CN115236228B - Construction method and quality detection method of she medicine ginseng stem characteristic spectrum - Google Patents
Construction method and quality detection method of she medicine ginseng stem characteristic spectrum Download PDFInfo
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- 239000003208 petroleum Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 201000003068 rheumatic fever Diseases 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- G—PHYSICS
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- 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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a construction method and a quality detection method of a she medicine treelet characteristic map, wherein the quality detection method comprises the following steps: (1) preparing a sample solution; (2) Detecting the contents of syringin and chlorogenic acid in the sample solution under the following high performance liquid chromatography conditions; (3) Detecting the content of isochlorogenic acid A and isochlorogenic acid C in the sample solution under the following high performance liquid chromatography conditions; (4) And comparing the content of the syringin, chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C with a preset value, and judging whether the quality of the sample meets the requirement according to the comparison result. The quality detection method is formulated in a targeted manner according to four most important effective components displayed in the characteristic spectrum, and has reasonable standard and accurate judgment result.
Description
Technical Field
The invention belongs to the technical field of quality detection, and particularly relates to a construction method and a quality detection method of a she medicine ginseng stem characteristic map.
Background
The tree ginseng is shrub or arbor of the Morus genus Dendropanax dentiger (Harms) Merr of Araliaceae family, has long administration history, has the effects of dispelling pathogenic wind, removing dampness, promoting blood circulation and relieving swelling, can be used for treating diseases such as rheumatism, arthritis, hemiplegia and the like, and is one of 30 she drugs with higher frequency of use in Jiangxi and Zhejiang countries in China.
The medicinal part with antirheumatic effect in radix Ginseng comprises root, stem, bark, such as radix Ginseng Rubra, and radix Ginseng Rubra
The Chinese patent of CN202010854436.2 discloses a trepang coumarin compound which is extracted from the root of the trepang and can be used for preparing medicines for treating rheumatoid arthritis or rheumatic arthritis diseases; the Chinese patent application of the invention with the application number of 201710584820.3 discloses a ginseng extract, wherein the raw material of the ginseng extract is Chinese medicinal decoction pieces cut from ginseng branches, and the ginseng extract can also be used for preparing medicaments for treating rheumatoid arthritis.
The composition and the content of the antirheumatic active ingredients contained in the she medicine tree ginseng are different under the influence of various factors such as medicinal part sources, production places, tree ages, growth environments and the like, so that the quality of the she medicine tree ginseng is uneven. Due to the lack of quality standard of the ginseng decoction pieces, not only is the ginseng decoction pieces not dared to be randomly applied clinically, but also the ginseng decoction pieces are widely applied to the medical preparation of maple lotus pears taking ginseng stems as main medicines for resisting rheumatism.
In order to make the she medicine trepang fully applied to benefit the majority of patients, it is imperative to establish a quality detection method of she medicine trepang and to formulate quality standards of she medicine trepang.
Disclosure of Invention
The invention aims to provide a construction method and a quality detection method of she medicine treelet finger print, which are used for solving the problems in the prior art.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the construction method of the she medicine tree ginseng stem characteristic map comprises the following steps:
(a) Preparing 60% ethanol extract of the stem of the ginseng into a test solution;
(b) Obtaining a high performance liquid chromatography of the test solution under the following high performance liquid chromatography conditions, and confirming 34 chromatographic peaks;
the conditions of high performance liquid chromatography include:
chromatographic column: waters canfire C 18 The specification is as follows: 4.6mm by 250mm,5 μm;
mobile phase: phase A is acetonitrile, phase B is a mixed aqueous solution containing 0.2% formic acid and 0.2% tetrahydrofuran;
gradient elution: 0 to 5min,4 to 9%A; 5-35 min, 9-14% of A; 35-50 min,14% A;50 to 60min,14 to 20 percent of A,60 to 80min,20 to 30 percent of A;
detection wavelength: 256nm;
column temperature: 30 ℃;
volume flow rate: 1.0 mL/min -1 ；
Analysis time: 80min;
sample injection amount: 5. Mu.L;
(c) Taking the peak area of each chromatographic peak in the high performance liquid chromatography as an independent variable, taking the cell proliferation inhibition rate as a dependent variable, performing partial least squares regression analysis, calculating the standardized regression coefficient and the VIP value of 34 chromatographic peaks and the cell proliferation inhibition rate, and screening out 19 chromatographic peaks with the VIP value larger than 1;
(d) Taking the cell proliferation inhibition rate as a reference sequence, taking the peak area of each chromatographic peak as a comparison sequence, carrying out dimensionless treatment by adopting a mean change method, respectively calculating correlation coefficients corresponding to each comparison sequence and the reference sequence, taking a sequence average value to obtain gray correlation degree of 34 chromatographic peaks to the cell proliferation inhibition rate, and screening 7 chromatographic peaks with gray correlation degree larger than 0.9;
(e) And 7 characteristic peaks with VIP value greater than 1 and gray correlation degree greater than 0.9 are screened out, and the characteristic map of the she medicine tree ginseng stems is obtained.
The 7 characteristic peaks in the characteristic spectrum are sequentially as follows:
peak No. 7, retention time 14.0min;
peak 11, retention time 16.3min, and corresponding component of syringin;
peak 17, retention time of 20.0min, and chlorogenic acid as the corresponding ingredient;
peak No. 24, retention time 36.9min;
peak No. 27, retention time 47.6min, corresponding ingredient Saikolignanoside A;
peak 33, retention time 67.3min, corresponding component is isochlorogenic acid A;
peak 34, retention time 71.2min, corresponding component was isochlorogenic acid C.
According to the invention, a high performance liquid chromatography is adopted to obtain the chromatogram of the ginseng stem extract, a Partial Least Squares Regression (PLSR) method and a gray correlation (GRA) method are adopted to jointly analyze the relation between chromatographic peaks and the anti-inflammatory effect of the ginseng, 7 characteristic peaks are screened out, the material basis of the effect of the ginseng stem is revealed, and the foundation is laid for reasonable formulation of quality standards and development and application of the she medicine.
Of the 7 characteristic peaks of the characteristic spectrum, the No. 7 peak and the No. 24 peak correspond to unknown components, and the other five characteristic peaks correspond to known components. After the characteristic spectrum is obtained, the characteristic spectrum can be used as a reference, and after the chromatogram of the test sample is obtained by adopting the same method, the chromatogram is compared with the characteristic spectrum, and then the quality of the test sample is obtained according to the comparison result.
