmeric, performs content determination on three representative turmeric varieties (high-content turmeric, rhizoma curcumae longae and curcuma longa), and provides reference for quality control of turmeric medicines.

In order to achieve the purpose, the invention adopts the following technical scheme:

a determination method for curcumin compound content specifically comprises the following steps:

(1) preparation of Standard solutions

Dissolving curcumin compound standard substance in methanol to prepare single stock solution or mixed standard stock solution, and diluting to obtain standard solution for later use;

(2) preparation of sample solution

Firstly, cutting rhizomes of a turmeric sample into slices, drying, crushing 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; 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 extracting solutions, fixing the volume, filtering to obtain a sample solution, and storing for later use;

(3) establishment of regression equation and content determination

The method comprises the steps of firstly 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.

Further, in the step (1), the curcumin compound is at least one of curcumin, demethoxycurcumin, dideoxy curcumin, dihydrocurcumin and tetrahydrocurcumin.

The curcumin compound selected by the invention has the effects of antioxidation, anti-inflammation, free radical removal, anticancer and the like.

Further, in the step (1), the concentration of the single stock solution was 10. mu.g.mL^-1(ii) a The concentration of the mixed standard stock solution was 1. mu.g.mL^-1(ii) a The concentrations of the standard solutions were 2ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL and 500ng/mL, respectively.

Further, in the step (2), the turmeric sample is at least one of high-content turmeric collected in Yunnan of institute of medicinal plants of medical sciences of western double-banna, Yunnan province, Sichuan castrated turmeric and Yunnan turmeric.

The technical scheme has the beneficial effect that the curcumin compound content in the turmeric sample selected by the invention is higher.

Further, in the step (2), the drying temperature is 35 ℃; the mesh number of the screened mesh is 60 meshes.

The further technical scheme has the beneficial effects that through drying, the moisture in the rhizome slices of the turmeric sample can be quickly removed; through crushing and sieving, the curcumin compound is beneficial to extraction.

Further, in the above step (2), the mass volume ratio of the turmeric powder, 80% methanol aqueous solution, 50% methanol aqueous solution, and 100% methanol was 0.2 g: 10mL of: 10mL of: 10 mL.

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 method has the advantages of high efficiency, low cost and the like.

Further, in the step (2), the shaking time is 1 min; the ultrasonic extraction time is 10 min; the rotation speed of the centrifugation was 12000rpm for 5 min.

The technical scheme has the beneficial effects that the turmeric powder can be rapidly and uniformly dispersed in 80% methanol water solution by shaking; by ultrasonic extraction, on one hand, the extraction time can be effectively shortened, on the other hand, the active ingredients of the curcumin can be protected from being damaged, and the ultrasonic extraction equipment is convenient to maintain, simple to operate, low in operation cost and obvious in economic benefit; by centrifugation, the supernatant and the residue can be separated quickly.

Further, in the step (2), the filter membrane for filtration is a 0.22 μm microporous filter membrane.

The method has the advantages that residues in the supernatant and the twice extracting solution can be further removed through filtering, and the accuracy and the sensitivity of the determination method are guaranteed.

Further, in the step (3), the chromatographic conditions of the liquid chromatography-tandem mass spectrometry are as follows: a 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 water solution, 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; the flow rate is 0.3 mL/min; the column temperature is 40 ℃; the sample size was 2. mu.L.

Further, in the step (3), the mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry are as follows: ESI ion source, scanning mode is positive ion mode; the relevant parameters include air curtain air, atomizing air and auxiliary air which are respectively 30psi, 50psi and 50 psi; the spraying voltage is 5500V, and the ion source temperature is 550 ℃; the outlet voltage of the collision cell is 10V, quantitative analysis is carried out by adopting a multi-reaction monitoring mode, and the residence time is 50 ms.

