CN111562251A - Method for rapidly detecting ganoderic acid B in ganoderma lucidum spore powder - Google Patents

Method for rapidly detecting ganoderic acid B in ganoderma lucidum spore powder Download PDF

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CN111562251A
CN111562251A CN202010455703.9A CN202010455703A CN111562251A CN 111562251 A CN111562251 A CN 111562251A CN 202010455703 A CN202010455703 A CN 202010455703A CN 111562251 A CN111562251 A CN 111562251A
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ganoderma lucidum
spore powder
ganoderic acid
sample
lucidum spore
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张兰天
张岩
郭美娟
张斌
张会军
刘�东
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Hebei food inspection research institute
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q

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Abstract

The invention relates to a rapid detection technology of ganoderic acid B in ganoderma lucidum spore powder, in particular to a detection method adopting a Raman spectrum characteristic peak signal, which is suitable for a qualitative detection method of ganoderic acid B in ganoderma lucidum spore powder. Selecting 550cm by using Raman spectrum characteristic peak information‑1(±3cm‑1)、1485cm‑1(±3cm‑1) As a characteristic Raman peak, the method has accurate qualitative property, the whole detection process is completed within 15 minutes, and the rapid qualitative, convenient and low-cost detection is realized.

Description

Method for rapidly detecting ganoderic acid B in ganoderma lucidum spore powder
Technical Field
The invention relates to a rapid detection technology of ganoderic acid B in ganoderma lucidum spore powder, in particular to a detection method adopting a Raman spectrum characteristic peak signal, which is suitable for a rapid qualitative detection method of ganoderic acid B in ganoderma lucidum spore powder.
Background
Ganoderic acid is a triterpene substance contained in Ganoderma, and has more than 100 kinds separated, such as ganoderic acid A, B, C, D, E, F, G, I, L, ma, md, mg, etc. Especially ganoderic acid A, B, C, D. Ganoderic acid can inhibit release of cell histamine, enhance functions of various organs of digestive system, and has effects of reducing blood lipid, lowering blood pressure, protecting liver, and regulating liver function.
At present, in order to further understand the source and distribution rule of the triterpenic acid component in the ganoderma lucidum, the ganoderic acid B with bitter taste in the ganoderma lucidum is generally used as an index in an experiment, and various detection methods are applied to determine the ganoderic acid B in different parts. Wherein the concerned parts in Ganoderma are Ganoderma encarpium and Ganoderma sporophyte. Wherein the ganoderma lucidum fruiting body belongs to woody or herbaceous plants, commonly called ganoderma lucidum whole grass. The ganoderma lucidum sporophyte belongs to the seeds of ganoderma lucidum, is an extremely fine cell apparatus for mass transfer, and a single spore is extremely fine and is only a few microns to ten microns large and can not be seen clearly by naked eyes, so that people usually see that a plurality of spores are concentrated and are commonly called ganoderma lucidum spore powder.
A method for determining ganoderic acid content in Ganoderma product, high performance liquid chromatography (NY/T2278-2012) is adopted as industry standard of Ministry of agriculture, and comprises ultrasonic extracting ganoderic acid A and ganoderic acid B in Ganoderma fruiting body sample with methanol, blowing nitrogen to the extractive solution, dissolving with methanol, measuring with high performance liquid chromatography, and quantifying with retention time and external standard method. The method only aims at measuring the content of ganoderic acid A and ganoderic acid B of the lucid ganoderma sporocarp, but no research on qualitative and quantitative measurement of ganoderic acid A and ganoderic acid B of other lucid ganoderma products by adopting the method is reported at present.
Another method is a method for measuring triterpene and sterol in Ganoderma lucidum extract (T/CCCHMHPIE 1.13-2016): the triterpenoids in the sample are subjected to ultrasonic extraction by absolute ethyl alcohol, a certain amount of the triterpenoids is volatilized, under the action of perchloric acid, the triterpenoids are oxidized and dehydrated, react with a color developing agent vanillin to form a conjugated structure, and form a cationic salt under the action of acid to develop color. The solution has characteristic absorption at 546nm, the color intensity is in direct proportion to the content of triterpenes, and the content of triterpenes and sterols in the sample can be calculated by taking oleanolic acid as a reference substance. The method adopted by the standard is one of the most commonly used quantitative detection methods for the content of total triterpenes at present, but the error of the determination result is large because of a large number of interfering substances in the ganoderma lucidum, and the structural difference between the oleanolic acid serving as the standard and the triterpenes in the ganoderma lucidum is large, so that the method is questioned whether the components are proper as the index components. The method is mainly used for detecting the content of total triterpenes in the sample, and the analysis and detection of triterpenoid monomer components in the sample cannot be realized.
