AU2020101360A4 - Detection method for mold contamination in traditional chinese medicinal material - Google Patents
Detection method for mold contamination in traditional chinese medicinal material Download PDFInfo
- Publication number
- AU2020101360A4 AU2020101360A4 AU2020101360A AU2020101360A AU2020101360A4 AU 2020101360 A4 AU2020101360 A4 AU 2020101360A4 AU 2020101360 A AU2020101360 A AU 2020101360A AU 2020101360 A AU2020101360 A AU 2020101360A AU 2020101360 A4 AU2020101360 A4 AU 2020101360A4
- Authority
- AU
- Australia
- Prior art keywords
- mold
- mold contamination
- tcm
- solvent
- marker
- 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.)
- Ceased
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
- G01N30/90—Plate chromatography, e.g. thin layer or paper 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
- G01N2030/042—Standards
- G01N2030/045—Standards internal
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)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The present invention provides a detection method for mold contamination of a traditional
Chinese medicinal (TCM) material including the following steps: step 1. preparation of a mold
contamination marker: performing extraction of a mold contaminated TCM material or a mold grown
on a TCM material, filtration and solvent vaporization, adding a solvent for dissolution, loading into
a liquid chromatography, collecting target peak fractions, and concentrating to obtain a mold
contamination marker; step 2. preparation of a test sample solution: adding a solvent to a TCM
material or a powdered TCM preparation sample, performing extraction, filtration and filtrate
vaporization, adding a solvent to a residue for dissolution to obtain a test sample solution; step 3.
analysis of a sample to be tested for mold contamination: determining that a sample has mold
contamination if a chromatogram of a test sample shows a fluorescent spot having a color the same
as that of the mold contamination marker or a peak at a position corresponding to the mold
contamination marker. The present invention is effective in characterizing the quality of a TCM
material (not including fungus medicinal material) or a powdered TCM preparation, thus avoiding
use of mold contaminated TCM materials and improving safe clinical use of TCM materials. The
method of the present invention is advantageous as it is convenient, sensitive and cost-effective.
11/11
DADI Sig=365
4
Ref off(D Data-IelinIhtAnnmRhach201-10-23 Mold extract fromiHelhanthiAnrmRhachi012D
mA
0.4
0).3
0.2
0.1
0
10 20 30 40 50 m
DAD B Sig='2 54 ,4 Ref-off (D:\DataoHeliantlu Annui Rhab 201810-23 Mold extract from %Helianthi Ani Rhach s 012.D)
mAU:
2.5
2
1.5
1I
0.5
10 _ _ _ _ _ _ _ _ _ _ 2 0 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 4 0 _ _ _ _ _ _ _ _ _5 0 _ _ rn
Figure 11a
DAD I A. Si
g
=3
65
4 Refoff (:\DataoHeliathi Anm RhachQ2018S- 10-23 Mold extract from \HehanthiAnnmu Rhachs 011 .D)
mAU
120
100
80
60
40
20
20 40 60 80 100 mir
DAD IB. S g-254,A Re eff (DDaoHehanth Annn Rhacho'2018 -10-23 2Mod extct from Heanthi Annm Rhachu 0 1 .D)
mAU
500
400
300
200
100 - ~l'J: ~ a
20 40 60 80 100 mi
Figure 11b
Description
11/11
4 DADI Sig=365 Ref off(D Data-IelinIhtAnnmRhach201-10-23Mold extract fromiHelhanthiAnrmRhachi012D mA
0.4
).3
0.2
0.1
0 10 20 30 40 50 m DAD B Sig='2 54 ,4 Ref-off (D:\DataoHeliantlu Annui Rhab 201810-23 Mold extract from %Helianthi Ani Rhach s 012.D) mAU: 2.5
2
1.5
1I 0.5
10 __ _ _ _ _ _ _ _ _ 20 _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 40 _ _ _ _ _ _ _ _ _5 0 _ _ rn
Figure 11a g 65 DAD I A. Si =3 4 Refoff (:\DataoHeliathi Anm RhachQ2018S- 10-23 Mold extract from \HehanthiAnnmu Rhachs 011 .D) mAU
120 100 80 60 40 20
20 40 60 80 100 mir DAD IB. S g-254,A Re eff (DDaoHehanth Annn Rhacho'2018 -10-23 2Mod extct from Heanthi Annm Rhachu 0 1 .D) mAU 500
400
300
200
100 - ~l'J: ~ a
20 40 60 80 100 mi
Figure 11b
The present invention relates to a detection method for mold contamination in a substance, in particular to a detection method for mold contamination in a traditional Chinese medicinal (TCM) material.
TCM materials are widely used as they show a treatment effect through multiple ingredients aiming at multiple targets. With increasing research, TCM materials are accepted by more and more people. TCM materials have complex compositions which may easily result in mold contamination if they are stored, transported or treated improperly. Mold contamination is a predominant factor for safe use and efficacy of TCM materials. Pathogens (for example, those generating aflatoxin) commonly seen in TCM materials have potential damaging effect on human body and directly affect their safe use and clinical efficacy. Even if the molds on the surface of a mold contaminated medicinal material are removed, the material will have a change in its composition.
Currently, there are relatively in-depth studies on factors for mold contamination in TCM materials and measures for prevention and treatment of mold contamination. However, there is not yet a matured detection method described in a local standard or an international standard for mold contamination in TCM materials or relative preparations thereof. Detection by naked eyes, microscopy or aflatoxin test are the only methods available for mold contamination now. Since aflatoxin tests of some mold contaminated medicinal materials (for example, Citri Reticulatae Pericarpium) may show negative results, and traditional detection methods for mold contamination through observation of appearance and characteristics by eyes or microscopy cannot determine precisely whether there is slight contamination within the materials, there is a serious threat to the safe clinical use of TCM materials and relative preparations thereof.