The method has the advantages that the liquid chromatogram-tandem mass spectrometry adopts the liquid chromatogram as a separation system and the mass spectrum as a detection system, and a mass spectrogram is obtained by separating and ionizing the purified sample in the liquid chromatogram part and the mass spectrum part and using a detector; the method embodies the complementation of the advantages of the chromatogram and the mass spectrum, and combines the advantages of high separation capability of the chromatogram on the complex sample and high selectivity and high sensitivity of the mass spectrum, and can provide relative molecular weight and structural information.

According to the technical scheme, compared with the prior art, the invention has the following beneficial effects:

1. the purpose is as follows: the content of 5 curcumin compounds (curcumin, demethoxycurcumin, bisdemethoxycurcumin, dihydrocurcumin and tetrahydrocurcumin) in three turmeric varieties (high-content turmeric, rhizoma curcumae longae and yun turmeric) is determined by adopting a liquid chromatography-tandem mass spectrometry (LC-MS/MS) so as to evaluate the quality of the high-content turmeric and the application prospect thereof.

2. The method comprises the following steps: ultrasonic extracting 5 curcumin compounds in three turmeric varieties by using 80% methanol, 50% methanol and methanol in sequence, determining by LC-MS/MS in a Multiple Reaction Monitoring (MRM) mode, qualitatively and quantitatively analyzing two pairs of ions of each component, and statistically analyzing content variation among different varieties.

3. As a result: the content and component proportion of 5 curcumin compounds in the three turmeric varieties have larger difference, and the content of the index component curcumin in the high-content turmeric is the highest and reaches 3.23 percent, which is 2 times of the content of the Sichuan turmeric and 15 times of the content of the cloud turmeric.

4. And (4) conclusion: the contents of five flavin compounds in the high-content turmeric are all higher than that of the curcuma longa and the curcuma longa, and the evaluation on the accumulation of active ingredients of the curcumin is that the high-content turmeric variety is superior to the curcuma longa and the curcuma longa.

Drawings

FIG. 1 is a total ion flow graph of three species samples and a mixed standard (wherein A is the mixed standard, B is high-content turmeric, C is Sichuan turmeric, D is Yun 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 technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the following examples, the concentrations of the respective reagent aqueous solutions are volume concentrations.

Example 1

The determination method of the curcumin compound content specifically comprises the following steps:

(1) preparation of Standard solutions

Accurately weighing curcumin standard substance, dissolving in chromatographic grade methanol, and making into 10 μ g/mL solution^-1Diluting the single stock solution to obtain standard solutions with the concentrations of 2ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL and 500ng/mL respectively, and storing the standard solutions in a refrigerator at 4 ℃ for later use;

(2) preparation of sample solution

Sampling by adopting a five-point sampling method, namely cutting fresh roots and stems of high-content turmeric collected by Yunnan institute of medicinal plants of medical science institute of Xishuangbanna, Yunnan province into slices, drying in a drying oven at 35 ℃, crushing and sieving by a 60-mesh sieve to obtain turmeric powder; weighing Curcuma rhizome powder 0.2g, precisely weighing, placing in 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; extracting the residue with 10mL 50% methanol aqueous solution and 10mL 100% methanol for 1 time to obtain two extractive solutions; mixing the supernatant with the two extracting solutions, and adding methanol into 1mL of the mixture to be constant volume to 20 mL; finally, filtering through a 0.22-micron microporous filter membrane 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 determination

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 content of the curcumin compound in the sample solution;

wherein the chromatographic conditions of the liquid chromatography-tandem mass spectrometry are as follows: and (3) chromatographic column: waters ACQUITY UPLC HST 3(2.1 mm. times.100 mm, 1.8 μm); mobile phase: phase A is 0.1% formic acid water solution, 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; the flow rate is 0.3 mL/min; the column temperature is 40 ℃; the sample volume is 2 mu L;

the mass spectrum conditions of the liquid chromatography-tandem mass spectrometry are as follows: ESI ion source, scanning mode is positive ion mode; the relevant parameters include air curtain air, atomizing air and auxiliary air which are respectively 30psi, 50psi and 50 psi; the spraying voltage is 5500V, and the ion source temperature is 550 ℃; the outlet voltage of the collision cell is 10V, quantitative analysis is carried out by adopting a multi-reaction monitoring mode, and the residence time is 50 ms.

