CN113640448B - Quality control method and construction method of saffron - Google Patents

Quality control method and construction method of saffron Download PDF

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CN113640448B
CN113640448B CN202111023375.6A CN202111023375A CN113640448B CN 113640448 B CN113640448 B CN 113640448B CN 202111023375 A CN202111023375 A CN 202111023375A CN 113640448 B CN113640448 B CN 113640448B
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saffron
methanol
lovastatin
extract
quality control
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CN113640448A (en
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胡学民
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Tibet Yuewang Drugstore Ecological Tibetan Medicine Technology Co ltd
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    • 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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • 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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample

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Abstract

The invention discloses a quality control method and a construction method of saffron, wherein the saffron comprises the following active ingredients: nucleoside compounds, monascus pigment compounds, lovastatin, citrinin, and Moraxelin compounds; the quality control method comprises detecting the effective components of the saffron by UHPLC method. The quality control method of the saffron is characterized in that the method is used for exploring the conditions of liquid chromatography aiming at the component characteristics of the saffron, and can identify and detect 21 effective components in the saffron at one time, so that the detection efficiency is greatly improved, the chemical components and the quality characteristics of the saffron are comprehensively reflected, the quality control of the saffron is facilitated, the saffron can be effectively distinguished from the prior Chinese medicinal red yeast, and the homogenization is avoided.

Description

Quality control method and construction method of saffron
Technical Field
The invention relates to the technical field of detection of effective components of saffron, in particular to a quality control method and a construction method of saffron.
Background
The red rice is a common lipid-lowering medicament, is fermented by red rice bacteria, and 2 red rice medicaments which are clinically used at present are the traditional Chinese medicines of red rice and Tibetan medicine of Tibetan red rice. The traditional Chinese medicine "red rice" is prepared by taking the peduncles as a matrix, inoculating aspergillus purpureus Monascus purpureus Went (CGMCC, no.0272 strain) of Aspergillus and fermenting, and is also known as "red rice", and the red rice decoction pieces, the Chinese patent medicines of the Zhuezhikang capsules and the zhibituo capsules are clinically used; *** medicine "Tibetan red rice" (original name: highland barley red rice) is prepared by taking kernel of highland barley (special crop of Gramineae, hordeum vulgare var. Nudum hook. F.) as matrix, inoculating Aspergillus Monascus pilosus (No. YWG-1 strain) of Aspergillus, fermenting, and clinically using decoction pieces thereof.
The research reports that the active ingredients of the traditional Chinese medicine red yeast rice for reducing blood fat are lovastatin ingredients, and the standard of the lovastatin ingredients is attached to the "blood fat recovery capsule" strip of Chinese pharmacopoeia, and the limit of "fingerprint" and "lovastatin" is regulated (not less than 0.22%). The quality standards of the Tibetan red rice are recently reported, and in view of the differences of the Tibetan red rice and the traditional Chinese medicine red rice in the aspects of the strains, the fermentation matrixes, the production environments and the like, the composition components and the quality control of the Tibetan red rice are necessary to be researched, the standards reflecting the characteristics of the Tibetan red rice are established, and the homogenization is avoided.
Disclosure of Invention
The invention provides a quality control method and a construction method of saffron, which aim to solve the defects in the prior art, and the method can determine the types and the contents of the saffron components in one sample injection detection and distinguish the saffron from the prior Chinese medicinal red yeast by UHPLC detection.
The invention firstly provides a quality control method of saffron, and the saffron comprises the following active ingredients: nucleoside compounds, monascus pigment compounds, lovastatin, citrinin, and Moraxelin compounds; the quality control method comprises detecting the effective components of the saffron by UHPLC method.
Preferably, the chromatographic conditions of the UHPLC method are: gradient elution is carried out by adopting a phenanthrene door C18 chromatographic column with the column temperature of 30 ℃ and the mobile phase of methanol-water.
More preferably, during the gradient elution, the concentration of methanol in the mobile phase is as follows:
0-3 min,6% methanol;
3-7 min, 6-16% of methanol;
7-10 min, 16-16% methanol,
10 To 15min,16 to 60 percent of methanol,
15-25 Min, 60-85% methanol,
25 To 32min,85 to 85 percent of methanol,
32-34 Min, 85-100% methanol,
34-38 Min, 100-100% methanol,
38-40 Min, 100-6% methanol,
40-44 Min,6% methanol.
More preferably, the chromatographic conditions of the UHPLC process further comprise: the detection wavelength was 250nm.
More preferably, the flow rate of the mobile phase is 1mL/min.
More preferably, the chromatographic conditions of the UHPLC process further comprise: the sample injection amount at the time of detection was 10. Mu.L.
More preferably, the specification of the chromatographic column is: 4.6 mm. Times.150 mm,3.0 μm.
The invention also provides a fingerprint obtained by the quality control method, which comprises 21 chromatographic peaks numbered in sequence according to the peak-out time sequence, wherein the components represented by peaks 2, 3 and 4 are nucleoside substances, the components represented by peaks 13, 14 and 15 are monascus pigment substances, the component represented by peak 18 is lovastatin, the component represented by peak 10 is citrinin, and the components represented by other 13 chromatographic peaks are Moracolin compounds.
Preferably, the component represented by the peak No. 2 is uridine, the component represented by the peak No. 3 is guanosine, the component represented by the peak No. 4 is adenosine, the molecular formula of the component represented by the peak No. 13 is C 21H28O5, the molecular formula of the component represented by the peak No. 14 is C 21H26O5, and the molecular formula of the component represented by the peak No. 15 is C 20H28O4.