In addition to the traditional method, the invention also provides another quality detection method of the she-medicine ginseng stems, which comprises the following steps:
(1) Preparing a sample solution;
(2) Under the following high performance liquid chromatography conditions, the contents of syringin and chlorogenic acid in the sample solution are detected: the chromatographic column is Waters surfire C 18 The volume ratio of the mobile phase is 7:93, wherein the mixed aqueous solution contains 0.2 percent of formic acid and 1.0 percent of tetrahydrofuran; the detection wavelength is 264nm, the column temperature is 30 ℃, and the volume flow is 1.0 mL.min -1 The analysis time is 30min, and the sample injection amount is 5 mu L;
(3) Detecting the content of isochlorogenic acid A and isochlorogenic acid C in the sample solution under the following high performance liquid chromatography conditions: the chromatographic column is Waters surfire C 18 The volume ratio of the mobile phase is 20:80, wherein the mixed aqueous solution contains 0.2 percent of formic acid and 1.0 percent of tetrahydrofuran; the detection wavelength is 328nm, the column temperature is 30 ℃, and the volume flow is 1.0 mL.min -1 The analysis time is 25min, and the sample injection amount is 5 mu L;
(4) And comparing the content of the syringin, chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C with a preset value, and judging whether the quality of the sample meets the requirement according to the comparison result.
The quality detection method is formulated specifically according to four most important effective components (syringin, chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C) displayed in a characteristic map, wherein the quality of the four most important effective components is in accordance with the requirements when the content of the syringin in the sample is not less than 0.010%, the content of chlorogenic acid in the sample is not less than 0.075%, the content of isochlorogenic acid A is not less than 0.047% and the content of isochlorogenic acid C is not less than 0.020%, and the quality is better when the content is higher. The quality detection method is easy to implement, reasonable in standard and accurate in judgment result.
In the step (1) of the quality detection method of the she drug tree ginseng stems, the preparation method of the sample solution comprises the following steps: grinding the stem of the ginseng into powder, uniformly mixing with 60% ethanol, carrying out ultrasonic extraction, filtering, and taking filtrate to obtain the test sample solution.
Preferably, the preparation method of the sample solution comprises the following steps: the powder of the stems of the ginseng and 60% ethanol is mixed with 1g:25mL of the materials are mixed in proportion, the materials are weighed, then ultrasonic treatment is carried out for 1h under the conditions of power of 500W and frequency of 53kHz, the materials are cooled, then the weighed materials are weighed, 60% ethanol is used for supplementing the lost materials, and then a 0.45 mu m microporous filter membrane is used for filtering.
Besides the content indexes of the four effective components, the quality detection method of the invention also comprises the following four indexes: an alcohol-soluble extract content index, a water content index, a total ash content index and an authenticity identification index.
The patented experiments find that the anti-inflammatory effect of the 60% ethanol extract of the ginseng stem is optimal, and the anti-inflammatory effect of the ginseng stem is the result of the combined action of chemical component groups, so that the method for preparing the alcohol-soluble extract of the ginseng stem has important significance except for content measurement.
In the invention, the method for obtaining the content index of the alcohol soluble extract comprises the following steps: the method comprises the steps of taking 60% ethanol as a solvent, obtaining an alcohol-soluble extract of a test sample by adopting a thermal extraction method, weighing after drying, calculating the mass ratio of the alcohol-soluble extract to the test sample, comparing the mass ratio with a preset mass ratio, and judging whether the mass of the test sample meets the requirement or not according to a comparison result.
Preferably, the content of the alcohol-soluble extract is not less than 5.0% by mass based on the dry product (test product). The method for obtaining the moisture content index and the total ash content index comprises the following steps: the water content and the total ash content of the sample are detected according to the water content measuring method and the ash content measuring method recorded in Chinese pharmacopoeia, the water content and the total ash content are compared with the preset content respectively, and whether the quality of the sample meets the requirements is judged according to the comparison result.
Preferably, the moisture content is not more than 12.0% and the total ash content is not more than 5.0% by mass of the dry product (test product).
The authenticity identification is to determine whether the sample is a genuine trepang product by carrying out microscopic identification and thin layer identification on the sample.
Wherein, the characteristics of the genuine ginseng stem are as follows: cylindrical or quasi-cylindrical, 15-30 cm long, 3-8 cm diameter, grayish green to tan surface, transverse leather holes or a plurality of protrusions, coarse 'wrinkles' formed by long leather holes, brown to tan leather holes, gray yellow or gray brown color spots after lichen is removed on the surface, yellow or gray yellow sawn surfaces, and a few rings of annual rings can be seen, and the central marrow part is quasi-white, punctiform, firm, slightly gas, slightly astringent, pi Wei astringent and slightly bitter.
Microscopic identification: the powder of the stem of the ginseng is brown yellow. The wood fiber wall is thicker, the hole grooves and the tattoos are more obvious, and the tattoos are edge tattoos; wood plug tissue fragment cells are elliptic, rectangular or irregular, have wall thickness and contain brown matters; the calcium oxalate cluster crystals are more, yellow brown, blunt in edges and corners and 10-25 mu m in diameter; stone cells are scattered in groups like squares, ovals or polygons, the diameter is 15-30 mu m, the wall is thicker, the pore canal is fine, and the cell cavity is large; the catheter is provided with edge holes, the edge holes are round, and the arrangement of the holes is tight; bast fibers are elongated, have slightly pointed or oval ends and different wall thicknesses, have cell membranes, have deep grooves and have visible pits.
The thin layer identification comprises a thin layer identification method of syringin and a thin layer identification method of chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C, and two sets of thin layer identification systems are adopted to identify the existence of four effective components, so that the identification result is more accurate.
The thin-layer identification method of the syringin comprises the following steps: respectively dispensing the sample solution and syringin reference solution on the same silica gel GF 254 On the thin layer plate, chloroform, methanol and formic acid with volume ratio of 6:1.2:0.5 are used as developing agent, and after developing, the thin layer plate is taken out and dried, and is inspected under a 254nm ultraviolet lamp to see whether the color spectrum of the sample has bright blue fluorescent spots corresponding to the color spectrum position of the syringin reference substance.
The thin layer identification method of chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C comprises the following steps: the sample solution and the reference substance mixed solution are respectively spotted on the same silica gel H thin layer plate, butyl acetate, formic acid and water with the volume ratio of 7:2.5:2.5 are taken as developing agents, taken out after the developing, dried in the air, inspected under an ultraviolet light lamp with the wavelength of 365nm, and whether three bright blue fluorescent spots corresponding to the chromatographic positions of the reference substance appear in the chromatogram of the sample or not is checked.
Under the condition that the sample to be tested is identified as a genuine product according to the method, and the content index of the effective components, the content index of the alcohol soluble extract, the content index of the moisture and the content index of the total ash are all in accordance with the requirements, the quality of the sample to be tested is qualified; the greater the difference between each content index and the minimum or maximum value, the better the quality.
Compared with the prior art, the invention has the beneficial effects that:
(1) According to the invention, a high performance liquid chromatography is adopted to obtain the chromatogram of the ginseng stem extract, a Partial Least Squares Regression (PLSR) method and a gray correlation (GRA) method are adopted to jointly analyze the relation between chromatographic peaks and the anti-inflammatory effect of the ginseng, 7 characteristic peaks are screened out, the material basis of the effect of the ginseng stem is revealed, and the foundation is laid for reasonable formulation of quality standards and development and application of the she medicine.