A study carried out by Chinese medical academy of sciences and Chinese cooperative medical university shows that the mass fraction of ganoderic acid in wild Ganoderma lucidum is as high as 0.343%; the highest ganoderic acid content in artificially cultivated ganoderma is only 0.258%, the content of ganoderma spore powder is the lowest, and even the ganoderic acid cannot be detected due to the detection method.
The Raman spectrum is discovered in 1928, the Nobel prize of physics is obtained in 1930, the Nobel prize is universally existed in all molecules, the structure information of the molecules can be reliably provided, the Nobel prize is not influenced by various solvents, the laser Raman spectrum becomes an important analysis means of compounds along with the use of a laser light source, the Nobel prize is widely applied to the fields of criminal investigation identification, mineral analysis and the like, and the surface enhanced Raman scattering discovered in Fleischmann in 1974 enables the detection of trace substances to be possible. The Surface Enhanced Raman Spectroscopy (SERS) utilizes trace molecules to be adsorbed on the surfaces of Cu, Ag, Au and other metal sols and electrodes, and Raman spectrum signals of the SERS can be enhanced by 104～106And the defect of low sensitivity of the conventional Raman spectroscopy is overcome.
In order to overcome the defects of the existing detection method and technology, the invention provides a method for stably, quickly and qualitatively detecting ganoderic acid B in ganoderma lucidum spore powder based on a surface enhanced Raman spectroscopy technology.
Disclosure of Invention
The invention relates to a detection technology for qualitatively analyzing ganoderic acid B in ganoderma lucidum spore powder, which adopts a Raman spectrum characteristic peak signal and selects 550cm-1(±3cm-1)、1485cm-1(±3cm-1) As a characteristic Raman peak, the method has accurate qualitative property, the whole detection process is completed within 10 minutes, and the rapid qualitative, convenient and low-cost detection is realized.
The invention adopts absolute methanol ultrasonic wave to extract ganoderic acid B in ganoderma lucidum spore powder, and uses surface enhanced laser Raman spectrum characteristic peak signals to rapidly determine the ganoderic acid B in the ganoderma lucidum spore powder, wherein: the method comprises the following steps:
preparation of standard substance to-be-detected liquid
Accurately weighing 10mg of ganoderic acid B standard substance, and diluting to 10mL with anhydrous methanol to obtain 1000 μ g/mL standard solution to be detected.
(II) sample pretreatment Process
a. Weighing a proper amount of sample, adding absolute methanol, oscillating, and placing in an ultrasonic extractor for ultrasonic treatment for 10 minutes.
b. Centrifuging the extractive solution at a rotation speed of 1500r/min or more.
c. Taking the supernatant, adding concentrated sulfuric acid, and mixing to obtain the solution to be detected.
The proportion relation of the added anhydrous methanol and the sample is that V: m is 10-5:1 (mL/g); preferably 8-5:1 (mL/g);
the volume ratio of the concentrated sulfuric acid to the supernatant is 1: 1-5; preferably 1: 2.
(III) fast measurement and analysis of surface enhanced laser Raman spectrum
Taking a certain amount of liquid to be detected, sequentially adding surface enhancing reagent and acidifying agent, and recording for 550cm-1(±3cm-1)、1485cm-1(±3cm-1) The existence of characteristic peaks.
The surface enhancing reagent is any one of gold nano sol and silver nano sol, preferably the gold nano sol;
the acidifying agent is a coagulant and is a 2mol/L nitric acid solution;
the volume ratio of the surface enhancing reagent to the liquid to be detected is 1-10: 1; preferably 2-5: 1;
the volume ratio of the acidifying agent to the liquid to be detected is 1: 1-10; preferably 1: 1-5.
The invention adopts special software which is special and solidified in a portable Raman spectrometer to realize an ultra-fast, low-cost and on-site identification method of ganoderic acid B in ganoderma lucidum spore powder. The detection time of one sample is less than 15 minutes, the operation is simple, the operator does not need to have a strong professional background, the reading is visual, and the numerical value can be directly read from the instrument interface.
The invention applies the technology to the detection of the ganoderic acid B in the ganoderma lucidum spore powder for the first time, and determines the ganoderic acid B in the ganoderma lucidum spore powder through the Raman spectrum characteristic analysis of the ganoderic acid B, wherein the lowest detection limit of the detection method is 10 ppm. The test is completed within 15min, has the obvious characteristics of accuracy, rapidness, no damage and low cost, is suitable for field emergency detection, enterprise automatic control detection, large-batch sample screening detection and the like, effectively distinguishes problem ganoderma lucidum spore powder, and protects consumers' rights.