The invention also provides a construction method for controlling the quality of the saffron, which comprises the following steps:
Grinding the Tibetan red rice decoction pieces, performing osmotic extraction and reduced pressure concentration to obtain a Tibetan red rice extract;
Segmenting the saffron extract by chemical components to obtain segmented extract;
Detecting the saffron extract and the segmented extract by adopting the UHPLC chromatographic conditions;
And analyzing and comparing the UHPLC chromatograms obtained after detection, and confirming the characteristic peak information corresponding to the saffron.
Preferably, 70% ethanol is used as the extraction solvent in the osmotic extraction process.
Preferably, the chemical composition segmentation process adopts macroporous resin HPD-100 column chromatography.
More preferably, the chemical composition segmentation process specifically comprises the following steps:
washing the saffron extract with pure water to obtain water-soluble part;
washing the saffron extract with 30% ethanol to obtain a large polar fraction;
washing the saffron extract with 60% ethanol to obtain a medium polarity fraction;
Washing the saffron extract with 90% ethanol to obtain small polar fraction;
Preferably, the Moraxelin-based compound is distributed predominantly in the medium and low polarity sites.
Preferably, the mass content of lovastatin in the saffron is not less than 0.60%, and the lovastatin comprises lactone lovastatin and acid lovastatin.
Preferably, the mass content of the beta-glucan in the saffron is not less than 1.80%.
The beneficial effects of the invention are as follows:
1. According to the quality control method of the saffron, provided by the invention, the conditions of liquid chromatography are explored aiming at the component characteristics of the saffron, 21 effective components in the saffron can be identified and detected at one time, the detection efficiency is greatly improved, the chemical components and the quality characteristics of the saffron are comprehensively reflected, the quality control of the saffron is facilitated, the saffron can be effectively distinguished from the prior Chinese medicinal red yeast, and the homogenization is avoided.
2. The chromatographic condition of the UHPLC method is that a phenanthrene C18 chromatographic column is adopted, the column temperature is 30 ℃, and the mobile phase is methanol-water, so as to carry out gradient elution. The invention takes the richness of chromatographic peak information presented by a fingerprint and the extraction rate of lovastatin as main active ingredient as investigation indexes, and finally determines the elution condition of a chromatographic mobile phase, the column temperature and the detection wavelength.
3. The quality control method can accurately detect the effective components of the saffron, and has the advantages of high detection precision, good repeatability, strong stability, good sample adding and recycling, and good durability.
4. The fingerprint spectrum of the saffron can better reflect the quality and stability of the saffron product, and can identify the difference between the saffron decoction pieces and the clinically common monascus products; the blood fat recovery capsules have larger difference with the saffron in composition, and are all between 0.85 and 0.88; the other traditional Chinese medicine red rice samples have obvious difference in composition with the Tibetan red rice, the similarity is between 0.33 and 0.81, and the quality consistency of red rice decoction pieces produced by different manufacturers in the market is poor.
5. According to the construction method for controlling the quality of the saffron, disclosed by the invention, the saffron is better segmented according to the polarity size by segmenting the chemical components, and a foundation is provided for subsequent component separation and drug effect research.
6. According to the quality control construction method, under the chromatographic condition, the lovastatin content of 20 batches of saffron samples is measured, the total amount of lovastatin (lactone) and lovastatin (acid form) in the samples is 0.52% -0.87%, the average value is 0.75%, the average value is 20% and is 0.60%, 18 batches of samples in 20 batches are qualified, the qualification rate is 90%, and the total amount limit of lovastatin (lactone) and lovastatin (acid form) is not less than 0.60%, so that the quality control of lovastatin can be performed.
7. According to the quality control construction method, under the chromatographic condition, the content of beta-glucan in 20 batches of saffron samples is measured, the content of beta-glucan is 1.75% -3.17%, the average value is 2.31%, the average value is taken to float 20% to be 1.8%, 17 batches of 20 batches of samples are qualified, the qualification rate is 85.0%, and the content limit is 1.8% according to the measurement, so that the quality control of the beta-glucan can be performed.
Drawings
FIG. 1A sectional flow chart of chemical components of saffron extract
FIG. 2 UHPLC-UV chromatogram of example 1 (wherein S1: saffron total extract, S2: pure water rinse, S3:30% ethanol rinse, S4:60% ethanol rinse, S5:90% ethanol rinse)
FIG. 3 UHPLC-MS chromatogram of example 1 (1: saffron total extract, 2: pure water rinse, 3:30% ethanol rinse, 4:60% ethanol rinse, 5:90% ethanol rinse)
FIG. 4 example 1 common characteristic fingerprint peaks in the control fingerprint of saffron
FIG. 5A fingerprint analysis and full spectrum peak matching chart of 28 batches of saffron decoction pieces in example 3 (wherein R: control fingerprint, S1-S28: saffron decoction piece samples)
FIG. 6A comparative fingerprint of Zanghong Yeast in example 3 (wherein, peaks 1 to 10 are common characteristic fingerprint peaks, and peak 8 is defined as reference peak S)
FIG. 7 shows a full spectrum peak matching chart of Xuezhikang capsule sample and Zanghongqu reference fingerprint (wherein R is Zanghongqu reference fingerprint, S1-S12 are Xuezhikang capsule sample)
FIG. 8 shows a full spectrum peak matching chart of the traditional Chinese medicine red rice sample and the Tibetan medicine red rice decoction piece contrast fingerprint (wherein R is the Tibetan medicine red rice contrast fingerprint, S1-S10 is the traditional Chinese medicine red Qu Yangpin)
Detailed Description
The present invention will be described in further detail with reference to specific embodiments thereof in order to enable those skilled in the art to better understand the technical aspects of the invention.