(2) The invention also provides a quality detection method of the stem of the tree ginseng according to four most important active ingredients (syringin, chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C) shown in the characteristic map, wherein the quality of the stem of the tree ginseng meets the requirements when the content of the syringin in the sample is not less than 0.010%, the content of chlorogenic acid in the sample is not less than 0.075%, the content of isochlorogenic acid A is not less than 0.047% and the content of isochlorogenic acid C is not less than 0.020%, and the quality is better when the content is higher; the quality detection method is easy to implement, reasonable in standard and accurate in judgment result.
(3) Besides the content indexes of the four effective components, the quality detection method of the invention also comprises the following four indexes: an alcohol-soluble extract content index, a water content index, a total ash content index and an authenticity identification index; under the condition that the sample is identified as a genuine product and the content index of the effective components, the content index of the alcohol soluble extract, the content index of the water and the content index of the total ash are all in accordance with the requirements, the quality of the sample is qualified; the greater the difference between each content index and the minimum or maximum value, the better the quality.
Drawings
FIG. 1 is a high performance liquid chromatogram summary of 11 extracts obtained from the stems of Panax schinseng;
wherein S1 represents a water extract, S2 represents a 20% ethanol extract, S3 represents a 40% ethanol extract, S4 represents a 60% ethanol extract, S5 represents an 80% ethanol extract, S6 represents a 100% ethanol extract, S7 represents an n-butanol extract, S8 represents a water extract after ethanol extraction, S9 represents a chloroform extract, S10 represents an diethyl ether extract, and S11 represents an ethyl acetate extract; the following is the same;
FIG. 2 is a high performance liquid chromatogram of nine controls;
wherein, peak 11 is syringin, peak 17 is chlorogenic acid, peak 22 is sinapial glucoside, peak 23 is coniferyl alcohol, peak 27 is Saikolignanoside A, peak 28 is rutin, peak 32 is 3, 4-O-dicaffeoylquinic acid, peak 33 is 3, 5-O-dicaffeoylquinic acid (isochlorogenic acid A), and peak 34 is 4, 5-O-dicaffeoylquinic acid (isochlorogenic acid C);
FIG. 3 is an absorbance value after 11 trepang stem extracts were administered to cells;
wherein, the blank represents a blank control group, the MTX represents a positive methotrexate group, and the model represents a model group; the following is the same; * Represents P <0.01 compared to the blank; # represents P <0.01 compared to TNF- α model group;
FIG. 4 shows the measurement results of the inhibition rate of cell proliferation by 11 extracts of the stems of Panax ginseng C.A.Meyer;
wherein # denotes P <0.01 compared to MTX group, # denotes P <0.05 compared to MTX group;
FIG. 5 is a graph of PLSR normalization regression coefficients of a ginseng stem extract;
wherein Var ID (Primary) represents peak number and Coeffcs (Var-35) represents PLSR normalization regression coefficient; the following is the same;
FIG. 6 is a graph showing the VIP contribution of the extract of the stems of Panax ginseng;
FIG. 7 is a microscopic identification of the powder of the stems of Panax ginseng;
wherein 1 represents wood fiber, 2 represents wood plug tissue fragment cells, 3 represents calcium oxalate cluster crystals, 4 represents stone cells, 5 represents a catheter, and 6 represents bast fiber;
FIG. 8 is a thin-layer chromatography identification chart of the effective component syringin in the extract of the stem of the Panax ginseng C.A.Meyer,
wherein, 1 and 14 are syringin reference substance solutions, and 2-13 are sample solutions prepared from No. 1-12 of the stem sample of the radix Ginseng;
FIG. 9 is a thin layer chromatography identification chart of chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C as effective components in the stem extract of Panax ginseng;
wherein 1 and 14 are mixed reference substance solutions of chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C, and 2-13 are test substance solutions prepared from No. 1-12 of Ginseng radix stem samples;
FIG. 10 is a high performance liquid chromatogram of a mixed control solution of syringin and chlorogenic acid;
Wherein, peak 1 is syringin, peak 2 is chlorogenic acid, min represents retention time, and mAU represents milliabsorbance unit;
FIG. 11 is a high performance liquid chromatogram of a test solution prepared from a ginseng stem sample;
wherein, peak 1 is syringin, peak 2 is chlorogenic acid, min represents retention time, and mAU represents milliabsorbance unit;
FIG. 12 is a high performance liquid chromatogram of a mixed control solution of isochlorogenic acid A and isochlorogenic acid C;
wherein, peak 1 is isochlorogenic acid A, peak 2 is isochlorogenic acid C, min represents retention time, mAU represents milliabsorbance unit;
FIG. 13 is a high performance liquid chromatogram of a test solution prepared from a stem sample of Panax ginseng;
wherein, peak 1 is isochlorogenic acid A, peak 2 is isochlorogenic acid C, min represents retention time, mAU represents milliabsorbance unit.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to the attached drawings and the detailed description.
The instruments and reagents used in the examples of the present invention are as follows:
instrument: 1260 high performance liquid chromatograph (agilent, DAD detector); XS105DU type electronic balance (mertrer); IX71 fluorescence inverted microscope (Olinbas); MK3 microplate reader (sameifer); 3111 carbon dioxide incubator (sameifer); TC20 type cell counter (berle); s105DU type electronic balance (mertrehler switzerland); DM2500 upright microscope (laika germany); a membert UNE400 natural convection oven (membert inc, germany); SK8210HP ultrasonic cleaner (Shanghai Kochia Utility ultrasonic instruments Co., ltd.); SXL-1008 program controlled box electric furnace (Shanghai macro laboratory equipment Co., ltd.); silica gel H thin layer plates and silica gel G254 thin layer plates were all purchased from Qingdao ocean chemical Co.
Control: syringin reference (DST 200912-012, content 98.54%), chlorogenic acid reference (DSTDL 002102, content 99.56%), isochlorogenic acid A reference (DSTDY 003601, content 98.56%), isochlorogenic acid C reference (DSTDY 003802, content 99.58%), sinapine aldehyde glucoside reference (DST 210311-292, content not less than 95%) were purchased from Desmot Biotechnology Co., ltd; rutin control (100080-201409, 91.9% content) was purchased from China food and drug testing institute; saikolignanoside A control (103203, content > 95%) was purchased from Jiangsu Yongjian medical science and technology Co., ltd; coniferyl alcohol reference substance (wkq 20052602, content not less than 98%) was purchased from vickers biotechnology limited, sichuan province.
And (3) cells: HFLS-OA (third generation) was purchased from Beijing along-path technology Co.
Reagent: acetonitrile, formic acid and tetrahydrofuran are chromatographic purity; chloroform, petroleum ether, diethyl ether, ethyl acetate, n-butanol, and methanol are analytically pure; the water is ouha purified water; methotrexate injection (MTX, lot number: DL6898, specification: 2mL:50 mg) was purchased from Pfizer (Perth) Pty Limited; TNF- α was purchased from Nanjing Jinsri biotechnology Co., ltd; high sugar medium (DMEM), australian priority Fetal Bovine Serum (FBS), dimethyl sulfoxide (DMSO) were all purchased from Gibco company in the united states; thiazole blue reagent (MTT) was purchased from amerco corporation, usa; double antibody (penicillin-streptomycin 100X) was purchased from Shanghai source culture Biotech Co., ltd; trypsin (EDTA) was purchased from kinoform biomedical technologies limited.