Thus, there is a need to establish a detection method for mold contamination in a TCM material to improve the quality estimation system of TCM materials and relative preparations, thus to ensure the safe use of TCM materials.
To overcome the defects in the prior art, the present invention provides a detection method for mold contamination in a TCM material (not including fungus medicinal material) or a powdered TCM preparation which is highly sensitive, efficient and cost-effective. Using the method of the present invention, the quality of a TCM material (not including fungus medicinal material) or a powdered TCM preparation can be controlled to ensure safe clinical use and efficacy.
To achieve the above objective, the present invention provides a detection method for mold contamination in a TCM material, including the following steps:
step 1. preparation of a mold contamination marker
performing extraction of a mold contaminated TCM material or a mold grown on a TCM material, filtration and solvent vaporization to dry, adding a solvent for dissolution, loading into a liquid chromatography, collecting target peak fractions, and concentrating to obtain a mold contamination marker;
step 2. preparation of a test sample solution
adding a solvent to a TCM material or a powdered TCM preparation sample, performing extraction, filtration and filtrate steaming to dry, adding a solvent to a residue for dissolution to obtain a test sample solution;
step 3. analysis of a sample to be tested for mold contamination
determining that a sample has mold contamination if a chromatogram of a test sample shows a fluorescent spot having a color the same as that of the mold contamination marker, or a peak at a position corresponding to the mold contamination marker.
In step 1, the mold contaminated TCM material or the mold grown on a TCM material is added with 5-50 folds of solvent for extraction for 10-180 min, with the solvent selected from one or more of methanol, ethanol, acetone, or ethyl acetate, and the extraction selected from ultrasonic extraction, continuous reflux extraction, reflux extraction, or immersion extraction.
In step 1, after the solvent vaporization, a column is packed with petroleum ether as solvent, a sample is loaded and elution is carried out, then the mold contamination marker is traced by observation, eluted solutions containing the mold contamination marker are collected and combined, and the solvent is recycled.
The mold contamination marker is traced with thin layer chromatography (TLC) by spotting eluted solutions to a silica gel G TLC plate and positioning under an ultraviolet (UV) light for observation, where a developing solvent is used and selected from a mixed solvent of toluene, ethyl acetate or chloroform, formic acid or acetic acid or phosphoric acid in a ratio of (5-10):(3-0.7):(0.5 0.1), or a mixture of petroleum ether and ethyl acetate in a ratio of (20-1):1.
The elution is carried out using petroleum ether, a mixed solution of petroleum ether and ethyl acetate in a ratio of 20:1 or 40:1, and a mixed solution of petroleum ether and ethyl acetate in a ratio of 10:1 or 20:1 successively.
In step 1, the solvent for dissolution is methanol or ethanol in a volume of 1/30-1/2 of the weight of the mold contaminated TCM material or the mold grown on a TCM material.
In step 2, the TCM material or the powdered TCM preparation sample was added with 5-50 folds of solvent for extraction for 10-180 min, with the solvent selected from one or more of methanol, ethanol, acetone, or ethyl acetate, and the extraction selected from ultrasonic extraction, continuous reflux extraction, reflux extraction, or immersion extraction, and the residue is dissolved with 0.5-10 ml of the solvent used for extraction.
In step 3, the analysis of a sample to be tested for mold contamination is carried out with TLC by drawing 1-20 [ of the mold contamination marker or the test sample solution respectively, spotting on a same silica gel G TLC plate, using a developing agent which is a mixed solvent of toluene, ethyl acetate or chloroform, formic acid or acetic acid or phosphoric acid in a ratio of (5 ):(3-0.7):(0.5-0.1), or a mixture of petroleum ether and ethyl acetate in a ratio of (20-1):1, and positioning under an UV light at 365 nm for observation.
In step 3, the analysis of a sample to be tested for mold contamination is carried out with a high performance liquid chromatography (HPLC) method by drawing the mold contamination marker or the test sample solution respectively, injecting into an HPLC and recording the chromatogram.
The HPLC method is carried out by drawing precisely 5-20 [ of the mold contamination marker or the test sample solution respectively, injecting into an HPLC using acetonitrile or methanol and water in a ratio of 95:5-5:95 as a mobile phase, a flow rate of 0.7-1.2 ml/min, and a column temperature of 20-35°C, and measuring at two wavelengths of 254nm and 365nm.
A preparation method for a mold contamination marker established by the present invention can be used to obtain a mold contamination marker which has a specific recognition ability, further to determine whether a sample to be tested is contaminated by mold through comparison of results of the marker and the sample. The detection method for mold contamination in a TCM material (not including fungus medicinal material) or a powdered TCM preparation of the present invention is effective in characterizing the quality of a TCM material (not including fungus medicinal material) or a powdered TCM preparation. Thus, use of mold contaminated TCM materials can be avoided and safe clinical use of TCM materials is improved. The method of the present invention is advantageous as it is convenient, sensitive and cost-effective.
FIG. 1 is a picture comparing TLC observation results of mold contaminated samples from different production regions.
FIG. 2 is a picture comparing TLC observation results of Helianthi Annui Rhachis with or without mold contamination.
FIG. 3 is a picture comparing TLC observation results of mold contamination to different extents.
FIG. 4 is a TLC of mold contamination markers.
FIG. 5 is a TLC showing the minimum detectable amount of a mold contamination marker.
FIGs. 6-7 are TLC showing different medicinal materials before and after mold contamination.
FIGs. 8-9 are three-dimensional chromatograms showing an ingredient with green fluorescence in mold contaminated Citri Reticulatae Pericarpium.
FIG. 10 is a TLC of a TCM preparation, Er Chen Wan.
FIGs. 11A-11B are liquid chromatograms of a mold contamination marker and a mold contaminated sample.