The conventional red yeast rice is prepared by parasitizing mycelia of Aspergillus ruber Monascus purpureus Went of Aspergillus on semen oryzae sativae. Distributed in Hebei, jiangxi, zhejiang, taiwan, fujian, guangdong, etc. Has effects of invigorating spleen, resolving food stagnation, promoting blood circulation, and removing blood stasis. It is often indicated for food stagnation, abdominal distention, red and white diarrhea, postpartum lochiorrhea and traumatic injury.
The Tibetan monascus takes Tibetan highland barley as a raw material, combines the effects of antioxidation, blood lipid reduction and the like of the monascus with rich nutrient substances (such as rich beta-glucan, low fat, high fiber, high protein and high vitamin) of the highland barley through the highland barley monascus fermentation technology, so that the effective components of the highland barley monascus and the highland barley monascus are fully utilized, and the quality is improved.
The invention provides a quality control method and a construction method of saffron, which are used for establishing a quality standard reflecting the characteristics of the saffron and avoiding homogenization with traditional Chinese medicine, wherein the method can simultaneously determine the types and the contents of the components in one sample injection detection by UHPLC (ultra high performance liquid chromatography) detection and distinguish the components from the traditional Chinese medicine, namely the monascus.
In order to achieve the above object, the present invention firstly provides a quality control method of saffron, the effective components of the saffron include: nucleoside compounds, monascus pigment compounds, lovastatin, citrinin, and Moraxelin compounds; the quality control method comprises detecting the effective components of the saffron by UHPLC method.
The saffron contains lovastatin component and beta-glucan with various biological activities, which is possibly related to the fermentation production of the saffron in highland (pizza) by taking highland barley as a matrix, wherein the saffron selects monascus strains adapting to the plateau environment. Because of detecting and identifying a plurality of active ingredients in the saffron, in order to effectively separate various active ingredients to obtain characteristic peaks with better separation degree, the invention explores the chromatographic conditions of liquid chromatography, so that the peaks with better separation degree can be obtained by single detection.
The invention takes the richness of chromatographic peak information presented by a fingerprint and the extraction rate of lovastatin as a main effective component as investigation indexes, and finally determines the chromatographic mobile phase elution condition, the column temperature and the detection wavelength. In the present invention, the chromatographic conditions of the UHPLC method are: gradient elution is carried out by adopting a phenanthrene door C18 chromatographic column with the column temperature of 30 ℃ and the mobile phase of methanol-water.
In the gradient elution process, the concentration of methanol in the mobile phase is as follows:
0-3 min,6% methanol;
3-7 min, 6-16% of methanol;
7-10 min, 16-16% methanol,
10 To 15min,16 to 60 percent of methanol,
15-25 Min, 60-85% methanol,
25 To 32min,85 to 85 percent of methanol,
32-34 Min, 85-100% methanol,
34-38 Min, 100-100% methanol,
38-40 Min, 100-6% methanol,
40-44 Min,6% methanol.
The chromatographic conditions of the UHPLC method further comprise: the detection wavelength was 250nm.
The flow rate of the mobile phase was 1mL/min.
The chromatographic conditions of the UHPLC method further comprise: the sample injection amount at the time of detection was 10. Mu.L.
The specification of the chromatographic column is as follows: 4.6 mm. Times.150 mm,3.0 μm.
The invention also provides a fingerprint obtained by the quality control method, which comprises 21 chromatographic peaks numbered in sequence according to the peak-out time sequence, wherein the components represented by peaks 2,3 and 4 are nucleoside substances, the components represented by peaks 13, 14 and 15 are monascus pigment substances, the component represented by peak 18 is lovastatin, the component represented by peak 10 is citrinin, and the components represented by other 13 chromatographic peaks are Moracolin compounds. Further, the component represented by the peak No. 2 is uridine, the component represented by the peak No. 3 is guanosine, the component represented by the peak No. 4 is adenosine, the molecular formula of the component represented by the peak No. 13 is C 21H28O5, the molecular formula of the component represented by the peak No. 14 is C 21H26O5, and the molecular formula of the component represented by the peak No. 15 is C 20H28O4.
The invention also provides a construction method for controlling the quality of the saffron, which comprises the following steps:
Grinding the Tibetan red rice decoction pieces, performing osmotic extraction and reduced pressure concentration to obtain a Tibetan red rice extract;
Segmenting the saffron extract by chemical components to obtain segmented extract;
Detecting the saffron extract and the segmented extract by adopting the UHPLC chromatographic conditions;
And analyzing and comparing the UHPLC chromatograms obtained after detection, and confirming the characteristic peak information corresponding to the saffron.
In the technical scheme of the invention, as the effective components of the saffron are more, special consideration is needed for the selection of the extraction solvent and the extraction method. In the invention, the richness of chromatographic peak information presented by the fingerprint and the extraction rate of lovastatin as a main effective component are taken as investigation indexes, the solvent selection of the preparation method is investigated, and finally the quality control construction method is determined, so that the components from small polarity to large polarity in the saffron can be comprehensively reflected.