The ginseng stem samples are prepared by mixing 12 batches of ginseng stem samples in equal quantity, and the 12 batches of ginseng stem samples are identified as ginseng Dendropanax dentiger of Araliaceae plant by Li Jianliang major Chinese medicinal pharmacist. The sources are shown in Table 1.
TABLE 1 Ginseng Stem extract sample Source
| Sample numbering | Harvesting land | Date of collection |
| 1 | Lishui city lotus district Pang Shancun | 20180702 |
| 2 | Huang Ducun of Qingdan county in Lishui city | 20180707 |
| 3 | Lishui city cloud and county Zhu Cun | 20180709 |
| 4 | Dam town of Pingyang county in Lishui city | 20180709 |
| 5 | Grass carp pond in Jing Ning county in Lishui city | 20180714 |
| 6 | Zhang Cun of Qingdan county in Lishui city | 20180719 |
| 7 | Lishuilongquan city | 20190321 |
| 8 | Fujian province and Dengde City | 20190321 |
| 9 | Eucalyptus and village in Sunchang county in Lishui city | 20200410 |
| 10 | Nanping City, Fujian Province | 20200427 |
| 11 | Zhang Cun of Qingdan county in Lishui city | 20200524 |
| 12 | Lishui city Qingdan county upward Tianhu | 20200614 |
Example 1
Extracts of the trepang stem samples are obtained by adopting different solvents, and anti-inflammatory effects of the extracts are researched to construct a characteristic map of the trepang stem. The specific process is as follows:
1. preparing the extract of the stem of the ginseng into a test solution:
taking 100g of dried ginseng stem powder (equal amount of 12 batches of samples are mixed) passing through a second sieve, heating and reflux-extracting with 10 times of pure water for 3 times each time for 2 hours, mixing the extracting solutions, concentrating under reduced pressure, and then drying in a reduced pressure drying oven to obtain a water extract (S1); weighing 5 parts of dried powder (mixed in equal amounts by 12 batches of samples) of the ginseng stems of the second sieve, wherein each 300g of powder is respectively subjected to heating reflux extraction for 3 times by taking 8 times of 20%, 40%, 60%, 80% and 100% ethanol as solvents, each time for 2 hours, merging the extracting solutions, concentrating under reduced pressure until no alcohol smell exists, and drying in a reduced pressure drying box to obtain ethanol extracts with corresponding concentrations (S2-S6 respectively); adding 10 times of pure water into the medicinal material powder after 80% ethanol extraction, heating and refluxing for extraction for 3 times, each time for 2 hours, combining the extracting solutions, concentrating under reduced pressure, and drying in a reduced pressure drying oven to obtain an ethanol extracted water extract (S8); weighing 5g of 80% ethanol extract, suspending in 50mL of water, transferring to a separating funnel, sequentially adding chloroform, diethyl ether, ethyl acetate and n-butanol, extracting for 3 times, respectively collecting the extracted components, and drying under reduced pressure to obtain corresponding solvent extracts (S9, S10, S11 and S7 respectively);
Weighing the extract equivalent to 5g of the crude drug, dissolving with an extraction solvent, fixing the volume to a 5mL volumetric flask, and filtering to obtain a test solution.
2. Preparing a mixed reference substance solution:
accurately weighing appropriate amount of standard substance into 10mL volumetric flask, dissolving in methanol, and fixing volume to obtain a solution containing 618.0 mg.L of syringin per 1mL -1 621.0 mg.L chlorogenic acid -1 Sinapine aldehyde glucoside 361.0 mg.L -1 579.0 mg.L of coniferyl alcohol -1 、Saikolignanoside A 400.0mg·L -1 Rutin 402.7 mg.L -1 Isochlorogenic acid A554.0 mg.L -1 Isochlorogenic acid B572.0mg.L -1 Isochlorogenic acid C506.0 mg.L -1 Standard stock solution of (2); and respectively and precisely transferring 1.5, 1, 0.5, 0.2, 1mL of the standard substance stock solution into the same 15mL volumetric flask, and adding methanol to a fixed volume to a scale to obtain a mixed reference substance solution.
3. High performance liquid chromatography detection:
11 parts of test solution and 1 part of mixed reference solution were analyzed under the following conditions of high performance liquid chromatography:
chromatographic column: waters canfire C 18 The specification is as follows: 4.6mm by 250mm,5 μm;
mobile phase: phase A is acetonitrile, phase B is a mixed aqueous solution containing 0.2% formic acid and 0.2% tetrahydrofuran;
gradient elution: 0 to 5min,4 to 9%A; 5-35 min, 9-14% of A; 35-50 min,14% A;50 to 60min,14 to 20 percent of A,60 to 80min,20 to 30 percent of A;
Detection wavelength: 256nm; column temperature: 30 ℃; volume flow rate: 1.0 mL/min -1 The method comprises the steps of carrying out a first treatment on the surface of the Analysis time: 80min;
sample injection amount: 5. Mu.L; analysis results fig. 1 and 2.
Comparing the chromatograms of 11 test solutions, determining 34 obvious characteristic peaks and recording the corresponding peak areas, and identifying 9 components by a reference substance comparison method according to the identification result of the chemical components of the ginseng stems.
4. Anti-inflammatory action study:
1) Cell culture: inoculating HFLS-OA cells into DMEM medium containing 10% foetal calf serum and 1% diabody, placing at 37deg.C and 5% CO 2 And (3) carrying out stationary culture in an incubator, carrying out passage when the cell grows to 80% -90%, and inoculating the cell into a culture bottle or a culture plate for culture.
2) Molding and grouping: HFLS-OA cell modeling was stimulated with TNF- α and cells were divided into blank groups (DMEM broth), model groups (final added mass concentration of 10 ng.mL) -1 TNF- α solution of (c); positive methotrexate group (MTX, added to a final mass concentration of 20mg.L) -1 And 10 ng.mL -1 Mixed solution of TNF-alpha solution); different extracts of the stem of the ginseng (S1-S11, added with the final mass concentration of 31.25 mg.mL) -1 And 10ng mL of the extract solution -1 Mixed solution of TNF-alpha solution).
3) Cell proliferation inhibition assay: measuring cell proliferation activity by MTT method, taking logarithmic phase cell, digesting with pancreatin containing 0.25% EDTA, and measuring 800rpm min -1 Centrifuging for 5min, and inoculating into 96-well culture plates with 100 μl per well; blowing and mixing with disposable straw to obtain single cell suspension, placing at 37deg.C and 5% CO 2 Culturing in incubator until cell fusionAfter 80% of the cells were dosed in a preset group, 100. Mu.L of each well was subjected to 6 multiplex wells per concentration, after 24 hours of incubation, 20. Mu.L of MTT solution was added to each well, after 4 hours of incubation at 37℃the absorbance value (A) of each well was measured at 490nm using a microplate reader, and the cell proliferation inhibition ratio was calculated as cell proliferation inhibition ratio (%) = (TNF- α model group A value-drug group A value)/TNF- α model group A value X100%, and repeated 3 times.
