CN101732364B - Method for identifying lucid ganoderma alkaloids substance finger-print - Google Patents
Method for identifying lucid ganoderma alkaloids substance finger-print Download PDFInfo
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Abstract
The invention discloses a method for identifying lucid ganoderma alkaloids substance finger-print, comprising the following steps: adopting 5-10 batches of dried lucid ganoderma fruit body fragments from known standard variety samples produced in different places; taking the same mass of crude medicine in each batch for producing test article solution; and carrying out high performance liquid chromatography analysis on the test article solution; taking common absorption peaks of the high performance liquid chromatography as characteristic chromatographic peaks; choosing chromatographic peaks with uniformization peak area exceeding 1% as fingerprint peaks; obtaining standard lucid ganoderma alkaloids substance finger-print; taking the sample to be tested: dried lucid ganoderma fruit body fragments, obtaining high performance liquid chromatography of the sample to be tested: the lucid ganoderma alkaloids substance by the same method. The high performance liquid chromatography of the sample to be tested: the lucid ganoderma alkaloids substance has characteristic peaks found in the standard finger-print of the lucid ganoderma alkaloids substance, the sample to be tested is a standard variety. With the method of the invention adopted, different lucid ganoderma varieties can be identified.
Description
Technical field
The invention belongs to edible fungi and Chinese crude drug starting material alkaloids substance quality analyzing technique and identify the field, be specifically related to the construction method and the analysis discrimination method of a kind of efficient liquid-phase chromatograph finger print atlas of the biological active component alkaloids substance in the glossy ganoderma.
Background technology
Glossy ganoderma (Ganoderma Lucidum) is described as the miraculous cure that the mankind promote longevity since ancient times, nourishes the celestial grass of beneficial gas.In China's first this agrostology monograph Shennong's Herbal, just think that it has the beneficial motive, pacifies smart soul, the effect of tonifying liver benefit gas, hard muscles and bones.Li Shizhen (1518-1593 A.D.) has also been done high evaluation to the food therapy health effect of glossy ganoderma in Compendium of Material Medica: " red sesame has another name called red sesame, and smell is bitter flat, nontoxic.Cure mainly knot in the heart, the beneficial motive, bowl spares increases wisdom, does not forget.Food of a specified duration is made light of one's life by commiting suicide not old, and the angle prolongs life ".In recent decades, scientist is through various researchs, and prove from many aspects: glossy ganoderma has very the disease-relieving and physique enhancing of " spirit ", the health-care effect of strengthening the body resistance to consolidate the constitution.Especially biological immune and traditional treatment tumour had peculiar effect.
A large amount of in recent years chemistry and pharmacology analysis show: glossy ganoderma specifically have antitumor, alleviate pain, enhance immunity that cancer or other diseases cause, delay senility, multiple effect such as protecting liver and detoxication, treatment hypertension, hypoglycemic, antitussive and antiasthmatic, radioresistance and antiallergy.Especially at anti-tumor aspect, many famous medical research mechanisms reach a conclusion after deliberation both at home and abroad: glossy ganoderma has very significant curative effect to kinds of tumors diseases such as cancer of the stomach, lung cancer, malignant lymphoma, liver cancer, breast cancer, nasopharyngeal carcinoma, intestinal cancer, cancer of the esophagus, leukaemia.Why glossy ganoderma has so significant medical health effect, and in the active substance wherein, lucid ganoderma alkaloids (like Diterpenes, triterpenes, fast quinoline alkali, unknown alkali etc.) is a kind of important basic effective component in the glossy ganoderma, and its effect mainly comprises: protect the liver; Antitumor; Hypoglycemic; Anti-HIV-1 and HIV-1 proteinase activity; Suppress histamine release; Suppress ACE; Suppress cholesterol and synthesize the effect of grade.Therefore, lucid ganoderma alkaloids has important medicines and health protection and is worth, and glossy ganoderma has been developed to the plurality kinds of health care article through different processing modes at present, and lucid ganoderma alkaloids substance is a kind of product wherein.
Present domestic glossy ganoderma kind has a lot; Like red sesame, black sesame, purple sesame, Han Zhi, Bai Zhi etc.; Wherein in the majority with red sesame kind, because cultivation of glossy ganoderma is different with difference, planting type, the kind in weather conditions, zone, the biological active component alkaloids substance has certain difference in the glossy ganoderma; How to differentiate biological active component alkaloids substance contained in the glossy ganoderma, the quality of glossy ganoderma is differentiated and scientific evaluation has the meaning of value lever.At present, to different glossy ganoderma kinds, with the kind zones of different, with the discriminating of the biological active component alkaloids substance of the different cultural methods of kind and the evaluation method that a science is not also set up in quality assessment, there is not the method report of related fields yet.