Example 1
Step 1: preparation of a mold contamination marker
1 g of mold grown on a mold contaminated TCM material was added with 50 folds of ethyl acetate for ultrasonic extraction for 10 min, then filtered and spin dried. A small amount of ethyl acetate was added for dissolution, an appropriate amount of silica gel (100-200 meshes) was added with well mixing, and solvent was vaporized to dry using a water bath. A column was wet packed with petroleum ether as solvent, and then a sample was dry loaded. Elution was carried out using petroleum ether, a mixed solution of petroleum ether and ethyl acetate in a ratio of 20:1, and a mixed solution of petroleum ether and ethyl acetate in a ratio of 10:1 successively. Eluted solutions were spotted on a silica gel G TLC plate, developed with toluene-ethyl acetate-formic acid in a ratio of 24:7:1 as a developing solvent, and positioned under a UV light (at 365 nm) for observation. A mold contamination marker was traced, eluted solutions containing target ingredients were collected and combined, the solvent was recycled and a crude extraction of mold contamination marker was obtained.
The crude extraction was added with 0.5 ml of methanol for dissolution, filtered with a 0.22 rm membrane, loaded into an Agilent 1260 HPLC (provided with a DAD detector) using acetonitrile and water in a ratio of 35:65 as a mobile phase, a flow rate of 1.0 ml/min, and a column temperature of 25°C, and measured at two wavelengths of 254 nm and 365 nm. Target peak fractions were collected and concentrated to obtain a mold contamination marker.
Step 2: preparation of a test sample solution
10 g of powdered sample to be tested was added with 5 folds of ethyl acetate for ultrasonic treatment for 20 min, and then filtered. A filtrate was evaporated to dry, and a residue was added with 10ml of ethyl acetate for dissolution to obtain a test sample solution.
Step 3: analysis of a sample for mold contamination with TLC
Test sample solutions were prepared according to step 2 using a sample without mold contamination and 10 mold contaminated samples collected from different regions. 10 1 of each of the mold contamination marker and test sample solutions was separately spotted onto a same silica gel G TLC plate and developed with toluene-ethyl acetate-formic acid in a ratio of 24:7:1 as a developing solvent. Then the plate was taken out, air dried and positioned under a UV light (at 365 nm) for observation. All the mold contaminated samples showed fluorescent spots in green, the same color as that of the mold contamination marker, on the thin layer chromatograms. Results were shown in FIG. 1 where 1-10 represented different batches of mold contaminated Helianthi Annui Rhachis samples, 11 represented a Helianthi Annui Rhachis sample without mold contamination, and 12 represented the mold contamination marker.
Example 2
Step 1: preparation of a mold contamination marker
50 g of mold contaminated sample powder was added with 15 folds of ethanol, subjected to continuous reflux extraction for 60 min, filtered and spin dried. 5 ml of ethanol was added for dissolution, filtered with a 0.22 m membrane, loaded into an Agilent 1260 HPLC (provided with a DAD detector) using acetonitrile and water in a ratio of 35:65 as a mobile phase, a flow rate of 0.9 ml/min and a column temperature of 30°C, and measured at two wavelengths of 254 nm and 365 nm. Target peak fractions were collected and concentrated to obtain a mold contamination marker.
Step 2: preparation of a test sample solution
20 g of powdered sample to be tested was added with 10 folds of ethanol for continuous reflux extraction for 60 min, and then filtered. A filtrate was evaporated to dry, and a residue was added with 10ml of ethanol for dissolution to obtain a test sample solution.
Step 3: analysis of a sample for mold contamination with TLC
Test sample solutions were prepared according to step 2 using samples with or without mold contamination. 1 1 of each of the mold contamination marker and test sample solutions was spotted on a same silica gel G TLC plate and developed with toluene-ethyl acetate-acetic acid in a ratio of :0.7:0.1 as a developing solvent. Then the plate was taken out, air dried and positioned under a UV light (at 365 nm) for observation. All the mold contaminated samples showed fluorescence spots in green, the same color as that of the mold contamination marker, on the thin layer chromatogram.
Example 3
Step 1: preparation of a mold contamination marker
100 g of powdered mold contaminated sample was added with 5 folds of ethanol for ultrasonic extraction for 60 min, then filtered and spin dried. A small amount of ethanol was added for dissolution, an appropriate amount of silica gel (100-200 meshes) was added with well mixing, and solvent was vaporized to dry using a water bath. A column was wet packed with petroleum ether as solvent, and then a sample was dry loaded. Elution was carried out using petroleum ether, a mixed solution of petroleum ether and ethyl acetate in a ratio of 20:1, and a mixed solution of petroleum ether and ethyl acetate in a ratio of 10:1 successively. Eluted solutions were spotted on a silica gel G TLC plate, developed with benzene-chloroform-phosphoric acid in a ratio of 5:3:0.5 as a developing solvent, and positioned under a UV light (at 365 nm) for observation. A mold contamination marker was traced, eluted solutions containing target ingredients were collected and combined, the solvent was recycled and a crude extraction of mold contamination marker was obtained.
The crude extraction was added with 10ml of ethanol for dissolution, filtered with a 0.22 m membrane, loaded into an Agilent 1260 HPLC (provided with a DAD detector) using acetonitrile and water in a ratio of 30:70 as a mobile phase, a flow rate of 0.9 ml/min, and a column temperature of 30°C, and measured at two wavelengths of 254 nm and 365 nm. Target peak fractions were collected and concentrated to obtain a mold contamination marker.
Step 2: preparation of a test sample solution
5 g of powdered sample to be tested was added with 10 folds of ethanol for immersion extraction for 180 min, and then filtered. A filtrate was evaporated to dry, and a residue was added with 5ml of ethanol for dissolution to obtain a test sample solution.
Step 3: analysis of a sample for mold contamination with TLC
Test sample solutions were prepared according to step 2 using samples with or without mold contamination. 5 1 of each of the mold contamination marker and test sample solutions were spotted on a same silica gel G TLC plate and developed with toluene-chloroform-phosphoric acid in a ratio of 5:3:0.5 as a developing solvent. Then the plate was taken out, air dried and positioned under a UV light (at 365 nm) for observation. All the mold contaminated samples showed fluorescence spots in green, the same color as that of the mold contamination marker, on the thin layer chromatogram.