4) Statistical analysis: the data were analyzed using SPSS 23.0 software, one-way analysis of variance was selected, dunnett test was used for comparison with the reference group, tukey test was used for comparison between groupsRepresenting the data.
As a result, it was found that TNF-. Alpha.model cells proliferated significantly as compared with the blank group (P<0.01 A) is provided; compared with the model group, the proliferation of cells of each administration group is obviously inhibited except the water extract after the S8 alcohol extraction (P<0.05 A) is provided; the extract of the stems of the ginseng tree is 20 mg.L relative to the extract of the stems of the ginseng tree -1 The positive MTX groups have obvious differences, the HFLS-OA proliferation inhibition rates of S1, S2, S3, S4 and S7 are higher, the range is 41.9-42.9%, and the positive MTX groups have no obvious differences; under the same conditions, the secondary conditions are that the inhibition rate of the HFLS-OA is 37.8-39.6%, the inhibition rate of the HFLS-OA of the S11 is 32.3% and the S8 has no obvious inhibition effect. The results are shown in detail in FIGS. 3 and 4.
5. And (3) constructing a characteristic map:
1) PLSR method analysis
The peak area of each chromatographic peak in the chromatogram is taken as an independent variable, the cell proliferation inhibition rate is taken as a dependent variable, the partial least squares regression analysis is carried out by adopting Simca-P14.1 software, standard deviation standardization treatment is selected for data, the variable dimension difference is eliminated, and standardized regression coefficients (figure 5) and VIP values (figure 6) of 34 characteristic peaks and inhibition rates are obtained through calculation.
When VIP >1, the independent variable has significance in interpreting the dependent variable; as can be seen from fig. 6, the VIP values of peaks 34, 21, 22, 17, 11, 27, 7, 13, 24, 8, 4, 31, 14, 9, 33, 29, 16, 3, 10 are all greater than 1 (ranging from large to small VIP values), and the regression coefficients thereof are all positive correlations, indicating that the substances corresponding to these peak numbers have a significant effect on the anti-inflammatory effect of cells.
2) GRA assay
Taking the cell proliferation inhibition rate as a reference sequence, taking the peak area of each chromatographic peak in the chromatogram as a comparison sequence, carrying out dimensionless treatment by adopting a mean change method, respectively calculating the correlation coefficient corresponding to each comparison sequence and the reference sequence, and taking a sequence average value to obtain the gray correlation degree (r) of 34 chromatographic peaks to the inhibition rate, wherein the gray correlation degree (r) is shown in table 2.
TABLE 2 Gray correlation results of 34 chromatographic peaks versus inhibition rate
The greater the degree of association, the greater the effect of the substance on the efficacy. As can be seen from table 2, the r values of the other peaks, except peaks 12, 26, were all greater than 0.8; wherein the first 7 peaks with maximum anti-inflammatory effect contribution and r.gtoreq.0.9 are 34, 17, 11, 7, 24, 33, 27, respectively.
3) PLSR and GRA combined comparison
The chromatographic peaks with VIP >1 and r more than or equal to 0.9 are integrated to obtain overlapped chromatographic peaks, the correlation with the anti-inflammatory action is from peak 34>17>11>7>24>33>27 in a sequence from top to bottom, which shows that the components corresponding to the peak numbers have important influence on the anti-inflammatory action of the treelet stems.
Thus, the characteristic spectrum of the ginseng stem is obtained, and the characteristic spectrum contains:
peak No. 7, retention time 14.0min, unknown corresponding composition;
peak 11, retention time 16.3min, and corresponding component of syringin;
peak 17, retention time of 20.0min, and chlorogenic acid as the corresponding ingredient;
peak No. 24, retention time 36.9min, unknown corresponding composition;
peak No. 27, retention time 47.6min, corresponding ingredient Saikolignanoside A;
peak 33, retention time 67.3min, corresponding component is isochlorogenic acid A;
peak 34, retention time 71.2min, corresponding component was isochlorogenic acid C.
Example 2
On the basis of the characteristic spectrum constructed in the embodiment 1, the embodiment provides a quality detection method of the she drug ginseng stems, which comprises the following steps:
1. authentication and verification
The characteristics of the genuine ginseng stem are as follows: cylindrical or quasi-cylindrical, 15-30 cm long, 3-8 cm diameter, grayish green to tan surface, transverse leather holes or a plurality of protrusions, coarse 'wrinkles' formed by long leather holes, brown to tan leather holes, gray yellow or gray brown color spots after lichen is removed on the surface, yellow or gray yellow sawn surfaces, and a few rings of annual rings can be seen, and the central marrow part is quasi-white, punctiform, firm, slightly gas, slightly astringent, pi Wei astringent and slightly bitter.
(1) Microscopic authentication (as in fig. 7): the powder of the stem of the ginseng is brown yellow. The wood fiber wall is thicker, the hole grooves and the tattoos are more obvious, and the tattoos are edge tattoos; wood plug tissue fragment cells are elliptic, rectangular or irregular, have wall thickness and contain brown matters; the calcium oxalate cluster crystals are more, yellow brown, blunt in edges and corners and 10-25 mu m in diameter; stone cells are scattered in groups like squares, ovals or polygons, the diameter is 15-30 mu m, the wall is thicker, the pore canal is fine, and the cell cavity is large; the catheter is provided with edge holes, the edge holes are round, and the arrangement of the holes is tight; bast fibers are elongated, have slightly pointed or oval ends and different wall thicknesses, have cell membranes, have deep grooves and have visible pits.
(2) Thin layer authentication:
(1) taking 1g of test sample powder, extracting with 25mL of 60% ethanol by ultrasonic wave (with power of 500W and frequency of 53 kHz) for 1 hour, evaporating the extract to dryness, and dissolving with 2mL of ethanol to obtain a test sample solution; and adding methanol into syringin reference substance to obtain solution containing 1mg of syringin per 1mL, and taking the solution as reference substance solution.
According to thin layer chromatography (general rule 0502), sucking 5-10 μl of sample solution and 5 μl of reference solution, respectively spotting on the same siliconGel GF 254 On the thin layer plate, chloroform-methanol-formic acid (volume ratio of 6:1.2:0.5) is used as developing agent, and the thin layer plate is developed, taken out, dried and inspected under an ultraviolet lamp (254 nm); if the color spectrum of the sample shows a bright blue fluorescence spot corresponding to the color spectrum position of the syringin reference substance, the sample is a true sample (as shown in figure 8).
(2) Taking 1g of the product powder, extracting with 25mL of 60% ethanol by ultrasonic wave (with the power of 500W and the frequency of 53 kHz) for 1 hour, evaporating the extracting solution to dryness, and dissolving with 2mL of ethanol to obtain a sample solution; and adding methanol into chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C to obtain mixed solution containing 1mg chlorogenic acid, 1mg isochlorogenic acid A and 1mg isochlorogenic acid C per 1mL, and making into reference solution.
According to thin layer chromatography (general rule 0502) test, sucking 5-10 μl of sample solution and 5 μl of reference solution, respectively spotting on the same silica gel H thin layer plate, spreading with butyl acetate-formic acid-water (volume ratio of 7:2.5:2.5) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm); if the sample has bright blue fluorescence spots corresponding to chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C in the control, the sample is true (see figure 9).