Summary of the invention
The objective of the invention is in order to set up the analysis discrimination method of glossy ganoderma starting material alkaloids substance; Preliminary qualitative, quantitative attributional analysis to the control of glossy ganoderma alkaloid crudy, starting material real and fake discrimination and alkaloids substance provides a kind of effective method, thereby the material quality of glossy ganoderma is carried out science appraisal.
The technical scheme that the present invention adopts is:
A kind of method for identifying lucid ganoderma alkaloids substance finger-print, described method may further comprise the steps:
(a) producing of standard finger-print: 5～10 batches, the every batch crude drug amount of getting equal in quality of ganoderma lucidum fruitbody fragment of getting the known standard kind sample drying that the place of production has nothing in common with each other is tested respectively as follows: the alcohol reflux that adds dry 25～35 times of quality of ganoderma lucidum fruitbody fragment of known sample; Obtain the known sample extract after removing by filter filter residue; The known sample extract removes through decompression distillation and desolvates; Known sample alkaloid medicinal extract liquid, add methanol constant volume and get known sample solution, controlling in the described known sample solution crude drug amount content is/100 milliliters of 2～7 grams; Known sample solution is through the organic membrane filtration of 0.45 μ m; Get the known sample need testing solution, get the known sample need testing solution and carry out efficient liquid phase chromatographic analysis, obtain known sample lucid ganoderma alkaloids substance high-efficient liquid phase chromatogram; The condition of said high performance liquid chromatography is: being the chromatographic column of filler with the octadecylsilane chemically bonded silica, is that moving phase is carried out gradient elution with own nitrile, 1vol% aqueous acetic acid, and the detection wavelength is 254nm; Get that the total absorption peak of high-efficient liquid phase chromatogram is the characteristic chromatographic peak in each batch, select among the figure normalization peak area greater than 1% characteristic chromatographic peak as fingerprint peaks, obtain the standard finger-print of lucid ganoderma alkaloids substance;
(b) the testing sample high-efficient liquid phase chromatogram is produced: get the dry ganoderma lucidum fruitbody fragment of testing sample; The alcohol reflux that adds dry 25～35 times of quality of ganoderma lucidum fruitbody fragment of testing sample obtains the testing sample extract after removing by filter filter residue, and the testing sample extract removes through decompression distillation and desolvates; Get testing sample alkaloid medicinal extract liquid; Add methanol constant volume and get testing sample solution, it is identical to control in the described testing sample solution in the crude drug amount content and step (a) the crude drug amount content of known sample solution, and testing sample solution is through the organic membrane filtration of 0.45 μ m; Get the testing sample need testing solution; Get the testing sample need testing solution and carry out efficient liquid phase chromatographic analysis, the condition of said high performance liquid chromatography is: being the chromatographic column of filler with the octadecylsilane chemically bonded silica, is that moving phase is carried out gradient elution with own nitrile, 1vol% aqueous acetic acid; The detection wavelength is 254nm, obtains testing sample lucid ganoderma alkaloids substance high-efficient liquid phase chromatogram;
Characteristic chromatographic peak contrast in the standard finger-print of the lucid ganoderma alkaloids substance that testing sample lucid ganoderma alkaloids substance high-efficient liquid phase chromatogram that (c) step (b) is obtained and step (a) obtain; Testing sample lucid ganoderma alkaloids substance high-efficient liquid phase chromatogram has characteristic chromatogram cutting edges of a knife or a sword all in the standard finger-print of lucid ganoderma alkaloids substance, and then testing sample is a standard variety.
Comparatively concrete; In the producing of said step (a) standard finger-print, the operation that every batch of ganoderma lucidum fruitbody fragment of getting the known standard kind sample drying of equal in quality crude drug amount makes known sample lucid ganoderma alkaloids substance high-efficient liquid phase chromatogram may further comprise the steps:
(1) takes by weighing the dry ganoderma lucidum fruitbody fragment of known sample; Add 95wt% ethanol, the mass ratio of the ganoderma lucidum fruitbody fragment that said 95wt% ethanol and known sample are dry is 25～35: 1, preferred 33: 1; Under little reflux state that boils; Extracted 1～2 hour, remove by filter filter residue after, the known sample extract;
(2) the known sample extract removes through decompression distillation and desolvates; Get known sample alkaloid medicinal extract liquid; Add methanol constant volume and get known sample solution; Controlling in the described known sample solution crude drug amount content is/100 milliliters of 2～7 grams, and known sample solution is through the organic membrane filtration of 0.45 μ m, the known sample need testing solution;
(3) the known sample need testing solution carries out efficient liquid phase chromatographic analysis; Get known sample lucid ganoderma alkaloids substance high-efficient liquid phase chromatogram, said chromatographic condition is: being the chromatographic column of filler with the octadecylsilane chemically bonded silica, is that moving phase is carried out gradient elution with own nitrile, 1vol% aqueous acetic acid; Flow rate of mobile phase is 1mL/min; The detection wavelength is 254nm, sample size 10 μ L, and be 40min analysis time; The gradient elution program is: 0～8min, the 1vol% aqueous acetic acid of 10vol% and the acetonitrile of 90vol%; 8～10min, the 1vol% aqueous acetic acid of 15vol% and the acetonitrile of 85vol%; 10～15min, the 1vol% aqueous acetic acid of 30vol% and the acetonitrile of 70vol%; 15～20min, the 1vol% aqueous acetic acid of 40vol% and the acetonitrile of 60vol%; 20～30min, the 1vol% aqueous acetic acid of 45vol% and the acetonitrile of 55vol%; 30～40min, the 1vol% aqueous acetic acid of 50vol% and the acetonitrile of 50vol%.