The invention has the advantages that:
1. the method is simple and convenient, has low cost, is beneficial to field operation, and can quickly give out a detection result;
2. qualitative analysis of a large number of samples can be realized simultaneously;
3. the using amount of chemical reagents is greatly reduced, the harm to operators is reduced, and the pollution of wastes to the environment is reduced;
4. high speed (within 15 min), low detection limit, low cost (about 5 yuan for each sample inspection), and easy operation.
Drawings
FIG. 1 Raman spectrum of Ganoderma spore powder containing ganoderic acid B
Detailed Description
The present invention will be described in further detail with reference to examples. But not to limit the invention thereto.
Firstly, preparing standard substance to-be-detected liquid
Accurately weighing 10mg of ganoderic acid B standard substance, and diluting to 10mL with anhydrous methanol to obtain 1000 μ g/mL standard solution to be detected.
Second, sample preparation
a. Weighing a proper amount of sample, adding absolute methanol, oscillating, and placing in an ultrasonic extractor for ultrasonic treatment for 10 minutes.
b. Centrifuging the extractive solution at a rotation speed of 1500r/min or more.
c. Taking the supernatant, adding concentrated sulfuric acid, and mixing to obtain the solution to be detected.
Third, qualitative determination
Conditions of the apparatus
a) Laser power: 200 mW.
b) Integration time: 2 s.
c) Average times: 2.
d) smoothing parameters: 4.
taking a certain amount of liquid to be detected, sequentially adding surface enhancing reagent and acidifying agent, and recording for 550cm-1(±3cm-1)、1485cm-1(±3cm-1) And (3) evaluating whether a characteristic peak exists or not: if the sample does not have a distinct characteristic Raman peak, the content of the ganoderic acid B in the sample is below the detection limit.
Example 1 Rapid qualitative analysis of Ganoderic acid B in Ganoderma spore powder
Sample preparation: is obtained from commercial Ganoderma spore powder A.
a. 0.2g of spore powder is weighed, added with 2mL of anhydrous methanol for oscillation, and placed in an ultrasonic extractor for 10 minutes.
b. Centrifuging the extractive solution at a rotation speed of 1500r/min or more.
c. Taking 200 mu L of supernatant, adding 100 mu L of concentrated sulfuric acid, and uniformly mixing to obtain a solution to be detected.
And (3) qualitative analysis:
taking 100 μ L of solution to be detected, adding 400 μ L of gold nano enhancer, adding 100 μ L of 2mol/L nitric acid solution, shaking up, detecting according to 550cm-1(±3cm-1)、1485cm-1(±3cm-1) Evaluating whether a characteristic Raman peak exists or not:
the solution to be measured is 550cm-1(±3cm-1)、1485cm-1(±3cm-1) If there is a characteristic peak (see fig. 1), it indicates that the sample contains ganoderic acid B.
Example 2 Rapid qualitative analysis of Ganoderic acid B in Ganoderma lucidum spore powder
Sample preparation: is obtained from commercial Ganoderma spore powder B.
a. 0.2g of spore powder is weighed, added with 1mL of anhydrous methanol for oscillation, and placed in an ultrasonic extractor for 10 minutes.
b. Centrifuging the extractive solution at a rotation speed of 1500r/min or more.
c. And adding 50 mu L of concentrated sulfuric acid into 200 mu L of supernatant liquid, and uniformly mixing to obtain a solution to be detected.
And (3) qualitative analysis:
taking 100 μ L of solution to be detected, adding 800 μ L of gold nano-enhancer, adding 50 μ L of acidifying agent, shaking, detecting according to 550cm-1(±3cm-1)、1485cm-1(±3cm-1) Evaluating whether a characteristic Raman peak exists or not:
the solution to be measured is 550cm-1(±3cm-1)、1485cm-1(±3cm-1) If no characteristic peak exists, the sample does not contain ganoderic acid B.
Example 3 Rapid qualitative analysis of Ganoderic acid B in Ganoderma lucidum spore powder
Sample preparation: is obtained from commercial Ganoderma spore powder C.
a. 0.2g of spore powder is weighed, added with 1.6mL of anhydrous methanol for oscillation, and placed in an ultrasonic extractor for 10 minutes.
b. Centrifuging the extractive solution at a rotation speed of 1500r/min or more.
c. And taking 200 mu L of supernatant, adding 200 mu L of concentrated sulfuric acid, and uniformly mixing to obtain the solution to be detected.
And (3) qualitative analysis:
taking 100 μ L of solution to be detected, adding 300 μ L of gold nano-enhancer, adding 25 μ L of acidifying agent, shaking, detecting according to 550cm-1(±3cm-1)、1485cm-1(±3cm-1) Evaluating whether a characteristic Raman peak exists or not:
the solution to be measured is 550cm-1(±3cm-1)、1485cm-1(±3cm-1) If there is a characteristic peak (see fig. 1), it indicates that the sample contains ganoderic acid B.