2. Moisture and total ash content detection
The water content of the test sample is detected according to the second method (general rule 0832) of the water content measuring method of the Chinese pharmacopoeia (2020 edition), and the total ash content of the test sample is detected according to the ash content measuring method (general rule 2302) of the Chinese pharmacopoeia (2020 edition), and the detection result is shown in table 3.
3. Alcohol soluble extract content detection
According to the extract measurement method (general rule 2201) of Chinese pharmacopoeia (2020 edition), 60% ethanol is used as solvent, and the alcohol-soluble extract content of the test sample is detected by adopting a hot dipping method, and the detection result is shown in Table 3.
TABLE 3 results of the moisture, total ash and alcohol soluble extract content measurements for each sample
| Sample numbering | Moisture content (%) | Total ash (%) | Extract (%) |
| 1 | 9.9 | 1.0 | 5.9 |
| 2 | 9.8 | 2.1 | 5.0 |
| 3 | 9.5 | 1.5 | 6.4 |
| 4 | 10.0 | 1.7 | 6.6 |
| 5 | 9.6 | 2.0 | 5.9 |
| 6 | 10.1 | 1.9 | 5.7 |
| 7 | 9.0 | 4.1 | 9.8 |
| 8 | 10.6 | 1.4 | 7.9 |
| 9 | 8.2 | 2.4 | 5.8 |
| 10 | 9.5 | 2.8 | 5.6 |
| 11 | 9.8 | 3.4 | 5.8 |
| 12 | 8.9 | 1.8 | 5.1 |
The water content is not more than 12.0%, the total ash content is not more than 5.0% and the alcohol-soluble extract content is not less than 5.0% according to the mass percentage of the dry product (the sample), thereby meeting the requirements of the quality detection of the invention.
4. Detection of content of effective components
(1) Preparing a sample solution and a mixed reference substance solution;
taking 2g of ginseng stem powder (sieving with a fourth sieve), precisely adding 50mL of 60% ethanol, weighing, performing ultrasonic treatment under the conditions of power of 500W and frequency of 53kHz for 1h, cooling, weighing again, supplementing the lost weight with 60% ethanol, and filtering with a 0.45 mu m microporous filter membrane to obtain a sample solution;
Precisely weighing appropriate amounts of syringin reference substance and chlorogenic acid reference substance, and diluting with 50% methanol to obtain mixed reference substance solutions containing 35.74 μg of syringin and 77.52 μg of chlorogenic acid per 1mL (step (2);
precisely weighing appropriate amounts of isochlorogenic acid A and isochlorogenic acid C reference substances, and diluting with 50% methanol to obtain mixed reference substance solutions containing each 1mL of isochlorogenic acid A44.56 μg and isochlorogenic acid C24.68 μg (step (3);
(2) Content determination of syringin and chlorogenic acid
(1) System adaptability test
Under the following high performance liquid chromatography conditions, the contents of syringin and chlorogenic acid in the sample solution and the mixed reference solution are detected:
the chromatographic column is Waters surfire C 18 The volume ratio of the mobile phase is 7:93, wherein the mixed aqueous solution contains 0.2 percent of formic acid and 1.0 percent of tetrahydrofuran; the detection wavelength is 264nm, the column temperature is 30 ℃, and the volume flow is 1.0 mL.min -1 The analysis time was 30min and the sample injection amount was 5. Mu.L.
The chromatograms are shown in fig. 10 and 11. As shown in fig. 10 and 11, under the chromatographic conditions, syringin, chlorogenic acid and other components can be separated from each other at a baseline, the separation degree is more than 1.5, the theoretical plate numbers are more than 10000, and the separation effect is good.
(2) Linear relationship investigation
Precisely weighing appropriate amount of syringin reference substance and chlorogenic acid reference substance into the same 25mL volumetric flask, dissolving with 50% methanol, and diluting to 0.1787 mg/mL -1 、0.3876mg·mL -1 Is prepared from the mixed reference substance solution I.
Precisely sucking the mixed reference substance solution I into a volumetric flask with 5mL to 25mL, and diluting with 50% methanol to scale to obtain a mixed reference substance solution II.
Precisely sucking the mixed reference substance solutions I1, 3 and 5 mu L, injecting the mixed reference substance solutions II 2, 3 and 5 mu L into a liquid chromatograph, measuring the peak area, taking the sample injection quantity (mu g) as an abscissa and the peak area as an ordinate, taking a standard curve, and calculating by a least square method to obtain a syringin regression equation: y=1829.1x+3.5032 (r=0.9998), and the result shows that the sample injection amount is in the range of 0.0357-0.894 mug, and the sample injection amount has good linear relation; chlorogenic acid regression equation: y= 531.13x-5.3732 (r=0.9998), and the result shows that the sample injection amount is in the range of 0.0775-1.938 mug, and the sample injection amount has good linear relation.
(3) Precision test
The sample solution prepared by adopting the reference number 11 ginseng stem sample is continuously sampled for 6 times according to the chromatographic conditions, the RSD of the syringin peak area is calculated to be 0.32%, the RSD of the chlorogenic acid peak area is calculated to be 0.12%, and the result shows that the precision is good.
(4) Stability test
Sample solutions prepared by adopting the reference number 11 ginseng stem samples are taken and respectively injected at 0, 2, 4, 8, 12 and 24 hours according to the chromatographic conditions, the calculated RSD of the syringin peak area is 1.99%, and the RSD of the chlorogenic acid peak area is 0.32%, and the result shows that the sample solutions have good stability in 24 hours.
(5) Repeatability test
Taking a sample solution prepared by adopting a reference stem sample with the number of 11, precisely weighing 6 parts of the sample solution, sampling according to the chromatographic conditions to determine the content, and calculating the content of syringin according to the dry product to be 1.00 mg.g -1 (rsd=0.61%) and the content of chlorogenic acid is 4.35 mg.g -1 (rsd=0.58%), the result showed good reproducibility.
(6) Recovery test
6 parts of a ginseng stem sample (number 11) with known syringin and chlorogenic acid content is precisely weighed, a syringin reference substance and a proper amount of chlorogenic acid reference substance are precisely added, a sample solution is prepared in the same manner, high performance liquid chromatography is carried out, and the recovery rate is calculated. The results showed an average recovery of syringin of 98.5% (rsd=0.6%) and chlorogenic acid of 99.8% (rsd=0.6%). The results are detailed in Table 4.
TABLE 4 results of sample recovery test of Syringin and chlorogenic acid
(7) Content determination
The contents of syringin and chlorogenic acid are measured according to the chromatographic conditions after the samples of the stems of the tree ginseng with different numbers are prepared into test solutions, and the contents of the syringin and the chlorogenic acid are calculated according to the dried products, and the results are shown in Table 6.
According to the determination result, the content of syringin is not less than 0.010% and the content of chlorogenic acid is not less than 0.075% of the dry product, wherein the higher the content is, the better the quality is.