In the invention, 70% ethanol is selected as an extraction solvent in the osmotic extraction process. Specifically, the preparation method comprises the steps of grinding the Tibetan monascus decoction pieces, adding 70% ethanol with the mass of 0.8 times of the Tibetan monascus decoction pieces, moistening for 12 hours, adding the layers into a percolating cylinder, percolating and extracting by 70% ethanol to obtain a percolate, collecting the percolate, and concentrating under reduced pressure by a large-scale rotary evaporator.
The chemical component segmentation process adopts macroporous resin HPD-100 column chromatography, as shown in figure 1, and specifically comprises the following steps: washing the saffron extract with pure water to obtain water-soluble part; washing the saffron extract with 30% ethanol to obtain a large polar fraction; washing the saffron extract with 60% ethanol to obtain a medium polarity fraction; washing the saffron extract with 90% ethanol to obtain small polar fraction. The Moraxelin compound is mainly distributed in the medium polar part and the small polar part.
The content of lovastatin in the saffron of the present invention is not less than 0.60% of the total amount of lovastatin (lactone) and lovastatin (acid form).
The content of beta-glucan in the saffron is not less than 1.80 percent.
The above is a detailed description of the present invention, and the following examples of the present invention, in which the saffron is from *** yue wang biotechnology limited.
Example 1 construction method for quality control of saffron sample
Test instrument: KQ-300DE type digital control ultrasonic cleaner (power 300W, frequency adjustable, kunshan ultrasonic instrument Co., ltd.); milli-Q ultra-pure water machine (Miibo, USA); electrothermal constant temperature blast drying oven (Shanghai Xinmiao medical instruments manufacturing Co., ltd.); an alcohol meter; BT25S electronic analytical balance (beijing certolis instruments systems limited); r-210 rotary film evaporator (BUCHI, switzerland); LC-20ATXR island body fluid phase chromatograph (shimadzu instrument); AB sicex TOF5600+ mass spectrometer (Ebola imaginative power and creativeness instruments, USA); disposable sterile syringe (with needle) holy light medical products, inc; disposable 0.22, 0.45 μm filter membrane, borui scientific instruments ltd, guangzhou; YMC 18 column (japan YMC technology);
Mass spectrometry conditions: electrospray ionization, positive and negative 2 ion mode detection, nitrogen as sheath gas, auxiliary gas and purge gas. In positive ion mode: the spraying voltage is 3.5kV, the sheath gas is 3.5MPa, the auxiliary gas is 785kPa, and the purge gas is 0Pa; the capillary temperature was 270 ℃, the capillary voltage was 10V, and the lens voltage was 80V. In the negative ion mode: spraying voltage is 5kV, sheath gas is 2.7MPa, auxiliary gas is 785kPa, and purging gas is 0Pa; the capillary temperature was 270 ℃, the capillary voltage was-10V, and the lens voltage was-100V. Full scan mass range: m/z is 100-1500. The secondary mass spectrum data is collected in a mode dependent on the mode, 3 peaks with highest responsivity intensity in the full-scan spectrum are used for secondary mass spectrum analysis, high-purity helium is used as collision gas, and the collision energy is 35eV.
Test reagent: the ethanol for sample preparation is analytically pure and purchased from the company of the chemical industry, inc.; methanol used in the liquid chromatography was purchased as chromatographic purity (lot number: 654655-5456, U.S. heaven and earth), and water was ultrapure water produced by a laboratory Miybbo pure water meter.
The saffron quality control construction method in the embodiment adopts the following steps:
1. grinding the Tibetan monascus decoction pieces, adding 70% ethanol with the mass of 0.8 times of the Tibetan monascus decoction pieces, moistening for 12 hours, adding the layers into a percolating cylinder, percolating with 70% ethanol to obtain a percolate, collecting the percolate, and concentrating under reduced pressure by a large-scale rotary evaporator while collecting the percolate to obtain the Tibetan monascus extract.
2. The saffron extract is subjected to chemical component segmentation by adopting macroporous resin HPD-100 column chromatography, and the method specifically comprises the following steps: washing the saffron extract with purified water to obtain a water-soluble fraction; washing the saffron extract with 30% ethanol to obtain a highly polar fraction; washing the saffron extract with 60% ethanol to obtain a medium polarity fraction; washing the saffron extract with 90% ethanol to obtain a small polar fraction; obtaining each segmented extract.
3. Detecting the saffron extract and the segmented extract by adopting UHPLC; and analyzing and comparing the UHPLC chromatograms obtained after detection, and confirming the characteristic peak information corresponding to the saffron.
The chromatographic conditions of the UHPLC are as follows: the chromatographic column is a Fei-Roman C18 chromatographic column (luna, 4.6 mm. Times.150 mm,3.0 μm), the column temperature is 30deg.C, the flow rate is 1mL/min, the detection wavelength is 250nm, and the sample injection amount is 10. Mu.L. Mobile phase methanol-water, gradient elution procedure:
0-3 min,6% methanol;
3-7 min, 6-16% of methanol;
7-10 min, 16-16% methanol,
10 To 15min,16 to 60 percent of methanol,
15-25 Min, 60-85% methanol,
25 To 32min,85 to 85 percent of methanol,
32-34 Min, 85-100% methanol,
34-38 Min, 100-100% methanol,
38-40 Min, 100-6% methanol,
40-44 Min,6% methanol.