Comparatively concrete, the operation that the testing sample high-efficient liquid phase chromatogram is produced in the said step (b) may further comprise the steps:
(1) takes by weighing the dry ganoderma lucidum fruitbody fragment of testing sample; Add 95wt% ethanol, the mass ratio of the ganoderma lucidum fruitbody fragment that said 95wt% ethanol and testing sample are dry is 25～35: 1, preferred 33: 1; Under little reflux state that boils; Extracted 1～2 hour, remove by filter filter residue after, the testing sample extract;
(2) the testing sample extract removes through decompression distillation and desolvates; Get testing sample alkaloid medicinal extract liquid; Add methanol constant volume and get testing sample solution; It is identical to control in the described testing sample solution in the crude drug amount content and step (a) the crude drug amount content of known sample solution, and testing sample solution is through the organic membrane filtration of 0.45 μ m, the testing sample need testing solution;
(3) the testing sample need testing solution carries out efficient liquid phase chromatographic analysis, gets high-efficient liquid phase chromatogram, and said chromatographic condition is: be the chromatographic column of filler with the octadecylsilane chemically bonded silica; With own nitrile, 1vol% aqueous acetic acid is that moving phase is carried out gradient elution, and flow rate of mobile phase is 1mL/min, and the detection wavelength is 254nm; Sample size 10 μ L; Be 40min analysis time, and the gradient elution program is: 0～8min, the 1vol% aqueous acetic acid of 10vol% and the acetonitrile of 90vol%; 8～10min, the 1vol% aqueous acetic acid of 15vol% and the acetonitrile of 85vol%; 10～15min, the 1vol% aqueous acetic acid of 30vol% and the acetonitrile of 70vol%; 15～20min, the 1vol% aqueous acetic acid of 40vol% and the acetonitrile of 60vol%; 20～30min, the 1vol% aqueous acetic acid of 45vol% and the acetonitrile of 55vol%; 30～40min, the 1vol% aqueous acetic acid of 50vol% and the acetonitrile of 50vol%.
The dry ganoderma lucidum fruitbody fragment of known sample according to the invention be with the section of known sample ganoderma lucidum fruitbody, dry, be crushed to 40～80 orders and obtain.
The dry ganoderma lucidum fruitbody fragment of testing sample according to the invention be with the section of testing sample ganoderma lucidum fruitbody, dry, be crushed to 40～80 orders and obtain.
Of the present invention under little reflux state that boils, extracted 1～2 hour, remove by filter filter residue after, the known sample extract, be preferably under little reflux state that boils, extracted 1 hour, remove by filter filter residue after, must the known sample extract; More preferably: adopting the microwave-assisted extraction method, is under the microwave condition of 800W at power, and microwave auxiliary reflux extracts 17min, remove by filter filter residue after, the known sample extract.
Of the present invention under little reflux state that boils, extracted 1～2 hour, remove by filter filter residue after, the testing sample extract, be preferably under little reflux state that boils, extracted 1 hour, remove by filter filter residue after, must the testing sample extract; More preferably: adopting the microwave-assisted extraction method, is under the microwave condition of 800W at power, and microwave auxiliary reflux extracts 17min, remove by filter filter residue after, the testing sample extract.
The present invention can be to different glossy ganoderma kinds, with the different places of production of kind, carry out discriminatory analysis with the biological active component alkaloids substance of kind different planting; Can be with reference to collection of illustrative plates through the alkaloid chromatogram of setting a certain lucidum variety; Coupling and calculated by peak area through chromatographic fingerprints of Chinese materia medica similarity evaluation system; Automatically calculate the similarity that has peak composition and sample in the red ganoderma sample of different producing regions; Size and calculation of similarity degree value with reference to peak area; Carry out data analysis and can more scientifically differentiate the contained alkaloidal quality situation of different producing regions red ganoderma, thereby differentiate that tentatively which kind of different classes of kind has more similarity and otherness, the quality of contained alkaloids is better.