(3) Determination of the content of isochlorogenic acid A and isochlorogenic acid C
(1) System adaptability test
Detecting the content of isochlorogenic acid A and isochlorogenic acid C in the sample solution and the mixed reference solution under the following high performance liquid chromatography conditions;
the chromatographic column is Waters surfire C 18 The volume ratio of the mobile phase is 20:80, wherein the mixed aqueous solution contains 0.2 percent of formic acid and 1.0 percent of tetrahydrofuran; the detection wavelength is 328nm, the column temperature is 30 ℃, and the volume flow is 1.0 mL.min -1 The analysis time was 25min and the sample loading was 5. Mu.L.
The chromatograms are shown in fig. 12 and 13.
As can be seen from fig. 12 and 13, under the chromatographic conditions, isochlorogenic acid a and isochlorogenic acid C can be separated from other components by baseline, the separation degree is more than 1.5, the theoretical plate number is more than 10000, and the separation effect is good.
(2) Linear relationship investigation
Precisely weighing isochlorogenic acid A and isochlorogenic acid C reference substances into the same 100mL volumetric flask, dissolving with 50% methanol, and diluting to 0.1114 mg/mL -1 、0.0617mg·mL -1 Is a mixed pair of (2)Illumination solution I.
Precisely sucking the mixed reference substance solution I into a volumetric flask with 5mL to 25mL, and diluting with 50% methanol to scale to obtain a mixed reference substance solution II.
Precisely sucking the mixed reference substance solution I1 and 5 mu L, injecting the mixed reference substance solution II 2, 3, 5 and 10 mu L into a liquid chromatograph, measuring the peak area, taking the sample injection quantity (mu g) as an abscissa and the peak area as an ordinate, taking a standard curve, and calculating by a least square method to obtain the isochlorogenic acid A regression equation: y=3296.4x+0.3842 (r=0.9999), and the result shows that the sample injection amount is in the range of 0.0223-1.114 μg, and the sample injection amount has good linear relation; isochlorogenic acid C regression equation: y= 3493.8x-13.212 (r=0.9999), and the result shows that the sample injection amount is in the range of 0.0123-0.617 mug, and the sample injection amount has good linear relation.
(3) Precision test
The sample solution prepared by adopting the stem sample of the American ginseng with the number 11 is continuously sampled for 6 times according to the chromatographic conditions, the RSD of the A peak area of the isochlorogenic acid is calculated to be 0.03%, the RSD of the C peak area of the isochlorogenic acid is calculated to be 0.14%, and the result shows that the precision is good.
(4) Stability test
Sample injection is carried out at 0, 2, 4, 8, 12 and 24 hours according to the chromatographic conditions, the RSD of the A peak area of the isochlorogenic acid is calculated to be 0.04%, the RSD of the C peak area of the isochlorogenic acid is calculated to be 0.15%, and the result shows that the sample solution has good stability in 24 hours.
(5) Repeatability test
6 parts of a sample solution prepared by adopting a reference stem sample of the reference tree of the number 11 are precisely weighed, the content is measured by sampling according to the chromatographic conditions, the content of the isochlorogenic acid A is 1.67mg g < -1 > calculated according to the dry product (RSD=0.62%), the content of the isochlorogenic acid C is 0.36mg g < -1 > (RSD=0.93%), and the result shows that the repeatability is good.
(6) Recovery test
6 parts of a ginseng stem sample (number 11) with known syringin and chlorogenic acid content is precisely weighed, an isochlorogenic acid A reference substance and an isochlorogenic acid C reference substance with proper amounts are precisely added, a sample solution is prepared in the same way, high performance liquid chromatography determination is carried out, and recovery rate is calculated. The results showed that the average recovery of isochlorogenic acid a was 98.3% (rsd=0.9%) and the average recovery of isochlorogenic acid C was 99.4% (rsd=1.0%). The results are detailed in Table 5.
TABLE 5 results of sample recovery test of isochlorogenic acid A and isochlorogenic acid C
(7) Content determination
The contents of the isochlorogenic acid A and the isochlorogenic acid C are measured according to the chromatographic conditions after the test sample solution is prepared by taking the stem samples of the tree ginseng with different numbers, and the contents of the isochlorogenic acid A and the isochlorogenic acid C are calculated according to the dry product, and the results are shown in Table 6.
TABLE 6 summary of the results of measuring the content of active ingredients
According to the determination result, the tested product with the content of the isochlorogenic acid A not lower than 0.047% and the content of the isochlorogenic acid C not lower than 0.020% accords with the quality standard of the invention, and the higher the content is, the better the quality is.
Under the condition that the sample to be tested is identified as a genuine product according to the method, and the content index of the effective components, the content index of the alcohol soluble extract, the content index of the moisture and the content index of the total ash are all in accordance with the requirements, the quality of the sample to be tested is qualified; the greater the difference between each content index and the minimum or maximum value, the better the quality.
Claims (8)
1. The quality detection method of the she medicine tree ginseng stems is characterized by constructing a characteristic map of the she medicine tree ginseng stems by adopting a high performance liquid chromatography method, and comprises the following steps of:
(a) Preparing 60% ethanol extract of the stem of the ginseng into a test solution;
(b) Obtaining a high performance liquid chromatography of the test solution under the following high performance liquid chromatography conditions, and confirming 34 chromatographic peaks;
The conditions of high performance liquid chromatography include:
chromatographic column: waters canfire C 18 The specification is as follows: 4.6mm by 250mm,5 μm;
mobile phase: phase A is acetonitrile, phase B is a mixed aqueous solution containing 0.2% formic acid and 0.2% tetrahydrofuran;
gradient elution: 0 to 5min,4 to 9%A; 5-35 min, 9-14% of A; 35-50 min,14% A; 50-60 min, 14-20% of A, 60-80 min, 20-30% of A;
detection wavelength: 256nm;
column temperature: 30 ℃;
volume flow rate: 1.0 mL/mm -1 ；
Analysis time: 80min;
sample injection amount: 5. Mu.L;
(c) Taking the peak area of each chromatographic peak in the high performance liquid chromatography as an independent variable, taking the cell proliferation inhibition rate as a dependent variable, performing partial least squares regression analysis, calculating the standardized regression coefficient and the VIP value of 34 chromatographic peaks and the cell proliferation inhibition rate, and screening out 19 chromatographic peaks with the VIP value larger than 1;
(d) Taking the cell proliferation inhibition rate as a reference sequence, taking the peak area of each chromatographic peak as a comparison sequence, carrying out dimensionless treatment by adopting a mean change method, respectively calculating correlation coefficients corresponding to each comparison sequence and the reference sequence, taking a sequence average value to obtain gray correlation degree of 34 chromatographic peaks to the cell proliferation inhibition rate, and screening 7 chromatographic peaks with gray correlation degree larger than 0.9;
(e) Screening 7 characteristic peaks with VIP value greater than 1 and gray correlation degree greater than 0.9 to obtain a characteristic map of the she medicinal tree ginseng stems;
the 7 characteristic peaks in the characteristic spectrum are sequentially as follows:
peak No. 7, retention time 14.0min;
peak 11, retention time 16.3min, and corresponding component of syringin;
peak 17, retention time of 20.0min, and chlorogenic acid as the corresponding ingredient;
peak No. 24, retention time 36.9min;
peak No. 27, retention time 47.6min, corresponding ingredient Saikolignanoside A;
peak 33, retention time 67.3min, corresponding component is isochlorogenic acid A;
peak 34, retention time 71.2min, corresponding component is isochlorogenic acid C;
detecting the contents of syringin, chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C in the characteristic spectrum, comprising the following steps:
(1) Preparing a sample solution;
(2) Under the following high performance liquid chromatography conditions, the contents of syringin and chlorogenic acid in the sample solution are detected: the chromatographic column is Waters surfire C 18 The volume ratio of the mobile phase is 7:93, wherein the mixed aqueous solution contains 0.2 percent of formic acid and 1.0 percent of tetrahydrofuran; the detection wavelength is 264nm, the column temperature is 30 ℃, and the volume flow is 1.0 mL/mm -1 The analysis time is 30min, and the sample injection amount is 5 mu L;
(3) Detecting the content of isochlorogenic acid A and isochlorogenic acid C in the sample solution under the following high performance liquid chromatography conditions: the chromatographic column is Waters surfire C 18 The volume ratio of the mobile phase is 20:80, wherein the mixed aqueous solution contains 0.2 percent of formic acid and 1.0 percent of tetrahydrofuran; the detection wavelength is 328nm, the column temperature is 30 ℃, and the volume flow is 1.0 mL/mm -1 The analysis time is 25min, and the sample injection amount is 5 mu L;
(4) And comparing the content of the syringin, chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C with a preset value, and judging whether the quality of the sample meets the requirement according to the comparison result.