Example 2

(1) preparation of Standard solutions

Accurately weighing curcumin, demethoxycurcumin, bisdeoxycurcumin, dihydrocurcumin and tetrahydrocurcumin, respectively, dissolving in chromatographic grade methanol to obtain 10 μ g/mL solution^-1Then mixing the stock solutions to a concentration of 1. mu.g.mL^-1Diluting the mixed standard stock solution to obtain standard solutions with the concentrations of 2ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL and 500ng/mL respectively, and storing the standard solutions in a refrigerator at 4 ℃ for later use;

(2) preparation of sample solution

Sampling by adopting a five-point sampling method, namely cutting high-content turmeric, Sichuan castration turmeric and fresh rhizome of Yunnan turmeric collected by Yunnan institute of medicinal plant research of western Banna Chinese medical science institute of Yunnan province into slices, drying in a drying oven at 35 ℃, crushing and sieving by a 60-mesh sieve to obtain turmeric powder; weighing Curcuma rhizome powder 0.2g, precisely weighing, placing in 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; extracting the residue with 10mL 50% methanol aqueous solution and 10mL 100% methanol for 1 time to obtain two extractive solutions; mixing the supernatant with the two extracting solutions, and adding methanol into 1mL of the mixture to fix the volume to 20 mL; finally, filtering through a 0.22-micron microporous filter membrane 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 determination

wherein the chromatographic conditions of the liquid chromatogram-tandem mass spectrometry are as follows: a chromatographic column: waters ACQUITY UPLC HST 3(2.1 mm. times.100 mm, 1.8 μm); mobile phase: phase A is 0.1% formic acid water solution, 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; the flow rate is 0.3 mL/min; the column temperature is 40 ℃; the sample volume is 2 mu L;

the mass spectrum conditions of the liquid chromatogram-tandem mass spectrometry are as follows: ESI ion source, scanning mode is positive ion mode; the relevant parameters comprise air curtain air, atomizing air and auxiliary air which are respectively 30psi, 50psi and 50 psi; the spraying voltage is 5500V, and the ion source temperature is 550 ℃; the outlet voltage of the collision cell is 10V, quantitative analysis is carried out by adopting a multi-reaction monitoring mode, and the residence time is 50 ms.

Performance testing

1 test materials

1.1 test samples

In 2016, in the research of turmeric germplasm, an inventor collects a turmeric cultivar with orange red rhizome from Guizhou copper kernel, the curcumin content of the turmeric cultivar is obviously higher than that of other germplasms, the turmeric cultivar is tentatively named as 'high-content turmeric', and the turmeric cultivar is cultivated in a southern Yunnan pharmaceutical park of institute of medicinal plants of Chinese academy of medical sciences for three continuous generations, so that the turmeric cultivar has stable agronomic characters and quality characters. The invention takes high-content turmeric collected by Yunnan institute of medicinal plants of the national academy of medical sciences of western Banna, Yunnan province, Sichuan castration and Yunnan turmeric samples as test materials, three turmeric varieties are identified as turmeric Curcuma longa L by Zhangxia researchers of Yunnan institute of medicinal plants of the national academy of medical sciences, and are collectively called high-content turmeric (GJH), rhizoma curcumae longa (CJH) and Yunnan turmeric (YJH) unless specially stated in the invention.

1.2 reagents

Curcumin, demethoxycurcumin, bisdeoxycurcumin, dihydrocurcumin and tetrahydrocurcumin standards were purchased from Kyorman Biotech Co., Ltd and Shanghai-source leaf Biotech Co., Ltd (HPLC purities were more than 98%). Methanol and acetonitrile (chromatographic grade) were purchased from Fisher Scientific (Fair town, NJ, USA); formic acid was purchased from guangdong sialon chemical industry, china; purified water (Wahaha, Hangzhou, China).