The analysis results obtained are shown in fig. 2 (S1: saffron total extract, S2: pure water washing section, S3:30% ethanol washing section, S4:60% ethanol washing section, S5:90% ethanol washing section)) and fig. 3 (1: saffron total extract, 2: pure water flushing section, 3:30% ethanol flushing section, 4:60% ethanol flushing section and 5:90% ethanol flushing section, the difference of the components of each section is obvious, and the saffron ingredients are better segmented according to the polarity size.
The 21 common characteristic peaks in the saffron contrast fingerprint are subjected to preliminary identification through ultraviolet of chromatographic peaks and mass spectrum fragment information, the components are identified together, the numbers of the peaks are shown in figure 4, and the mass spectrum related information of the identification results of the compounds is shown in table 1. Among the identified components, the components represented by peaks 2, 3 and 4 are nucleoside substances, the components represented by peaks 13, 14 and 15 are monascus pigment substances, the peak 18 is lovastatin (Monacolin K) which is the main active ingredient of the saffron decoction pieces, and the peak 10 is citrinin (Cirinin) which is the main toxic ingredient; the other 13 chromatographic peaks represent Mo Lake lin (monacolins) compounds, and the results show that the saffron decoction pieces contain various Monacolin (statin) components, and lovastatin, namely Monacolin K, is a representative component with higher content in the components.
Table 1 mass spectrum identification information table of components in saffron extract
Example 2 HPLC fingerprint methodology investigation of saffron sample
Precision test: according to the quality control construction conditions of example 1, 10. Mu.L of lovastatin (lactone) reference solution (0.1054 mg/ml) and 10. Mu.L of lovastatin (acid) reference solution (0.1243 mg/ml) were respectively sucked precisely, injected into a liquid chromatograph, measured, and the sample was repeatedly injected 6 times for each reference solution, and the peak area was recorded. The results of the precision test are shown in Table 2.
TABLE 2 results of precision test
The results show that: the peak areas RSD of the above-mentioned measured lovastatin (lactone) and lovastatin (acid form) controls were 0.93% and 0.19%, respectively, which are less than 2.0%, respectively, indicating that the system precision is good.
Repeatability test: about 0.5g of saffron samples with different batch numbers are taken, 6 parts are precisely weighed, the saffron samples are placed in a conical flask with a plug, 25ml of 30% methanol is precisely added, the mixture is sealed, the mixture is weighed, the mixture is subjected to ultrasonic treatment (power 140W and frequency 42 kHz) for 1.5 hours, the mixture is cooled, the weighed mixture is weighed again, the reduced weight is complemented by the methanol, the extract is centrifuged at a high speed, the supernatant is filtered by a 0.45 mu ml filter head, and the subsequent filtrate is obtained, wherein the quality control construction conditions, sample introduction, measurement and repeatability investigation results are shown in tables 3-5 are carried out according to example 1.
TABLE 3 repeatability test results (sample lot number 20170317)
Table 4 repeatability test results (sample lot number 20170612)
Table 5 repeatability test results (sample lot number 20181101)
The results show that the average contents of the three batches of samples are 0.8208%, 0.7119% and 0.8680%, respectively; RSD was 1.90%, 1.86% and 0.46%, respectively, indicating good reproducibility of the process.
Stability test: samples of different batches of saffron are taken, prepared according to the quality control construction conditions of the embodiment 1, sample injection is carried out at intervals of two hours, measurement is carried out at intervals of 0h, 2h, 4h, 6h, 8h and 12h respectively, peak areas are recorded, and stability investigation results are shown in tables 6-8.
Table 6 stability investigation results (sample lot 20170317)
Table 7 stability test results (sample lot 20170810)
Table 8 stability study results (sample lot 20180605)
The results showed that the lovastatin (lactone) and lovastatin (acid form) peak areas RSD were 0.17% and 0.12%, 0.01% and 0.69%, 0.07% and 0.27%, respectively, as measured for the above three batches. Indicating that the test solution was stable for 12 hours under the conditions of the present invention.
Sample addition recovery test: 6 parts of each of the saffron samples with different batch numbers are taken, each part is about 0.5g, precisely weighed, placed in a conical flask with a plug, and respectively precisely added with 25ml of lovastatin (lactone) reference solution with the concentration of 0.0635mg/ml and 25ml of lovastatin (acid) reference solution with the concentration of 0.0434mg/ml, and the recovery rate is calculated according to the quality control construction conditions of the example 1. The results are shown in tables 9 to 11.
TABLE 9 sample recovery test (sample lot number 20170317)
Table 10 sample recovery test (sample batch number 20170612)
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TABLE 11 sample recovery test (sample lot 20181101)
The results showed that the average recovery of lovastatin was 103.44%, 101.86% and 101.87% for the three batches of samples, and RSD (n=6) was 1.52%, 1.83% and 1.28%, respectively. The sample recovery rate is between 95% and 105%, and the sample recovery is good.
Durability test: samples of different batches of saffron were prepared according to the quality control construction conditions of example 1, using the Agilent 1260 Infinity HPLC instrument system (Agilent, usa) and the Essentia-16 HPLC instrument system (shimadzu), respectively, and were measured by sample injection according to the chromatographic conditions of example 1. The results are shown in tables 12-14.
Table 12 durability test results (sample lot number 20170317)
Table 13 durability test results (sample lot number 20170612)
Table 14 durability test results (sample lot number 20181101)
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The results show that the RSD content of the three batches of samples measured by different instruments is 1.68%, 0.94% and 0.54%, respectively, and the method has better durability.