The present invention preferably carries out pre-treatment with the microwave auxiliary reflux extraction method; Optimized the alkaloid method for distilling; Adopt the C18 post, with own nitrile, 1vol% aqueous acetic acid be moving phase carry out the binary gradient elution fingerprint map analyzing of alkaloids substance in the glossy ganoderma is differentiated have quick, easy, stable, directly perceived, favorable reproducibility, with strong points, alkaloid composes that the peak number order is many, the advantage of good separating effect and easy operating.Utilization the inventive method is to different glossy ganoderma kinds, with the different places of production of kind, very desirable with the identification result of the biological active component alkaloids substance of kind different planting, and the quality of the biological active component alkaloids substance of glossy ganoderma is differentiated the scientific basis that has proposed preliminary qualitative, quantitative first.
Figure of description
The finger-print of alkaloids substance in 1 No. a kind red ganodermas of accompanying drawing sample
The alkaloids substance chromatographic peak contrast of 29 kinds of different producing regions of accompanying drawing red ganoderma sample refers to collection of illustrative plates; The total absorption peak of getting these 9 kinds of high-efficient liquid phase chromatograms is the characteristic chromatographic peak; Select among the figure normalization peak area greater than 1% characteristic chromatographic peak as fingerprint peaks, obtain the standard finger-print of red ganoderma alkaloids substance.
35 kinds of red sesames of different genera of accompanying drawing, Han Zhi, purple sesame, black sesame, the contrast of Wild ganoderma alkaloids substance chromatographic peak refer to that collection of illustrative plates, the red sesame of red expression, Korea Spro represent Han Zhi, the purple sesame of purple expression, the black sesame of black expression, wild expression Wild ganoderma.
Embodiment:
With specific embodiment the present invention is further specified below, but protection scope of the present invention is not limited thereto.
Embodiment 1 sets up the finger-print of alkaloids substance in the glossy ganoderma
1. pulverising step: with No. a kind red ganoderma sample entity section, dry, be crushed to about 40 orders, the glossy ganoderma dry product.
2. extraction step: take by weighing 5g glossy ganoderma dry product, add 95wt% ethanol at 1: 33, under little reflux state that boils, adopt 800W power with solid-liquid ratio, microwave refluxing extraction 17min, remove by filter filter residue after, extract.
3. extract except that desolvating, gets alkaloid medicinal extract liquid through decompression distillation, adds methanol constant volume to 100mL, gets sample solution, and red ganoderma dry product content is/100 milliliters of 5 grams in the sample solution: sample solution gets need testing solution through the organic membrane filtration of 0.45 μ m;
4. need testing solution carries out efficient liquid phase chromatographic analysis, gets high-efficient liquid phase chromatogram.Chromatographic condition is: the Waters1525 high performance liquid chromatograph, and binary pump, UV-detector, Bress software, octadecylsilane chemically bonded silica are the chromatographic column C of filler
18(model: Symmetry250 * 4.6mm I.D.5 μ m, U.S. Waters company produces) is that moving phase is carried out gradient elution with own nitrile, water (containing 1% acetic acid v/v); Flow rate of mobile phase is 1mL/min; The detection wavelength is 254nm, sample size 10 μ L, and be 40 minutes analysis time; The gradient elution program is: 0～8min, the 1vol% aqueous acetic acid of 10vol% and the acetonitrile of 90vol%; 8～10min, the 1vol% aqueous acetic acid of 15vol% and the acetonitrile of 85vol%; 10～15min, the 1vol% aqueous acetic acid of 30vol% and the acetonitrile of 70vol%; 15～20min, the 1vol% aqueous acetic acid of 40vol% and the acetonitrile of 60vol%; 20～30min, the 1vol% aqueous acetic acid of 45vol% and the acetonitrile of 55v ol%; 30～40min, the 1vol% aqueous acetic acid of 50vol% and the acetonitrile of 50vol%.
5. fingerprint map construction: in high-efficient liquid phase chromatogram, select the normalization peak area greater than 1% chromatographic peak as fingerprint peaks; Finally selected 38 fingerprint peakses (accounting for more than 90% of total peak area) to analyze collection of illustrative plates as the unimodal alkaloids of glossy ganoderma; See Fig. 1, be fingerprint peaks among the figure shown in the bullet.But have the alkaloidal kind of glossy ganoderma like this in the qualitative discrimination red ganoderma sample, simultaneously according to the normalized area computing method, roughly differentiate accordingly and go out peak retention time section, which kind of alkaloid is higher.
The preliminary qualitative, quantitative of embodiment 2 different producing regions red ganoderma alkaloids is discriminatory analysis relatively
Gather 9 kinds of different producing regions red ganoderma samples and carry out the alkaloids analysis, experiment condition and method of operating obtain 9 kinds of red ganoderma sample alkaloidss and analyze collection of illustrative plates with embodiment 1; Adopt chromatographic fingerprints of Chinese materia medica similarity evaluation system then; Choose a kind of producing region red ganoderma sample as with reference to spectrum peak (R), and carry out chromatographic peak multiple spot method of correcting, the chromatographic peak of 8 samples is mated and generates contrast automatically refer to collection of illustrative plates; See accompanying drawing 2; Through the coupling and the calculated by peak area of chromatographic fingerprints of Chinese materia medica similarity evaluation system, calculate the similarity that has peak composition and sample in the red ganoderma sample of different producing regions automatically, see table 1, table 2 respectively.