1.3 Instrument

The LC-MS/MS analysis system consisted of Shimadzu type LC-20A high performance liquid chromatography system (Shimadzu, Japan, including LC-20A infusion pump, SIL-20AC autosampler, CTO-20A column incubator, and DGU-20A3 degasser) and AB SCIEX

5500 systems (applied biosystems, usa, including a Turbo V ion source and a 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, ultrasonic instruments, Inc. of Kunshan; model LG16-B rebell high speed centrifuge, beijing rebell centrifuge ltd; TarginVX-02 model multitube vortex oscillator, Beijing Pedal technology Limited; DHG-9030A electrothermal blowing dry box, Shanghai-Hengchun scientific instruments Co., Ltd。

2 methods and results

2.1 preparation of Standard solutions

Accurately weighing 5 curcumin compounds standard substances respectively, and dissolving in chromatographic grade methanol to obtain final product with concentration of 10 μ g/mL^-1Of the individual stock solutions of (a). Then, the stock solutions were mixed to make 1. mu.g.mL^-1Mixing the standard stock solution to obtain a standard solution, and storing the standard solution in a refrigerator at 4 ℃ for later use.

2.2 preparation of sample solutions

Sampling by adopting a five-point sampling method, respectively taking more than 6 strains of high-content curcuma longa, curcuma longa and curcumalonga in each batch, uniformly mixing fresh rhizomes of the high-content curcuma longa, curcumalonga and curcumalonga, cutting the fresh rhizomes into uniform slices, drying the slices in a drying oven at 35 ℃, crushing the slices, and sieving the slices with a 60-mesh sieve. Weighing Curcuma rhizome powder 0.2g, precisely weighing, placing in 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 respectively; extracting the residue with 10mL 50% methanol aqueous solution and 10mL 100% methanol for 1 time respectively to obtain two extractive solutions; mixing the supernatant and the two extractive solutions, and adding methanol into 1mL to reach a constant volume of 20 mL. Finally, filtering through a 0.22 mu m microporous filter membrane to obtain a sample solution, and storing the sample solution in a sample injection bottle for sample injection analysis. All operations were protected from light in order to avoid photodegradation of curcumin.

2.3 LC-MS/MS analysis conditions

Chromatographic conditions are as follows: and (3) chromatographic column: waters ACQUITY UPLC HST 3(2.1 mm. times.100 mm, 1.8 μm); mobile phase: phase A is 0.1% formic acid water solution, 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; the flow rate is 0.3 mL/min; the column temperature is 40 ℃; the sample size was 2. mu.L.

Mass spectrum conditions: ESI ion source, scanning mode is positive ion mode. The relevant parameters included air curtain air (CUR), atomizing air (GS1) and assist air (GS2) were 30psi, 50psi and 50psi, respectively. The spray voltage (IS) was 5500V and the ion source Temperature (TEM) was 550 ℃. The outlet voltage (CXP) of the collision cell is 10V, the quantitative analysis is carried out by adopting a multi-reaction monitoring (MRM) mode, and the residence time is 50 ms. The mass spectral parameters of each target compound are shown in table 1.

TABLE 1 Mass Spectrometry acquisition parameters for each target Compound

Note: denotes the quantitative ion pair.

2.4 methodological validation

2.4.1 linearity and detection Limit

And diluting the mixed standard stock solution to obtain standard solutions of 2ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL and 500ng/mL respectively, and injecting and analyzing. Calibration curves were created using peak areas as dependent variables (Y-axis) and analyte concentrations as independent variables (X-axis), and correlation coefficients (r) of the calibration curves were used²) The linearity was evaluated. Gradually diluting the mixed standard solution, and taking the amount of each standard when the signal-to-noise ratio S/N is 3 and S/N is 10 as a detection Limit (LOD) and a quantification Limit (LOQ) respectively. The regression equation, linear range, detection limit, and quantitation limit for the five target compounds are shown in table 2.