Example 3 HPLC fingerprint analysis of Tibet monas decoction pieces
The detection conditions are the same as in example 1, 14 batches of saffron decoction piece samples are taken, 2 samples are taken for each batch of samples, chromatographic peaks with the retention time of more than 1000 in 2-38 min in chromatographic data of 28 samples are automatically integrated, one sample chromatogram with the batch number of 20080201 is taken as a reference chromatogram, a median generation method is adopted, the time width of 0.1min is selected, 28 sample chromatographic data are imported into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), 6 common characteristic component peaks are selected as marker peaks through comparison for multipoint correction, full spectrum peak matching is carried out to obtain peak matching fingerprints (figure 5, R is a reference fingerprint; S1-S28 is a saffron decoction piece sample), software generates the reference fingerprints (figures 6, 1-10 peaks are common characteristic fingerprints, 8 peaks are taken as reference peaks S), and similarity data of each batch of samples are obtained through software similarity analysis, and the results are shown in table 15.
TABLE 15 similarity analysis results of 28 samples of Tibetan Monascus decoction pieces and control patterns
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The results show that the similarity between the established control fingerprint of the saffron decoction pieces and 28 samples is over 0.95, which indicates that the quality stability of the samples of each batch of the saffron decoction pieces is good and the batch-to-batch difference is small.
Fingerprint spectrum comparison analysis of Tibetan monascus and Chinese patent medicine 'zhikang' capsule
The detection conditions were the same as in example 1, 6 Xuezhikang capsules were used, 2 samples were taken from each sample, the drug sources were shown in Table 16, the obtained chromatographic data and the saffron control fingerprint were introduced into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), the similarity was calculated, and the results were shown in FIG. 7 (R is the saffron control fingerprint, S1 to S12 are Xuezhikang capsule samples) and Table 17.
Table 16 lot number and source of Xuezhikang samples
Medicine Sample lot number Source(s)
Zhikang for curing blood fat 181205 North Daweixin
Zhikang for curing blood fat 171223 North Daweixin
Zhikang for curing blood fat 181221 North Daweixin
Zhikang for curing blood fat 190312 North Daweixin
Zhikang for curing blood fat 190406 North Daweixin
Zhikang for curing blood fat 190411 North Daweixin
Table 17 Xuezhikang Capsule and saffron crocus contrast fingerprint similarity analysis result
The results show that the similarity between each sample of the Xuezhikang capsule and the fingerprint of the saffron contrast is between 0.85 and 0.88, and the difference between the Xuezhikang capsule and the saffron decoction pieces in composition is shown.
Fingerprint spectrum comparison analysis of Tibetan monascus decoction pieces and other traditional Chinese medicine monascus samples
The detection conditions were the same as in example 1, 10 samples (2 samples were taken for each sample) were taken for 5 batches of the traditional Chinese medicine monascus samples, the medicine sources were shown in table 18, the obtained chromatographic data and the saffron decoction pieces reference fingerprint were introduced into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), the similarity was calculated, and the results were shown in fig. 8 (R is the saffron decoction pieces reference fingerprint; S1 to S10 are traditional Chinese medicine red Qu Yangpin) and table 19.
Sample lot number and source of red rice in Table 18
Medicine Sample lot number Source(s) Medicine
22 Red Qu Yinpian for treating diabetes 181122 Zhejiang tungjuntang traditional Chinese medicine
23 Red Qu Yinpian for treating diabetes 160407 Chinese medicine for chicken
24 Red Qu Yinpian for treating diabetes 1812152 Beijing shengshilong pharmaceutical industry
25 Red Qu Yinpian for treating diabetes 041812051 Beijing Mo Taili g pharmaceutical industry
26 Red Qu Yinpian for treating diabetes 1807249 Sichuan Qian prescription traditional Chinese medicine
Table 19 results of analysis of similarity of fingerprints of Monascus sample and Tibetan Monascus decoction pieces
The results show that the similarity between the traditional Chinese medicine red rice samples of the 5 factories and the saffron contrast fingerprint is 0.33 at the minimum and 0.81 at the maximum, which indicates that the traditional Chinese medicine red rice samples have great difference in composition with the saffron and the quality stability among samples of each batch is poor.
The fingerprint analysis of each sample of the saffron and other traditional Chinese medicine red yeast rice shows that the saffron contrast fingerprint can better reflect the quality and stability of the saffron decoction piece products, and can identify the difference between the saffron decoction pieces and the red yeast rice products commonly used in clinic; the blood fat recovery capsules have larger difference with the saffron in composition, and are all between 0.85 and 0.88; the other traditional Chinese medicine red rice samples have obvious difference in composition with the Tibetan red rice, the similarity is between 0.33 and 0.81, and the quality consistency of red rice decoction pieces produced by different manufacturers in the market is poor.
Example 4 quality control criteria for saffron
Lovastatin content determination
Taking 20 batches of saffron samples, respectively sampling about 0.5g of sample powder (passing through a third sieve), precisely weighing, placing into a 50ml volumetric flask with a plug, adding 30ml of 75% ethanol, sealing, performing ultrasonic treatment (with the power of 250W and the frequency of 28 kHz) for 50 minutes, cooling, adding 75% ethanol to be close to a scale, performing ultrasonic treatment for 10 minutes, cooling, using 75% ethanol to fix the volume to the scale, shaking uniformly, taking supernatant, filtering through a microporous filter membrane with the size of 0.45 mu m, and taking subsequent filtrate to obtain each sample solution. The peak area was recorded and the content was calculated as determined by the chromatographic conditions described in example 1 and the results are shown in Table 20.