Claims (7)

1. A method for rapidly detecting ganoderic acid B in ganoderma lucidum spore powder comprises the following steps: the method comprises the following steps:
preparation of standard substance to-be-detected liquid
Accurately weighing 10mg of ganoderic acid B standard substance, and diluting to 10mL with anhydrous methanol to obtain 1000 μ g/mL standard solution to be detected;
(II) sample pretreatment Process
a. Weighing a proper amount of sample, adding absolute methanol for oscillation, and placing the sample in an ultrasonic extractor for ultrasonic treatment for 10 minutes;
b. centrifuging the extractive solution at a rotation speed of 1500r/min or more;
c. taking the supernatant, adding concentrated sulfuric acid, and mixing uniformly to obtain a solution to be detected;
(III) fast measurement and analysis of surface enhanced laser Raman spectrum
Taking a certain amount of liquid to be detected, sequentially adding surface enhancing reagent and acidifying agent, and recording for 550cm-1(±3cm-1)、1485cm-1(±3cm-1) The existence of characteristic peaks.
2. The method for rapidly detecting ganoderic acid B in ganoderma lucidum spore powder according to claim 1, wherein in the pretreatment process of a sample, the proportion relation of the added anhydrous methanol to the sample is V: m-10-5: 1 (mL/g).
3. The method for rapidly detecting ganoderic acid B in ganoderma lucidum spore powder according to claim 1, wherein in the sample pretreatment process, the volume ratio of concentrated sulfuric acid to supernatant is 1: 1-5.
4. The method for rapidly detecting ganoderic acid B in ganoderma lucidum spore powder according to claim 1, wherein in the pretreatment process of a sample, the surface enhancing reagent is any one of gold nano sol and silver nano sol.
5. The method for rapidly detecting ganoderic acid B in ganoderma lucidum spore powder according to claim 1, wherein in the sample pretreatment process, the acidifying agent is 2mol/L nitric acid solution.
6. The method for rapidly detecting ganoderic acid B in ganoderma lucidum spore powder according to claim 1 or 4, wherein the volume ratio of the surface enhancing reagent to the liquid to be detected is 1-10: 1.
7. The method for rapidly detecting ganoderic acid B in ganoderma lucidum spore powder according to claim 1 or 5, wherein the volume ratio of the acidulant to the liquid to be detected is 1: 1-10.
CN202010455703.9A 2020-05-26 2020-05-26 Method for rapidly detecting ganoderic acid B in ganoderma lucidum spore powder Pending CN111562251A (en)

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