TABLE 2 regression equation, Linear Range, detection limits and quantitation limits for each target Compound

As shown in Table 2, the amount of each target compound was 2.0 to 500ng/mL^-1Within the mass concentration range, the linear relation is good, and the correlation coefficient (r)²) Are all larger than 0.999, and meet the requirement of quantitative analysis.

2.4.2 precision test

The same mixed reference solution was sampled continuously 6 times, and the peak areas of curcumin, demethoxycurcumin, dideoxy curcumin, dihydrocurcumin and tetrahydrocurcumin were recorded and the RSD (relative standard deviation) values of the precision (precision within day and precision between days) were calculated respectively. The results are shown in Table 3.

TABLE 3 results of precision test

As can be seen from Table 3, the RSD value for the intra-day precision is between 1.31% and 3.04%, and the RSD value for the inter-day precision is between 0.58% and 4.10%, indicating that the precision of the instrument is good.

2.4.3 recovery test

9 portions of turmeric sample with known content of the same batch, each portion is about 0.1g, precisely weighed, and every three portions are respectively added with 80% (low concentration), 100% (medium concentration) and 120% (high concentration) standard solution of each component content in the sample, the standard sample is added to prepare sample solution according to the method under 2.2, the target analyte is measured according to the chromatographic condition under 2.3, and the sample adding recovery rate and the RSD value are calculated. The results are shown in Table 4.

TABLE 4 recovery test results

As can be seen from Table 4, the recovery rate of the sample application is between 84.36% and 108.09%, and the RSD value of the recovery rate is between 1.39% and 5.16%, which meets the requirement of quantitative analysis.

2.4.4 repeatability test

The same batch of turmeric sample (6 parts) was taken in parallel, and the turmeric sample solution prepared according to the method under item 2.2 was added

2.3 under chromatographic conditions, recording the peak area of each target compound and calculating the RSD value. The results are shown in Table 5.

TABLE 5 results of the repeatability tests

Compound (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 for reproducibility were between 0.44% and 6.66%, indicating that the method was very reproducible.

2.4.5 stability test

A portion of the turmeric sample solution prepared by the method described in item 2.2 was taken, and measured once at each of the time points of 0, 2, 4, 8, and 24 under the chromatographic conditions described in item 2.3, and the RSD value was calculated. The results are shown in Table 6.

TABLE 6 stability test results

Compound (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 value of the stability is between 3.43% and 6.56%, indicating that the test solution is stable within 24 h.

2.6 determination of 5 curcuminoids in Curcuma rhizome samples

(1) The method comprises the steps of adopting a LC-QTRAP-MS/MS multi-reaction detection mode (MRM) to carry out data acquisition on high-content turmeric, rhizoma curcumae longae and curcuma yunnanensis samples, carrying out mass spectrum data processing by utilizing Analyst 1.6.2 software, and obtaining total ion flow graphs (TIC) of three variety samples and mixed standard products. The results are shown in FIG. 1.

As can be seen from FIG. 1, the target analytes such as tetrahydrocurcumin, dihydrocurcumin, curcumin, demethoxycurcumin and bisdemethoxycurcumin have high peak separation, good peak pattern and high correspondence.

(2) By comparing the retention time with that of a standard product, and comparing the abundance ratio of parent ions, daughter ions and ions, the content of 5 curcumin compounds in the three turmeric varieties is calculated by an external standard method. The results are shown in FIG. 2 and Table 7.