TABLE 20 results of lovastatin content measurement of samples of 20 batches of saffron
The results show that the total amount of lovastatin (lactone) and lovastatin (acid form) in 20 batches of saffron samples is 0.52% -0.87%, the average value is 0.75%, the average value is 20% lower than 0.60%, 18 batches of samples in 20 batches are qualified, the qualification rate is 90%, and the total amount of lovastatin (lactone) and lovastatin (acid form) is determined to be not less than 0.60%.
Beta-glucan content determination (ultraviolet-visible spectrophotometry (general rule 0401) determination)
1. Preparation of related reagents and solutions
(1) Preparation of 50U/mL lichenase solution
Taking lml lichenase solution (1000U/mL), adding sodium phosphate buffer (20 mmol/L, pH 6.5) to dilute to 20.0mL, equally dividing the enzyme solution into 4 parts, 5mL each, placing into polypropylene plastic bottles, and freezing at-20deg.C for later use. [ note: the units of lichenase activity are defined as: the amount of enzyme required to release a glucose reducing sugar equivalent of 1. Mu. Mol/L per minute from barley beta-glucan (10 mg/mL) at pH6.5 at 40℃was 1U. ]
(2) Preparation of 2U/mL beta-glucosidase solution
1ML of beta-glucosidase solution (40U/mL) is taken, sodium acetate buffer solution (50 mmol/L, ph=4.0) is added for dilution and volume fixation to 20mL, the enzyme solution is equally divided into 4 parts, 5mL each is placed in a polypropylene plastic bottle, and the polypropylene plastic bottle is frozen at the temperature of minus 20 ℃ for standby. [ note: the β -glucosidase activity units are defined as: the amount of enzyme required to release the equivalent of L. Mu. Mol/L of p-nitrophenol from p-nitrophenyl-beta-glucoside (10 mmol) per minute at Ph=4.0 and 40℃was 1U. ]
(3) Preparation of glucose oxidase-peroxidase-buffer solution mixture
Contains glucose oxidase (> 12000U/L), peroxidase (> 650U/L) and 4-amino-imidacloprid (0.4 mmol/L). [ note: the glucose oxidase activity units are defined as: the amount of enzyme required to oxidize 1mmol of glucose per minute at pH5.1 and 35 ℃ is 1U; the peroxidase activity units are defined as: the enzyme amount required for producing 1.0mg of red gallic acid from pyrogallic acid at pH6.0 and 20deg.C for 20 seconds is 1U
Preparing a buffer solution: taking 13.6g KH 2PO4, 4.2g NaOH and 3.0g 4-hydroxybenzoic acid, adding water for dissolving, fixing the volume to 100mL, and adjusting the pH to 7.4.
Preparation of glucose oxidase-peroxidase-buffer mixture: diluting 50mL of buffer solution to 1000mL; and (3) dissolving the glucose oxidase-peroxidase mixed reagent by using the dilution buffer solution to enable the glucose oxidase-peroxidase mixed reagent to reach the required concentration, thus obtaining the glucose oxidase-peroxidase mixed reagent.
(4) Preparing 50% ethanol solution: 50mL of absolute ethanol was taken and diluted to 100mL with water.
(5) Preparation of 20mmol/L sodium phosphate buffer solution with pH of 6.5
Dissolving 3.12g NaH 2P04·2H2, adding 900ml water, fixing volume to l000ml, and adjusting pH to 6.5.
(6) Preparation of sodium acetate buffer
200Mmol/L, pH4.0 sodium acetate buffer: taking 7.6ml of glacial acetic acid, adding water to dilute to 900ml, adding 4.8g of sodium acetate trihydrate, and shaking to dissolve; the pH was adjusted to 4.0 and the volume was set to l000ml.
50Mmol/L, pH4.0 sodium acetate buffer: 250mL 200mmol/L sodium acetate buffer, pH4.0, was diluted to 1000mL.
2. Preparation of D-glucose standard stock solution
Taking reference substance powder (purity is more than 99%) and drying at 100 ℃ and normal pressure for 2 hours; cooling in a drier, and sealing at room temperature. Taking about 0.1000g of dried glucose reference substance, precisely weighing, shaking to dissolve by 50mmol/L of sodium acetate buffer solution with pH of 4.0, and fixing the volume to 100 mL.
Note that: in the above steps, the enzyme preparation involved can be provided by Megazyme series-parallel beta-D glucan assay kit, megazyme being a trade name of the product of the company Megazyme, ireland.
3. Preparation and determination of test solutions
(1) Sampling about 0.1g of the sample, precisely weighing, placing at the bottom of a test tube, adding 0.5ml of 50% ethanol solution into the test tube of the sample to be tested after enzymolysis pretreatment, and oscillating and dispersing on a vortex mixer; 4.0mL of sodium phosphate buffer was added and sonicated for 5 minutes. The test tube is placed in a boiling water bath for 1 minute; taking out the test tube, and vigorously oscillating on a vortex mixer for a few seconds; the bath was kept for 2 minutes and then shaken again as before.