Logarithmic data is analyzed in table 1 chromatographic peak peak area and total peak match
| Retention time | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | R | Total peak number |
| 1.492 | 0 | 469.427 | 0 | 0 | 203.16 | 0 | 257.59 | 217.35 | 143.44 | 4 |
| 2.158 | 259.08 | 229.414 | 206.24 | 228.47 | 0 | 0 | 0 | 0 | 115.40 | 4 |
| 2.333 | 201.45 | 332.056 | 0 | 0 | 0 | 0 | 0 | 224.21 | 94.716 | 3 |
| 2.664 | 1576.5 | 2298.32 | 0 | 1882.2 | 1056.3 | 926.53 | 1530.7 | 1086.3 | 1294.6 | 7 |
| 3.537 | 1052.3 | 865.414 | 260.47 | 631.50 | 268.89 | 218.51 | 438.23 | 311.83 | 505.90 | 8 |
| 3.750 | 292.33 | 342.329 | 380.33 | 365.44 | 271.29 | 319.38 | 204.65 | 387.19 | 320.37 | 8 |
| 12.639 | 248.16 | 200.184 | 0 | 0 | 222.18 | 0 | 0 | 0 | 83.817 | 3 |
| 12.979 | 443.71 | 229.103 | 207.50 | 281.01 | 277.57 | 323.54 | 357.94 | 340.33 | 307.59 | 8 |
| 14.292 | 229.89 | 0 | 0 | 0 | 233.11 | 0 | 230.23 | 204.45 | 112.21 | 4 |
| 16.29 | 455.87 | 0 | 214.41 | 0 | 0 | 238.14 | 224.23 | 226.07 | 169.84 | 5 |
| 16.717 | 454.61 | 0 | 0 | 0 | 318.90 | 0 | 214.28 | 0 | 123.47 | 3 |
| 17.461 | 357.81 | 202.328 | 325.69 | 0 | 0 | 317.27 | 221.19 | 367.69 | 224.00 | 6 |
| 17.828 | 561.75 | 245.117 | 392.43 | 243.89 | 0 | 388.3 | 262.60 | 430.23 | 315.54 | 7 |
| 18.158 | 542.31 | 241.559 | 541.21 | 313.94 | 205.68 | 639.38 | 489.51 | 642.99 | 452.07 | 8 |
| 19.2 | 328.47 | 251.271 | 272.29 | 0 | 398.67 | 247.59 | 0 | 296.24 | 224.31 | 6 |
| 19.386 | 569.96 | 212.4 | 276.72 | 0 | 0 | 204.98 | 257.51 | 294.50 | 227.01 | 6 |
| 19.902 | 1299.1 | 790.102 | 1360.3 | 558.44 | 510.30 | 1368.7 | 652.80 | 1456.8 | 999.60 | 8 |
| 20.156 | 784.99 | 391.413 | 713.78 | 323.32 | 366.75 | 692.95 | 526.62 | 794.47 | 574.28 | 8 |
| 20.513 | 0 | 273.604 | 220.21 | 0 | 349.19 | 237.25 | 202.39 | 240.45 | 162.86 | 5 |
| 20.767 | 713.02 | 345.269 | 694.81 | 286.20 | 0 | 440.49 | 274.63 | 493.03 | 405.93 | 7 |
| 20.945 | 330.86 | 220.211 | 276.75 | 0 | 211.94 | 261.02 | 282.13 | 312.76 | 236.96 | 7 |
| 21.193 | 460.36 | 438.747 | 812.60 | 213.17 | 0 | 757.63 | 399.18 | 399.18 | 502.82 | 7 |
| 21.725 | 0 | 0 | 0 | 281.22 | 0 | 410.55 | 362.09 | 2034.0 | 385.98 | 4 |
| 21.805 | 548.94 | 342.893 | 0 | 0 | 0 | 1172.3 | 0 | 0 | 258.02 | 3 |
| 21.864 | 978.85 | 786.401 | 1611.2 | 328.31 | 520.39 | 0 | 628.33 | 0 | 606.68 | 6 |
| 22.571 | 202.39 | 0 | 0 | 0 | 249.58 | 0 | 209.33 | 204.57 | 108.23 | 4 |
| 22.933 | 258.10 | 0 | 204.16 | 0 | 0 | 214.48 | 0 | 0 | 84.594 | 3 |
| 23.916 | 253.83 | 307.435 | 228.67 | 0 | 590.15 | 0 | 0 | 275.26 | 206.92 | 5 |
| 24.238 | 469.04 | 407.614 | 671.56 | 0 | 203.71 | 569.73 | 244.59 | 760.22 | 415.81 | 7 |
| 24.847 | 523.59 | 457.65 | 563.65 | 0 | 283.83 | 479.07 | 219.82 | 640.13 | 395.97 | 7 |
| 25.33 | 320.14 | 0 | 323.08 | 0 | 248.33 | 221.79 | 241.58 | 303.52 | 207.30 | 6 |
| 25.669 | 217.50 | 293.771 | 207.22 | 0 | 621.66 | 356.39 | 310.96 | 282.77 | 286.28 | 7 |
| 27.166 | 388.86 | 303.588 | 295.68 | 0 | 280.62 | 271.10 | 0 | 370.27 | 238.76 | 6 |
| 19293. | 12530。 | 12615。 | 6172. | 8537. | 11277. | 9462.1 | 14617. | 113813 |
Annotate: Sx is the glossy ganoderma sample type
Table 2 chromatographic peak similarity value calculation
| S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | R | |
| S1 | 1 | 0.812 | 0.765 | 0.745 | 0.698 | 0.841 | 0.831 | 0.721 | 0.934 |
| S2 | 0.812 | 1 | 0.505 | 0.897 | 0.774 | 0.884 | 0.883 | 0.677 | 0.916 |
| S3 | 0.765 | 0.505 | 1 | 0.364 | 0.495 | 0.579 | 0.654 | 0.565 | 0.731 |