TABLE 7 content of 5 curcuminoids in different turmeric varieties

Note: the 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 differences in the contents of five curcumin compounds, namely tetrahydrocurcumin, dihydrocurcumin, curcumin, demethoxycurcumin and bisdemethoxycurcumin, in the three turmeric varieties are large, and the contents of 5 ingredients are the highest in the high-content turmeric, and the total content of 5 curcumin in the high-content turmeric is about 2.4 times that of chuanxiong turmeric and 28 times that of yun turmeric. Except for the yujin Concisa, the content of curcumin in the high-content turmeric and the chuangjin Concisa both accord with the specification that the content of curcumin in Chinese pharmacopoeia is not less than 1%, and the content of curcumin in the high-content turmeric reaches 3.23%, which is 2 times of that of the chuangjin Concisa and is 15 times of that of the chuangjin Concisa. Except for different contents, the content distribution proportions of the five compounds in three turmeric varieties are different, and the contents of the 5 curcumin compounds in the high-content turmeric are respectively tetrahydrocurcumin (1.76%), dihydrocurcumin (20.15%), curcumin (35.57%), demethoxycurcumin (17.73%) and bisdemethoxycurcumin (24.78%); the content of five curcumin compounds in the rhizoma curcumae longae is respectively 2.05%, 39.77%, 28.18%, 7.50% and 8.64%; the content ratio of 5 curcumin compounds in Curcuma longa is 6.25%, 21.87%, 56.25%, 9.38%, and 6.25%. The content of curcumin in Curcuma longa is 56.25%, and the content of dihydrocurcumin in Curcuma longa is 39.77%. In a word, compared with rhizoma curcumae longae and yun-huang, the curcumin compound in the high-content turmeric has larger content advantage, and the proportion difference of 5 ingredients in different varieties is larger.

Discussion of 3

3.1 optimization of extraction conditions

In order to obtain the optimum extraction conditions for the curcuminoid component, the extraction solvents (ethanol, methanol, 80% methanol aqueous solution and 50% methanol aqueous solution), the volumes of the extraction solvents (10mL, 20mL and 30mL) and the extraction times (10min, 20min and 30min) were optimized, respectively. Finally, the optimal extraction conditions of the effective components of the turmeric are determined by comparing chromatographic peaks of all components under different extraction conditions: 0.2g of each sample is added with 10mL of 80% methanol, ultrasonic extraction is carried out for 10min, and extraction of the residue is repeated for 1 time by using 10mL of 50% methanol and 10mL of pure methanol respectively. Under these conditions, the normalized recovery of 5 target compounds was within an acceptable range (84.36% -108.09%).

3.2 Mass Spectrometry Condition optimization

To obtain optimum mass spectrum condition for analyzing curcumin components, 100ng/mL is taken^-1Respectively injecting the standard solution into ESI by a peristaltic pump⁺And ESI^－Ionization monitoring was performed in mode. The comparison result shows that 5 curcumin components are subjected to ESI⁺There is a better response in the mode. Then subjecting the materials to ESI⁺Performing parent ion scanning in a mode to obtain an excimer ion [ M + H ] in a primary full-scanning mass spectrogram]⁺The abundance of parent ions is maximized by adjusting instrument parameters, secondary mass spectrum scanning is further performed on molecular ions to obtain fragment ion information, two pairs of daughter ions with strong abundance and small interference are selected as qualitative and quantitative ions, parameter conditions such as declustering voltage (DP) and collision voltage (CE) are optimized to achieve optimal sensitivity, parameters such as atomizing gas, drying gas, ion source temperature and electrospray voltage are optimized, and the detection limit of a target compound in 5 under the condition of optimized mass spectrum is 0.2 ng/mL^-1The established method has good sensitivity.