Example 4
Step 1: preparation of a mold contamination marker
300 g of mold grown on a mold contaminated TCM material was added with 30 folds of methanol for flux extraction for 90 min, then filtered and spin dried. A small amount of methanol was added for dissolution, an appropriate amount of silica gel (100-200 meshes) was added with well mixing, and solvent was vaporized to dry using a water bath. A column was wet packed with petroleum ether as solvent, and then a sample was dry loaded. Elution was carried out using petroleum ether, a mixed solution of petroleum ether and ethyl acetate in a ratio of 20:1, and a mixed solution of petroleum ether and ethyl acetate in a ratio of 10:1 successively. Eluted solutions were spotted on a silica gel G TLC plate, developed with petroleum ether and ethyl acetate in a ratio of 20:1 as a developing solvent, and positioned under a UV light (at 365 nm) for observation. A mold contamination marker was traced, eluted solutions containing target ingredients were collected and combined, the solvent was recycled and a crude extraction of mold contamination marker was obtained.
The crude extraction was added with 10ml of methanol for dissolution, filtered with a 0.22 m membrane, loaded into an Agilent 1260 HPLC (provided with a DAD detector) using acetonitrile and water in a ratio of 40:60 as a mobile phase, a flow rate of 1.2 ml/min and a column temperature of 35°C. Measurement was carried out at two wavelengths of 254 nm and 365 nm. Target peak fractions were collected and concentrated to obtain a mold contamination marker.
Step 2: preparation of a test sample solution
2 g of powdered sample to be tested was added with 20 folds of methanol for ultrasound extraction for 10 min, and then filtered. A filtrate was evaporated to dry, and a residue was added with 3 ml of ethanol for dissolution to obtain a test sample solution.
Step 3: analysis of a sample for mold contamination with TLC
Test sample solutions were prepared according to step 2 using samples with or without mold contamination. 10 1 of each of the mold contamination marker and test sample solutions was spotted on a same silica gel G TLC plate and developed with petroleum ether and ethyl acetate in a ratio of 20:1 as a developing solvent. Then the plate was taken out, air dried and positioned under a UV light (at 365 nm) for observation. All the mold contaminated samples showed fluorescent spots in green, the same color as that of the mold contamination marker, on the thin layer chromatogram.
Example 5
Step 1: preparation of a mold contamination marker
500 g of powdered mold contaminated sample was added with 50 folds of acetone for immersion extraction for 180 min, then filtered and spin dried. A small amount of acetone was added for dissolution, an appropriate amount of silica gel (100-200 meshes) was added with well mixing, and solvent was vaporized to dry using a water bath. A column was wet packed with petroleum ether as solvent, and then a sample was dry loaded. Elution was carried out using petroleum ether, a mixed solution of petroleum ether and ethyl acetate in a ratio of 40:1, and a mixed solution of petroleum ether and ethyl acetate in a ratio of 20:1 successively. Eluted solutions were spotted on a silica gel G TLC plate, developed with petroleum ether and ethyl acetate in a ratio of 1:1 as a developing solvent, and position under a UV light (at 365 nm) for observation. A mold contamination marker was traced, eluted solutions containing target ingredients were collected and combined, the solvent was recycled and a crude extraction of mold contamination marker was obtained.
An appropriate amount of the crude extraction was added with 20 ml of methanol for dissolution, filtered with a 0.22 m membrane, loaded into an Agilent 1260 HPLC (provided with a DAD detector) using acetonitrile and water in a ratio of 30:70 as a mobile phase, a flow rate of 1.0 ml/min and a column temperature of 25°C, and measured at two wavelengths of 254 nm and 365 nm. Target peak fractions were collected and concentrated to obtain a mold contamination marker.
Step 2: preparation of a test sample solution
g of powdered sample to be tested was added with 10 folds of acetone for ultrasound extraction for 60 min, and then filtered. A filtrate was evaporated to dry, and a residue was added with 1 ml of acetone for dissolution to obtain a test sample solution.
Step 3: analysis of a sample for mold contamination with TLC
Test sample solutions were prepared according to step 2 using samples with or without mold contamination. 20 1 of each of the mold contamination marker and test sample solutions was spotted on a same silica gel G TLC plate and developed with petroleum ether and ethyl acetate in a ratio of 1:1 as a developing solvent. Then the plate was taken out, air dried and positioned under a
UV light (at 365 nm) for observation. All the mold contaminated samples showed fluorescent spots in green, the same color as that of the mold contamination, on the thin layer chromatogram.
Example 6
Step 1: preparation of a mold contamination marker
750 g of powdered mold contaminated sample was added with 20 folds of ethyl acetate for ultrasonic extraction for 60 min, then filtered and spin dried. A small amount of ethyl acetate was added for dissolution, an appropriate amount of silica gel (100-200 meshes) was added with well mixing, and solvent was vaporized to dry using a water bath. A column was wet packed with petroleum ether as solvent, and then a sample was dry loaded. Elution was carried out using petroleum ether, a mixed solution of petroleum ether and ethyl acetate in a ratio of 40:1, and a mixed solution of petroleum ether and ethyl acetate in a ratio of 20:1 successively. Eluted solutions were spotted on a silica gel G TLC plate, developed with toluene-ethyl acetate-formic acid in a ratio of 24:5:0.5 as a developing solvent, and positioned under a UV light (at 365 nm) for observation. A mold contamination marker was traced, eluted solutions containing target ingredients were collected and combined, the solvent was recycled and a crude extraction of mold contamination marker was obtained.