| S4 | 0.745 | 0.897 | 0.364 | 1 | 0.665 | 0.874 | 0.859 | 0.598 | 0.847 |
| S5 | 0.698 | 0.774 | 0.495 | 0.665 | 1 | 0.804 | 0.649 | 0.551 | 0.792 |
| S6 | 0.841 | 0.884 | 0.579 | 0.874 | 0.804 | 1 | 0.888 | 0.739 | 0.941 |
| S7 | 0.831 | 0.883 | 0.654 | 0.859 | 0.649 | 0.888 | 1 | 0.797 | 0.944 |
| S8 | 0.721 | 0.677 | 0.565 | 0.598 | 0.551 | 0.739 | 0.797 | 1 | 0.83 |
| R | 0.934 | 0.916 | 0.731 | 0.847 | 0.792 | 0.941 | 0.944 | 0.83 | 1 |
Through above-mentioned table 1 data analysis; Calculate greater than 1% by the normalization peak area; 8 kinds of different producing regions red ganoderma samples; Maximum fingerprint of alkaloid peak numbers can reach 42 kinds, and minimum is merely 14 kinds, can be easy to differentiate the otherness of the alkaloidal kind of the different contained glossy ganodermas of producing region red ganoderma sample like this.
In addition; Can also distinguish the total finger-print characteristic peak number of lucid ganoderma alkaloids substance easily; 3.537,3.750,12.979,18.158,19.902,20.156min in above-mentioned 8 glossy ganoderma samples, the total finger-print characteristic peak number of alkaloid is 6, respectively in retention time:.Calculate through the normalized area integral method, also can differentiate easily, in same retention time section; Or to the common characteristic peak; Its retention time section, the peak area relative comparison peak-to-peak area of each sample is big or small, can differentiate the size of each sample alkaloid; The integral and calculating of total peak area that also can be through each sample and the total peak area of reference substance are differentiated the size of each sample alkaloid.
In addition, by above-mentioned table 2 data analysis, calculate through the chromatographic peak similarity of each sample; Can know S1, S2, S6; S7 glossy ganoderma sample has more similarity, and its similarity reaches more than 0.916, and the otherness of S3 is maximum; To the raw material quality control of glossy ganoderma sample, real and fake discrimination has the evaluation of scientific meaning like this.
Embodiment 3 adopts different genera glossy ganoderma sample to carry out the alkaloids qualitative analysis
Gather 5 kinds of red sesames of different genera, Han Zhi, purple sesame, black sesame, the Wild ganoderma sample respectively carries out the alkaloids analysis; Sampling amount, experiment condition and method of operating obtain 5 kinds of glossy ganoderma sample alkaloidss and analyze collection of illustrative plates with embodiment 1, adopt chromatographic fingerprints of Chinese materia medica similarity evaluation system then; Choose the red ganoderma sample as reference spectrum peak; And carry out chromatographic peak multiple spot method of correcting, and the chromatographic peak of 5 samples is mated and generates contrast automatically refer to collection of illustrative plates, see accompanying drawing 3; Through the coupling and the calculated by peak area of chromatographic fingerprints of Chinese materia medica similarity evaluation system, Wild ganoderma, black sesame, the more red sesame of Han Zhijun and purple sesame have higher alkaloids substance content.The lucid ganoderma alkaloids substance otherness of different genera is bigger, and red sesame, Han Zhi and Hei Zhi are slightly similar, and similarity only reaches about 0.806.Have only the standard finger-print of red sesame finger-print and red ganoderma alkaloids substance to fit like a glove.