3.2 analysis of assay results

The sample determination result shows that the content difference of five curcumin compounds in the three turmeric varieties is large, and the proportions of the components are different. The content of curcumin compounds and the proportion among the components may influence the appearance of the turmeric and the change of the drug effect. In the aspect of variety evaluation, the content of five flavin compounds in the high-content turmeric is higher than that of the turmeric, and the quality of the high-content turmeric is considered to be better than that of the turmeric from the aspect of accumulation of active ingredients of curcumin. The pharmacological activity researches of natural curcumin including curcumin, demethoxycurcumin, bisdemethoxycurcumin and the like show that the bisdemethoxycurcumin has higher activity in the aspects of anti-tumor, anti-inflammatory, anti-oxidation and the like than the demethoxycurcumin and the curcumin and is the curcumin with the highest activity; the tetrahydrocurcumin has better pharmacological activity in the aspects of oxidation resistance, blood sugar reduction, blood fat reduction and the like than curcumin. Although the five curcumin compounds have similar pharmacological effects, the activity is inconsistent. This shows that the curcumin compounds in the turmeric have different proportions, and the pharmaceutical effects of the curcumin compounds are different, and 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

1. A determination method for curcumin compound content is characterized by comprising the following steps:

(1) preparation of Standard solutions

(2) preparation of sample solution

Firstly, cutting rhizomes of a turmeric sample into slices, drying, crushing 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; extracting the residue with 50% methanol water solution and 100% methanol water solution for 1 time to obtain two extractive solutions; mixing the supernatant with the two extracting solutions, fixing the volume, filtering to obtain a sample solution, and storing for later use;

(3) establishment of regression equation and content determination

A liquid chromatography-tandem mass spectrometry method is adopted, a regression equation of the curcumin compound standard substance is determined and established, and then the curcumin compound content in the sample solution is determined and calculated.

2. The method for measuring the content of the curcuminoid according to claim 1, wherein in the step (1), the curcuminoid is at least one of curcumin, demethoxycurcumin, bisdeoxycurcumin, dihydrocurcumin, and tetrahydrocurcumin.

3. The method for determining the content of curcumin compounds according to claim 1, wherein in step (1), the concentration of said single stock solution is 10 μ g-mL^-1(ii) a The concentration of the mixed standard stock solution is 1 mug.mL^-1(ii) a The concentrations of the standard solutions were 2ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL and 500ng/mL, respectively.

4. The method as claimed in claim 1, wherein in step (2), the Curcuma rhizome sample is at least one of high-content Curcuma rhizome collected in Yunnan institute of medicinal plants, Sichuan castration and Yunnan Curcuma rhizome.

5. The method for determining the content of the curcumin compound according to claim 1, wherein in the step (2), the drying temperature is 35 ℃; the mesh number of the screened screen is 60 meshes.

6. The method for determining the content of curcumin compounds according to claim 1, wherein in the step (2), the mass volume ratio of the turmeric powder, 80% methanol aqueous solution, 50% methanol aqueous solution and 100% methanol is 0.2 g: 10mL of: 10mL of: 10 mL.

7. The method for measuring the content of the curcuminoid compound according to claim 1, wherein in the step (2), the shaking time is 1 min; the ultrasonic extraction time is 10 min; the rotation speed of the centrifugation is 12000rpm, and the time is 5 min.

8. The method for measuring the content of the curcuminoid compound in the claim 1, wherein, in the step (2), the filtering membrane is a 0.22 μm microporous filtering membrane.

9. The method for determining the content of the curcumin compounds according to claim 1, wherein in the step (3), the chromatographic conditions of the liquid chromatography-tandem mass spectrometry are as follows: and (3) 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 water solution, 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; the flow rate is 0.3 mL/min; the column temperature is 40 ℃; the sample size was 2. mu.L.

10. The method for determining the content of the curcuminoid compound according to claim 1, wherein in the step (3), the mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry are as follows: ESI ion source, scanning mode is positive ion mode; the relevant parameters comprise air curtain air, atomizing air and auxiliary air which are respectively 30psi, 50psi and 50 psi; the spraying voltage is 5500V, and the ion source temperature is 550 ℃; the outlet voltage of the collision cell is 10V, quantitative analysis is carried out by adopting a multi-reaction monitoring mode, and the residence time is 50 ms.