2. The quality detection method of she drug ginseng stems according to claim 1, wherein in the step (1), the preparation method of the sample solution is as follows: grinding the stem of the ginseng into powder, uniformly mixing with 60% ethanol, carrying out ultrasonic extraction, filtering, and taking filtrate to obtain the test sample solution.
3. The method for detecting the quality of she medicine ginseng stems according to claim 2, wherein the powder of the ginseng stems and 60% ethanol are mixed in an amount of 1g:25mL of the materials are mixed in proportion, weighed, subjected to ultrasonic treatment under the conditions of power of 500W and frequency of 53kHz for 1 h, cooled, weighed again, complemented with 60% ethanol, and filtered by a 0.45 mu m microporous filter membrane.
4. The quality detection method of she drug tree ginseng stems according to claim 1, further comprising: the method comprises the steps of taking 60% ethanol as a solvent, obtaining an alcohol-soluble extract of a test sample by adopting a thermal extraction method, weighing after drying, calculating the mass ratio of the alcohol-soluble extract to the test sample, comparing the mass ratio with a preset mass ratio, and judging whether the mass of the test sample meets the requirement or not according to a comparison result.
5. The quality detection method of she drug tree ginseng stems according to claim 1, further comprising: the water content and the total ash content of the sample are detected according to the water content measuring method and the ash content measuring method recorded in Chinese pharmacopoeia, the water content and the total ash content are compared with the preset content respectively, and whether the quality of the sample meets the requirements is judged according to the comparison result.
6. The quality detection method of she drug tree ginseng stems according to claim 1, further comprising: and carrying out microscopic identification and thin-layer identification on the test sample to determine whether the test sample is a genuine trepang product.
7. The method for detecting the quality of the stems of she-medicine trepang according to claim 6, wherein the thin layer identification comprises a thin layer identification method of syringin, the thin layer identification method of the syringin comprises: respectively dispensing the sample solution and syringin reference solution on the same silica gel GF 254 On the thin layer plate, chloroform, methanol and formic acid with volume ratio of 6:1.2:0.5 are used as developing agent, and after developing, the thin layer plate is taken out and dried, and is inspected under a 254nm ultraviolet lamp to see whether the color spectrum of the sample has bright blue fluorescent spots corresponding to the color spectrum position of the syringin reference substance.
8. The quality control method of she drug trepang stem according to claim 6, wherein the thin layer identification method comprises thin layer identification method of chlorogenic acid, isochlorogenic acid a and isochlorogenic acid C, the thin layer identification method of chlorogenic acid, isochlorogenic acid a and isochlorogenic acid C comprises: the sample solution and the reference substance mixed solution are respectively spotted on the same silica gel H thin layer plate, butyl acetate, formic acid and water with the volume ratio of 7:2.5:2.5 are taken as developing agents, taken out after the developing, dried in the air, inspected under an ultraviolet light lamp with the wavelength of 365nm, and whether three bright blue fluorescent spots corresponding to the chromatographic positions of the reference substance appear in the chromatogram of the sample or not is checked.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109709259A (en) * | 2019-02-01 | 2019-05-03 | 贵州医科大学 | A kind of discrimination method of Radix Mussaendae |
| CN112098613A (en) * | 2019-06-18 | 2020-12-18 | 贵州广济堂药业有限公司 | Quality detection method of wall-broken decoction pieces |
| CN113640292A (en) * | 2021-09-07 | 2021-11-12 | 石河子大学 | Quality detection method of phellodendron amurense medicinal material |
-
2022
- 2022-07-15 CN CN202210836446.2A patent/CN115236228B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109709259A (en) * | 2019-02-01 | 2019-05-03 | 贵州医科大学 | A kind of discrimination method of Radix Mussaendae |
| CN112098613A (en) * | 2019-06-18 | 2020-12-18 | 贵州广济堂药业有限公司 | Quality detection method of wall-broken decoction pieces |
| CN113640292A (en) * | 2021-09-07 | 2021-11-12 | 石河子大学 | Quality detection method of phellodendron amurense medicinal material |
Non-Patent Citations (6)
| Title |
|---|
| "一测多评"法测定平卧菊三七中绿原酸类成分;李小军;张妹砣;穆云妹;李婷婷;杨艳羚;曾紫璇;李玉桑;唐和斌;;中国医院药学杂志(22);第2.1.4节右栏第1段 * |
| 刘敏等.基于 LC-MS 树参叶指纹图谱的建立.中国现代应用药学.2020,第37卷(第1期),摘要，第1-3节，表1-2，图1-4. * |
| 基于 LC-MS 树参叶指纹图谱的建立;刘敏等;中国现代应用药学;第37卷(第1期);摘要，第1-3节，表1-2，图1-4 * |
| 女儿茶HPLC指纹图谱及清除自由基活性谱效关系;王乐;何婷;常艳丽;范书生;王秀环;王炎;李晓;王小萍;许啸;孙志蓉;折改梅;;中国实验方剂学杂志(23);全文 * |
| 小叶黄杨TLC鉴别及不同部位中紫丁香苷和香草酸HPLC测定;杨娟艳;姚文丽;郭婷;罗奕;王芳;许乾丽;茅向军;;药物分析杂志(02);第2.1节，图1 * |
| 武当山区引种"亚特红"良种金银花的品质分析;魏达亨;李志浩;曲嘉琛;仇雨情;张婧;;现代中药研究与实践(01);第1.3节，图1 * |
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