Height to the alkaloids substance content in the glossy ganoderma of different genera can carry out qualitative discrimination soon like this.
Claims (9)
1. method for identifying lucid ganoderma alkaloids substance finger-print is characterized in that described method may further comprise the steps:
(a) producing of standard finger-print: 5～10 batches, the every batch crude drug amount of getting equal in quality of ganoderma lucidum fruitbody fragment of getting the known standard kind sample drying that the place of production has nothing in common with each other is tested respectively as follows: the alcohol reflux that adds dry 25～35 times of quality of ganoderma lucidum fruitbody fragment of known sample; Obtain the known sample extract after removing by filter filter residue; The known sample extract removes through decompression distillation and desolvates; Known sample alkaloid medicinal extract liquid, add methanol constant volume and get known sample solution, controlling in the described known sample solution crude drug amount content is/100 milliliters of 2～7 grams; Known sample solution is through the organic membrane filtration of 0.45 μ m; Get the known sample need testing solution, get the known sample need testing solution and carry out efficient liquid phase chromatographic analysis, obtain known sample lucid ganoderma alkaloids substance high-efficient liquid phase chromatogram; The condition of said high performance liquid chromatography is: being the chromatographic column of filler with the octadecylsilane chemically bonded silica, is that moving phase is carried out gradient elution with acetonitrile, 1vol% aqueous acetic acid, and the detection wavelength is 254nm; Flow rate of mobile phase is 1mL/min, sample size 10 μ L, and be 40min analysis time, the gradient elution program is: 0～8min, the 1vol% aqueous acetic acid of 10vol% and the acetonitrile of 90vol%; 8～10min, the 1vol% aqueous acetic acid of 15vol% and the acetonitrile of 85vol%; 10～15min, the 1vol% aqueous acetic acid of 30vol% and the acetonitrile of 70vol%; 15～20min, the 1vol% aqueous acetic acid of 40vol% and the acetonitrile of 60vol%; 20～30min, the 1vol% aqueous acetic acid of 45vol% and the acetonitrile of 55vol%; 30～40min, the 1vol% aqueous acetic acid of 50vol% and the acetonitrile of 50vol%; Get that the total absorption peak of high-efficient liquid phase chromatogram is the characteristic chromatographic peak in each batch, select among the figure normalization peak area greater than 1% characteristic chromatographic peak as fingerprint peaks, obtain the standard finger-print of lucid ganoderma alkaloids substance;
(b) the testing sample high-efficient liquid phase chromatogram is produced: get the dry ganoderma lucidum fruitbody fragment of testing sample, add the alcohol reflux of dry 25～35 times of quality of ganoderma lucidum fruitbody fragment of testing sample, obtain the testing sample extract after removing by filter filter residue; The testing sample extract removes through decompression distillation and desolvates, and gets testing sample alkaloid medicinal extract liquid, adds methanol constant volume and gets testing sample solution; It is identical to control in the described testing sample solution in the crude drug amount content and step (a) the crude drug amount content of known sample solution; Testing sample solution gets the testing sample need testing solution through the organic membrane filtration of 0.45 μ m, gets the testing sample need testing solution and carries out efficient liquid phase chromatographic analysis; The condition of said high performance liquid chromatography is: be the chromatographic column of filler with the octadecylsilane chemically bonded silica; With acetonitrile, 1vol% aqueous acetic acid is that moving phase is carried out gradient elution, and the detection wavelength is 254nm, and flow rate of mobile phase is 1mL/min; Sample size 10 μ L; Be 40min analysis time, and the gradient elution program is: 0～8min, the 1vol% aqueous acetic acid of 10vol% and the acetonitrile of 90vol%; 8～10min, the 1vol% aqueous acetic acid of 15vol% and the acetonitrile of 85vol%; 10～15min, the 1vol% aqueous acetic acid of 30vol% and the acetonitrile of 70vol%; 15～20min, the 1vol% aqueous acetic acid of 40vol% and the acetonitrile of 60vol%; 2o～30min, the 1vol% aqueous acetic acid of 45vol% and the acetonitrile of 55vol%; 30～40min, the 1vol% aqueous acetic acid of 50vol% and the acetonitrile of 50vol%; Obtain testing sample lucid ganoderma alkaloids substance high-efficient liquid phase chromatogram;
Characteristic chromatographic peak contrast in the standard finger-print of the lucid ganoderma alkaloids substance that testing sample lucid ganoderma alkaloids substance high-efficient liquid phase chromatogram that (c) step (b) is obtained and step (a) obtain; Testing sample lucid ganoderma alkaloids substance high-efficient liquid phase chromatogram has characteristic chromatogram cutting edges of a knife or a sword all in the standard finger-print of lucid ganoderma alkaloids substance, and then testing sample is a standard variety.