The crude extraction was added with 30ml of methanol for dissolution, filtered with a 0.22 m membrane, loaded into an Agilent 1260 HPLC (provided with a DAD detector) using methanol and water in a ratio of 95:5 as a mobile phase, a flow rate of 0.7 ml/min and a column temperature of °C, and measured at two wavelengths of 254 nm and 365 nm. Target peak fractions were collected and concentrated to obtain a mold contamination marker.
Step 2: preparation of a test sample solution
0.5 g of powdered sample to be tested was added with 10 folds of ethyl acetate for ultrasonic extraction for 30 min, and then filtered. A filtrate was evaporated to dry, and a residue was added with 0.5 ml of ethyl acetate for dissolution to obtain a test sample solution.
Step 3: analysis of a sample for mold contamination with liquid chromatography
The test sample solution was filtered with a 0.22 m membrane. 20 1 of each of the mold contamination marker and test sample solutions was injected into an Agilent 1260 HPLC (provided with a DAD detector) using methanol and water in a ratio of 95:5 as a mobile phase, octadecyl silane bonded silica as a stationary phase, a flow rate of 0.7 ml/min and a column temperature of °C, and measured at two wavelengths of 254 nm and 365 nm. The chromatogram showed that, the test samples had a peak with the same retention time as that of the mold contamination marker.
Example 7
Step 1: preparation of a mold contamination marker
1000 g of a powdered mold contaminated sample was added with 40 folds of methanol for reflux extraction for 60 min, then filtered and spin dried. A small amount of methanol was added for dissolution, an appropriate amount of silica gel (100-200 meshes) was added with well mixing, and solvent was vaporized to dry using a water bath. A column was wet packed with petroleum ether as solvent, and then a sample was dry loaded. Elution was carried out using petroleum ether, a mixed solution of petroleum ether and ethyl acetate in a ratio of 20:1, and a mixed solution of petroleum ether and ethyl acetate in a ratio of 10:1 successively. Eluted solutions were spotted on a silica gel G TLC plate, developed with toluene-ethyl acetate-acetic acid in a ratio of 10:0.7:0.1 as a developing solvent, and positioned under a UV light (at 365 nm) for observation. A mold contamination marker was traced, eluted solutions containing target ingredients were collected and combined, the solvent was recycled and a crude extraction of mold contamination marker was obtained.
The crude extraction was added with 35 ml of methanol for dissolution, filtered with a 0.22 m membrane, loaded into an Agilent 1260 HPLC (provided with a DAD detector) using acetonitrile and water in a ratio of 5:95 as a mobile phase, a flow rate of 1.2 ml/min and a column temperature of 35°C, and measured at two wavelengths of 254 nm and 365 nm. Target peak fractions were combined and condensed to obtain a mold contamination marker.
Step 2: preparation of a test sample solution
4 g of powdered sample to be tested was added with 50 folds of methanol for continuous reflux extraction for 90 min, and then filtered. A filtrate was evaporated to dry, and a residue was added with 2 ml of ethanol for dissolution to obtain a test sample solution.
Step 3: analysis of a sample for mold contamination with liquid chromatography
The test sample solution was filtered with a 0.22 m membrane. 5 1 of each of the mold contamination marker and test sample solutions was injected into an Agilent 1260 HPLC (provided with a DAD detector) using acetonitrile and water in a ratio of 5:95 as a mobile phase, octadecyl silane bonded silica as a stationary phase, a flow rate of 1.2 ml/min and a column temperature of °C, and measured at two wavelengths of 254 nm and 365 nm. The chromatogram showed that, the test samples had a peak with the same retention time as that of the mold contamination marker.
Experimental Example 1: TLC observations before and after mold contamination
An appropriate amount of samples not contaminated or contaminated by mold was added with folds of methanol for ultrasound treatment for 30 min, and then filtered. A filtrate was evaporated to dry, and a residue was added with 1 ml of ethanol for dissolution to obtain test sample solutions. Test samples and mold contamination marker were drawn and spotted respectively on a same silica gel G TLC plate and developed with toluene-ethyl acetate-formic acid in a ratio of 24:7:1 as a developing solvent. Then the plates were taken out, air dried and positioned under a UV light (at 365 nm) for observation. Results were shown in FIG. 2 where 1 represented a Helianthi Annui Rhachis as a control medicinal material, 2 and 3 represented Helianthi Annui Rhachis which were not contaminated by molds and which were collected in Taonan downtown, 4 and 5 represented mold contaminated Helianthi Annui Rhachis collected in Taonan downtown, 6 and 7 represented Helianthi Annui Rhachis which were not contaminated by molds and which were collected in Erlong village, and 8 and 9 represented mold contaminated Helianthi Annui Rhachis collected in Erlong village. Spot behaviors on TLC were not consistent between samples contaminated and samples not contaminated by molds. Compared with samples not contaminated by molds, the mold contaminated samples had major spots with lighter color accompanied by green florescent spots. It was believed that occurrence of a green florescent spot correlated to mold contamination in a sample to certain extent.
Experimental Example 2: verification of correlation of a green florescent spot to mold contamination in a sample
In this experiment, samples contaminated by molds to different extents were prepared and observed with TLC as described in Experimental Example 1. Results were shown in FIG. 3 where 1 represented a Helianthi Annui Rhachis which was not contaminated by mold, 2-7 represented Helianthi Annui Rhachis samples which were contaminated by molds to different extents. As the extent of mold contamination was increasing, fluorescence intensity of a blue violet spot originally shown with a sample was decreasing but fluorescence intensity of a green florescent spot was increasing, demonstrating that the green florescent spot closely correlates to the mold contamination in a TCM material.