(2) The enzymolysis reaction is to put the test tube into a water bath with the temperature of 50 ℃ for 5 minutes, add 0.2ml of lichenase solution and shake vigorously for several seconds; covering the test tube plug, and keeping the temperature in water bath at 50 ℃ for 60min; during this period, the tube was removed and the tube was subjected to shaking treatment 3 to 4 times. Taking out the test tube, adding 5ml of 200mmol/l sodium acetate buffer solution, uniformly mixing, and cooling for 5-10 minutes at room temperature; centrifuging (1000 rad/min,10 min), and collecting supernatant. Accurately transferring 0.3ml of supernatant to the bottoms of 3 test tubes respectively, and adding 0.3ml of beta-glucosidase solution into 2 test tubes; another tube was filled with 0.3ml of 50mmol/l sodium acetate buffer as reaction blank; each tube was incubated at 50℃for 10 minutes.
(3) Glucose standard working solution: and (3) transferring 0.15ml of the standard stock solution of the D-glucose into 2 test tubes in parallel, and adding 0.15ml of 50mmol/l sodium acetate buffer solution respectively to obtain the glucose-free buffer solution.
(4) Reagent blank: transferring 0.3ml of 50mmol/l sodium acetate buffer into a test tube to obtain the final product.
(5) Display reaction 9.0ml of the glucose oxidase-peroxidase-buffer mixture was removed to test tubes (including 2 test samples, 1 reaction blank, 3D-glucose standard working solution, 1 reagent blank) and reacted at 50 ℃ for 20 minutes; the tube was removed and cooled to room temperature.
(6) The content measurement is carried out by using a reagent blank to carry out zero setting, and the absorbance value of other test solutions is measured at 510nm, so that the result is calculated. ( If the concentration of beta-glucan in the sample is greater than 10%, a higher absorbance than 100 μg of glucose standard working solution will be produced. At this time, after the oscillation treatment of the enzymolysis reaction, a proper amount of 50mmol/l sodium acetate buffer solution is taken to dilute the supernatant, and then the subsequent steps are continued, and the dilution factor is considered in calculation of the result. )
And (3) calculating results:
A1, measuring the absorbance value of the liquid by the sample;
a0-reaction blank absorbance;
A-D-glucose standard working solution absorbance;
C-D-glucose standard stock concentration in micrograms per milliliter (μg/ml);
Volume correction factor (0.15 mL removed from 9.7mL for analysis);
m-sample mass in grams (g);
0.9-dehydration conversion factor of D-glucose to beta-glucan.
4. Sample measurement
The measurement results of 20 batches of saffron decoction pieces are shown in Table 21.
TABLE 21 determination of beta-glucan content in saffron samples
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The result shows that the beta-glucan content of 20 batches of saffron decoction piece samples is 1.75% -3.17%, the average value is 2.31%, the average value is taken to float 20% to be 1.8%, 17 batches of samples in 20 batches are qualified, the qualification rate is 85.0%, and the content limit is determined to be 1.8%.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.

Claims (4)

1. A quality control method of Tibetan Monascus is characterized in that the Tibetan Monascus decoction pieces are crushed, and then subjected to osmotic extraction and reduced pressure concentration to obtain a Tibetan Monascus extract; segmenting the saffron extract by chemical components to obtain segmented extract; the chemical component segmentation process specifically comprises the following steps: washing the saffron extract with pure water to obtain water-soluble part; washing the saffron extract with 30% ethanol to obtain a large polar fraction; washing the saffron extract with 60% ethanol to obtain a medium polarity fraction; washing the saffron extract with 90% ethanol to obtain small polar fraction; detecting the saffron extract and the segmented extract by adopting a UHPLC (ultra high Performance liquid chromatography) method; analyzing, comparing and detecting UHPLC chromatograms obtained, and confirming the characteristic peak information corresponding to the saffron;
The effective components of the saffron include: nucleoside compounds, monascus pigment compounds, lovastatin, citrinin, and Moraxelin compounds; the chromatographic conditions of the UHPLC method are as follows: adopting a phenanthrene door C18 chromatographic column, wherein the column temperature is 30 ℃, the detection wavelength is 250 nm, the flow rate of a mobile phase is 1 mL/min, and the mobile phase is methanol-water, and performing gradient elution; in the gradient elution process, the concentration of methanol in the mobile phase is as follows:
0-3 min,6% methanol;
3-7 min, 6-16% of methanol;
7-10 min, 16-16% methanol;
10-15 min, 16-60% of methanol;
15-25 min, 60-85% of methanol;
25-32 min, 85-85% methanol;
32-34 min, 85-100% methanol;
34-38 min, 100-100% of methanol;
38-40 min, 100-6% methanol;
40-44 min,6% methanol;
The fingerprint obtained by the quality control method comprises 21 chromatographic peaks numbered in sequence according to the peak-out time sequence, wherein the components represented by peaks 2,3 and 4 are nucleoside substances, the components represented by peaks 13, 14 and 15 are monascus pigment substances, the component represented by peak 18 is lovastatin, the component represented by peak 10 is citrinin, and the components represented by other 13 chromatographic peaks are Moracolin compounds.
2. The quality control method of saffron according to claim 1, wherein 70% ethanol is used as the extraction solvent in the osmotic extraction process.
3. The quality control method of saffron according to claim 1, wherein the mass content of lovastatin in the saffron is not less than 0.60%, the lovastatin comprising lactone type lovastatin and acid type lovastatin.
4. The method for controlling the quality of saffron according to claim 1, wherein the mass content of beta-glucan in the saffron is not less than 1.80%.
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