Experimental Example 3: selection of a mold contamination marker
Molds on the surface of a mold contaminated material were stripped. Extract of mold, extract of a material without mold contamination and extract of a mold contaminated material were prepared according to Experimental Example 1and spotted with 10 [ each on a silica gel G TLC plate for observation. Results were shown in FIG. 4 where 1 and 2 represented mold contamination markers, 3 and 4 represented mold contaminated Helianthi Annui Rhachis and 5 represented a Helianthi Annui Rhachis without mold contamination. Molds showed a single green florescent spot at a position corresponding to mold contaminated TCM materials in a chromatogram, and mold contaminated samples showed other spots in addition to the spots corresponding to molds. It was deduced that one of the green florescent spots shown with mold contamination was specific to mold and determined as mold contamination marker, and the other green florescent spot may due to conversion of certain chemical ingredient in a medicinal material. Mold contaminated samples showed florescent spots in a color the same as that of the mold contamination marker while samples without mold contamination had no such spot, demonstrating that this method can be used to detect mold contamination in a TCM material.
Experimental Example 4: measurement of minimum detectable amount of a mold contaminated sample
A test sample solution was prepared according to Experimental Example 1 using 0.5 g of mold contaminated sample, gradually diluted and separately spotted onto a same silica gel G TLC plate and developed with toluene-ethyl acetate-formic acid in a ratio of 24:7:1 as a developing solvent. Then the plate was taken out, dried and positioned under a UV light (at 365 nm) for observation. The minimum detectable amount of a mold contaminated sample was determined by observing whether a mold contamination marker was detectable. Results showed that, the minimum detectable amount of mold contamination was 4.11 g which was sensitive. Results were shown in FIG. 5 and the contents of individual spots showing mold contamination were shown in Table 1. In FIG 5, 1-11 represented test samples of mold contaminated Helianthi Annui Rhachis having different concentrations.
Experimental Example 5: analysis of a sample for mold contamination with TLC
(1) 12 mold contaminated medicinal materials, for example, Bupleuri Radix, Alismatis Rhizoma, Puerariae Lobatae Radix and Cimicifugae Rhizoma, were collected and tested according to Experimental Example 1 using medicinal materials with or without mold contamination. Results showed that, all the mold contaminated samples of Puerariae Lobatae Radix, Astragali Radix, Polygonati Rhizoma, Sophorae Flos, Citri Reticulatae Pericarpium, Ginseng Radix et Rhizoma, Anemarrhenae Rhizoma and Platycodonis Radix had florescent spots in a color the same as that of a mold contamination marker while medicinal materials without mold contamination had no such spots. Samples of Cimicifugae Rhizoma, Bupleuri Radix, Alismatis Rhizoma and Poria with or without mold contamination had weak florescence compared with the mold contamination marker. It was deduced to be resulted by molds which cannot be easily observed by naked eyes and which appeared due to storage for too long. Poria was a dry sclerotium of Poria, a Polyporaceae fungus, and thus mold contamination marker may present in medicinal materials such as Poria originally. Thus, this method can be used to determine mold contamination of a TCM material which was not a fungus since it was sensitive and effective. Results were shown in FIGs. 6 and 7. In FIG. 6, 1 was Sophorae Flos without mold contamination, 2 was mold contaminated Sophorae Flos, 3 was Polygonati Rhizoma without mold contamination, 4 was mold contaminated Polygonati Rhizoma, 5 was Citri Reticulatae Pericarpium without mold contamination, 6 was mold contaminated Citri Reticulatae Pericarpium, 7 was Ginseng Radix et Rhizoma without mold contamination, 8 was mold contaminated Ginseng Radix et Rhizoma, 9 was Alismatis Rhizoma without mold contamination, 10 was mold contaminated Alismatis Rhizoma, 11 was Puerariae Lobatae Radix without mold contamination, 12 was mold contaminated Puerariae Lobatae Radix, and 13 was mold contamination marker. In FIG. 7, 1 was Cimicifugae Rhizoma without mold contamination, 2 was mold contaminated Cimicifugae Rhizoma, 3 was Bupleuri Radix without mold contamination, 4 was mold contaminated Bupleuri Radix, 5 was Platycodonis Radix without mold contamination, 6 was mold contaminated Platycodonis Radix, 7 was Anemarrhenae Rhizoma without mold contamination, 8 was mold contaminated Anemarrhenae Rhizoma, 9 was Astragali Radix without mold contamination, 10 was mold contaminated Astragali Radix, 11 was Poria without mold contamination, 12 was mold contaminated Poria, and 13 was mold contamination marker.
(2) Verification by three-dimensional chromatogram: an appropriate amount of mold contaminated Citri Reticulatae Pericarpium was added with 30 folds of ethyl acetate for ultrasonic extraction for 30 min, and filtered. A filtrate was evaporated to dry. A column was wet packed with silica gel (100-200 meshes) and then a sample was dry loaded. Elution was carried out using petroleum ether, a mixed solution of petroleum ether and ethyl acetate in a ratio of 20:1, and a mixed solution of petroleum ether and ethyl acetate in a ratio of 20:2 successively. Eluted solutions showing florescence at a position corresponding to that of a mold contamination marker traced by TLC were combined, condensed, and filtered with a 0.22 m membrane. Precisely 2 0 [ 1 of each of the mold contamination marker and test sample solution was injected into an Agilent 1260 HPLC (provided with a DAD detector) with acetonitrile and water in a ratio of 40:60 as a mobile phase, octadecyl silane bonded silica as a stationary phase, a flow rate of 1.0 ml/min and a column temperature of 25°C. The three-dimensional chromatograms of Citri Reticulatae Pericarpium and mold contamination marker showed the same trend in absorption curve, based on which it can basically be determined that both samples contained an ingredient of the same kind. Thus this method was accurate. The three-dimensional chromatograms were shown in FIGs 8 and 9.
Experimental Example 6: analysis of Er Chen Wan for mold contamination with TLC
Er Chen Wan samples with or without mold contamination were respectively prepared with Citri Reticulatae Pericarpium with or without mold contamination as raw material using a method described in volume 1 of 2015 Chinese Pharmacopoeia. The samples were pulverized and tested using a method as described in Experimental Example 1. Results showed that, mold contaminated
Citri Reticulatae Pericarpium samples showed florescent spots the same as that of mold contamination marker while samples without mold contamination had no such spot, demonstrating the method can be used to detect mold contamination in a TCM preparation. Results were shown in FIG. 10 where 1 and 2 were TLC chromatograms of Er Chen Wan prepared with mold contaminated Citri Reticulatae Pericarpium, and 3 and 4 were TLC chromatograms of Er Chen Wan prepared with Citri Reticulatae Pericarpium without mold contamination. Mold contamination in a TCM material or a relative preparation can be detected by comparison with the spots of a mold contamination marker.
Experimental Example 7: analysis of a sample for mold contamination with HPLC
A test sample solution was filtered with a 0.22 m membrane. Precisely 20[ 1of each of mold contamination marker and test sample solution was injected into an Agilent 1260 HPLC (provided with a DAD detector) using acetonitrile and water in a ratio of 40:60 as a mobile phase, octadecyl silane bonded silica as a stationary phase, a flow rate of 1.0 ml/min, a column temperature of 25°C and measured at two wavelengths of 254 nm and 365 nm. Results were shown in FIG. 11A showing a liquid chromatogram of the mold contamination marker and FIG. 11B showing a liquid chromatogram of a mold contaminated Helianthi Annui Rhachis with a peak at a position corresponding to retention time which was the same as that of the mold contamination marker, demonstrating that this method can be effectively used in determining whether a TCM material is mold contaminated.
Claims (10)
1. A detection method for mold contamination in a traditional Chinese medicinal (TCM) material, comprising the following steps:
step 1. preparation of a mold contamination marker
performing extraction of a mold contaminated TCM material or a mold grown on a TCM material, filtration and solvent vaporization, adding a solvent for dissolution, loading into a liquid chromatography, collecting target peak fractions, and concentrating to obtain a mold contamination marker;
step 2. preparation of a test sample solution
adding a solvent to a TCM material or a powdered TCM preparation sample, performing extraction, filtration and filtrate steaming to dry, adding a solvent to a residue for dissolution to obtain a test sample solution;
step 3. analysis of a sample to be tested for mold contamination
determining that a sample has mold contamination if a chromatogram of a test sample shows a fluorescent spot having a color the same as that of the mold contamination marker, or a peak at a position corresponding to the mold contamination marker.
2. The detection method for mold contamination in a TCM material according to claim 1, wherein, in step 1, the mold contaminated TCM material or the mold grown on a TCM material is added with 5-50 folds of solvent for extraction for 10-180 min, with the solvent selected from one or more of methanol, ethanol, acetone, or ethyl acetate, and the extraction selected from ultrasonic extraction, continuous reflux extraction, reflux extraction, or immersion extraction.
3. The detection method for mold contamination in a TCM material according to claim 1, wherein, in step 1, after the solvent vaporization, a column is packed with petroleum ether as solvent, a sample is loaded, elution is carried out, then the mold contamination marker is traced by observation, eluted solutions containing the mold contamination marker are collected and combined, and the solvent is recycled.
4. The detection method for mold contamination in a TCM material according to claim 3, wherein, the mold contamination marker is traced with thin layer chromatography (TLC) by spotting the eluted solutions to a silica gel G TLC plate, positioning under an ultraviolet (UV) light for observation, wherein a developing solvent is used and selected from a mixed solvent of toluene, ethyl acetate or chloroform, formic acid or acetic acid or phosphoric acid in a ratio of (5-10):(3 0.7):(0.5-0.1), or a mixture of petroleum ether and ethyl acetate in a ratio of (20-1):1.
5. The detection method for mold contamination in a TCM material according to claim 3, wherein, the elution is carried out using petroleum ether, a mixed solution of petroleum ether and ethyl acetate in a ratio of 20:1 or 40:1, and a mixed solution of petroleum ether and ethyl acetate in a ratio of 10:1 or 20:1 successively.
6. The detection method for mold contamination in a TCM material according to claim 1, wherein, in step 1, the solvent for dissolution is methanol or ethanol in a volume of 1/30-1/2 of the weight of the mold contaminated TCM material or the mold grown on a TCM material.
7. The detection method for mold contamination in a TCM material according to claim 1, wherein, in step 2, the TCM material or the powdered TCM preparation sample is added with 5-50 folds of solvent for extraction for 10-180 min with the solvent selected from one or more of methanol, ethanol, acetone, or ethyl acetate, and the extraction selected from ultrasonic extraction, continuous reflux extraction, reflux extraction, or immersion extraction, and then the residue is dissolved with 0.5-10 ml of the solvent used for extraction.
8. The detection method for mold contamination in a TCM material according to claim 1, wherein, in step 3, the analysis of a test sample for mold contamination is carried out by determining a fluorescent spot having a color the same as that of the mold contamination marker with TLC, that is, by drawing 1-20 1 of the mold contamination marker or the test sample solution respectively, spotting on a silica gel G TLC plate, using a developing solvent which is a mixed solvent of toluene, ethyl acetate or chloroform, formic acid or acetic acid or phosphoric acid in a ratio of (5-10):(3-0.7):(0.5-0.1), or a mixture of petroleum ether and ethyl acetate in a ratio of (20 1):1, positioning under an UV light at 365 nm for observation.
9. The detection method for mold contamination in a TCM material according to claim 1, wherein, in step 3, the analysis of a test sample for mold contamination is carried out by determining a peak at a position the same as that of the mold contamination marker with a high performance liquid chromatography (HPLC) method, that is, by drawing the mold contamination marker and the test sample solution respectively, injecting into an HPLC and recording a chromatogram.
10. The detection method for mold contamination in a TCM material according to claim 9, wherein, the HPLC method is carried out by drawing precisely 5-20 1 of each of the mold contamination marker and the test sample solution respectively, injecting into an HPLC using acetonitrile or methanol, and water in a ratio of 95:5-5:95 as a mobile phase, with a flow rate of 0.7 1.2 ml/min, and a column temperature of 20-35°C, and measuring at two wavelengths of 254 nm and 365 nm.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910644142.4 | 2019-07-17 | ||
| CN201910644142.4A CN110361469A (en) | 2019-07-17 | 2019-07-17 | A kind of Chinese medicine mildew detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU2020101360A4 true AU2020101360A4 (en) | 2020-08-20 |
Family
ID=68219962
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2020101360A Ceased AU2020101360A4 (en) | 2019-07-17 | 2020-07-14 | Detection method for mold contamination in traditional chinese medicinal material |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN110361469A (en) |
| AU (1) | AU2020101360A4 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114689783A (en) * | 2020-12-31 | 2022-07-01 | 四川新绿色药业科技发展有限公司 | Rapid thin-layer identification method for poria cocos, cassia twig, rhizoma atractylodis and licorice root decoction freeze-dried powder |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100363029C (en) * | 2005-10-14 | 2008-01-23 | 贵州益佰制药股份有限公司 | Quality control method of cbinese medicinal preparation |
| CN101144808A (en) * | 2007-11-02 | 2008-03-19 | 建德市大洋化工有限公司 | Trichodermisin determination method and its uses |
| CN101269097B (en) * | 2008-04-30 | 2010-12-29 | 昆明振华制药厂有限公司 | Alstonia-leaf medicinal materials and medicament testing method for its preparation |
| CN101445812A (en) * | 2008-12-12 | 2009-06-03 | 浙江中医药大学 | Method for obtaining new compound group of curcumin by monascus bioconversion |
| CN101949854B (en) * | 2010-09-14 | 2012-10-17 | 云南健牛生物科技有限公司 | Method for rapidly detecting content of aflatoxin |
| WO2014113784A1 (en) * | 2013-01-21 | 2014-07-24 | Xiu-Min Li | Compounds, extracts and methods from chinese medicinal herb sophora flavescens that inhibit airway contraction |
| CN103210964A (en) * | 2013-05-06 | 2013-07-24 | 甘肃中医学院 | Medicine for preventing traditional Chinese medicinal materials from being damaged by worms and mildew, and preparation method thereof |
| CN107298642B (en) * | 2017-07-19 | 2020-09-22 | 重庆大学 | Extraction and purification method of 6-shogaol |
| CN109959620A (en) * | 2017-12-22 | 2019-07-02 | 江苏威凌生化科技有限公司 | The detection method of ochratoxin A in a kind of Chinese medicine |
| CN108918750A (en) * | 2018-07-03 | 2018-11-30 | 西安国联质量检测技术股份有限公司 | The discrimination method of semen coicis in a kind of Chinese medicine compound prescription |
-
2019
- 2019-07-17 CN CN201910644142.4A patent/CN110361469A/en active Pending
-
2020
- 2020-07-14 AU AU2020101360A patent/AU2020101360A4/en not_active Ceased
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114689783A (en) * | 2020-12-31 | 2022-07-01 | 四川新绿色药业科技发展有限公司 | Rapid thin-layer identification method for poria cocos, cassia twig, rhizoma atractylodis and licorice root decoction freeze-dried powder |
| CN114689783B (en) * | 2020-12-31 | 2023-09-12 | 四川新绿色药业科技发展有限公司 | Quick thin-layer identification method for poria, cassia, rhizoma atractylodis and sweet soup freeze-dried powder |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110361469A (en) | 2019-10-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2020101360A4 (en) | Detection method for mold contamination in traditional chinese medicinal material | |
| CN101703611A (en) | Quality detection method of Chinese angelica oral liquid for benefiting blood | |
| CN107315061B (en) | A kind of detection method of alizarin root of Dahurian angelica Chinese materia medica preparation that treating uterus bleeding | |
| CN102552496A (en) | Quality detection method of compound stomachache treating capsules | |
| CN112730674B (en) | Quality detection method of momordica grosvenori tea | |
| CN101181339B (en) | Detection method of aidi injection preparations | |
| CN101336983B (en) | Oldenlandia and quality control method of preparation containing oldenlandia | |
| CN115837063B (en) | Preparation and detection methods of lung force cough pharmaceutical composition | |
| CN100408069C (en) | Quality control method of oral preparation for yin enriching kidney supplementing | |
| CN116626204A (en) | Method for controlling quality of prepared rehmannia root in rehmannia root pill formula preparation | |
| CN101317935B (en) | Rujietai formulation and quality detection method | |
| CN115097040B (en) | UPLC characteristic spectrum construction method and application of semen momordicae | |
| CN101991636B (en) | Detection method of Aidi injection preparation | |
| CN101491630B (en) | Quality control method of Fenqing Wulin pill | |
| CN114441685B (en) | Paris polyphylla standard decoction quality detection method | |
| CN104345108B (en) | Qualitative quantitative determination method for liver-heat-clearing tablet | |
| CN102429998A (en) | Detection method for blood benefiting paste | |
| CN100370253C (en) | Method for quality control of Qianbai biyan solid prepn. for treating rhinitis | |
| CN102000139B (en) | Detection method for Aidi injection | |
| CN115728404A (en) | Lanqin oral liquid contrast extract and preparation method and application thereof | |
| CN106841499A (en) | A kind of method of quality control of old iron stop dysentery soluble powder preparation | |
| CN106383194A (en) | Identification method of herba siegesbeckiae in anti-rheumatism medicinal liquor | |
| CN102068599B (en) | Detection method for phlegm eliminating and asthma relieving cough syrup | |
| CN114487159B (en) | Detection method for fritillary bulb lung-heat clearing syrup | |
| CN110907562A (en) | Quality detection method of stomach harmonizing and detoxifying capsules |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FGI | Letters patent sealed or granted (innovation patent) | ||
| MK22 | Patent ceased section 143a(d), or expired - non payment of renewal fee or expiry |