2. method for identifying lucid ganoderma alkaloids substance finger-print as claimed in claim 1; It is characterized in that in the producing of described method step (a) standard finger-print that the operation that every batch of ganoderma lucidum fruitbody fragment of getting the known standard kind sample drying of equal in quality crude drug amount makes known sample lucid ganoderma alkaloids substance high-efficient liquid phase chromatogram may further comprise the steps:
(1) take by weighing the dry ganoderma lucidum fruitbody fragment of known sample, add 95wt% ethanol, the mass ratio of the ganoderma lucidum fruitbody fragment that said 95wt% ethanol and known sample are dry is 25～35: 1; Under little reflux state that boils; Extracted 1～2 hour, remove by filter filter residue after, the known sample extract;
(2) the known sample extract removes through decompression distillation and desolvates; Get known sample alkaloid medicinal extract liquid; Add methanol constant volume and get known sample solution; Controlling in the described known sample solution crude drug amount content is/100 milliliters of 2～7 grams, and known sample solution is through the organic membrane filtration of 0.45 μ m, the known sample need testing solution;
(3) the known sample need testing solution carries out efficient liquid phase chromatographic analysis; Get known sample lucid ganoderma alkaloids substance high-efficient liquid phase chromatogram, said chromatographic condition is: being the chromatographic column of filler with the octadecylsilane chemically bonded silica, is that moving phase is carried out gradient elution with acetonitrile, 1vol% aqueous acetic acid; Flow rate of mobile phase is 1mL/min; The detection wavelength is 254nm, sample size 10 μ L, and be 40min analysis time; The gradient elution program is: 0～8min, the 1vol% aqueous acetic acid of 10vol% and the acetonitrile of 90vol%; 8～10min, the 1vol% aqueous acetic acid of 15vol% and the acetonitrile of 85vol%; 10～15min, the 1vol% aqueous acetic acid of 30vol% and the acetonitrile of 70vol%; 15～20min, the 1vol% aqueous acetic acid of 40vol% and the acetonitrile of 60vol%; 20～30min, the 1vol% aqueous acetic acid of 45vol% and the acetonitrile of 55vol%; 30～40min, the 1vol% aqueous acetic acid of 50vol% and the acetonitrile of 50vol%.
3. method for identifying lucid ganoderma alkaloids substance finger-print as claimed in claim 1 is characterized in that the operation that the testing sample high-efficient liquid phase chromatogram is produced in the described method step (b) may further comprise the steps:
(1) take by weighing the dry ganoderma lucidum fruitbody fragment of testing sample, add 95wt% ethanol, the mass ratio of the ganoderma lucidum fruitbody fragment that said 95wt% ethanol and testing sample are dry is 25～35: 1; Under little reflux state that boils; Extracted 1～2 hour, remove by filter filter residue after, the testing sample extract;
(2) the testing sample extract removes through decompression distillation and desolvates; Get testing sample alkaloid medicinal extract liquid; Add methanol constant volume and get testing sample solution; It is identical to control in the described testing sample solution in the crude drug amount content and step (a) the crude drug amount content of known sample solution, and testing sample solution is through the organic membrane filtration of 0.45 μ m, the testing sample need testing solution;
(3) the testing sample need testing solution carries out efficient liquid phase chromatographic analysis, gets high-efficient liquid phase chromatogram, and said chromatographic condition is: be the chromatographic column of filler with the octadecylsilane chemically bonded silica; With acetonitrile, 1vol% aqueous acetic acid is that moving phase is carried out gradient elution, and flow rate of mobile phase is 1mL/min, and the detection wavelength is 254nm; Sample size 10 μ L; Be 40min analysis time, and the gradient elution program is: 0～8min, the 1vol% aqueous acetic acid of 10vol% and the acetonitrile of 90vol%; 8～10min, the 1vol% aqueous acetic acid of 15vol% and the acetonitrile of 85vol%; 10～15min, the 1vol% aqueous acetic acid of 30vol% and the acetonitrile of 70vol%; 15～20min, the 1vol% aqueous acetic acid of 40vol% and the acetonitrile of 60vol%; 20～30min, the 1vol% aqueous acetic acid of 45vol% and the acetonitrile of 55vol%; 30～40min, the 1vol% aqueous acetic acid of 50vol% and the acetonitrile of 50vol%.
4. method as claimed in claim 2 is characterized in that in the described step (1) said being extracted as: under little reflux state that boils, extracted 1 hour, remove by filter filter residue after, the known sample extract.
5. method as claimed in claim 3 is characterized in that in the described step (1) said being extracted as: under little reflux state that boils, extracted 1 hour, remove by filter filter residue after, the testing sample extract.
6. method as claimed in claim 2, it is characterized in that in the described step (1) said being extracted as: power is under the microwave condition of 800W, microwave refluxing extraction 17min, remove by filter filter residue after, the known sample extract.
7. method as claimed in claim 3, it is characterized in that in the described step (1) said being extracted as: power is under the microwave condition of 800W, microwave refluxing extraction 17min, remove by filter filter residue after, the testing sample extract.
8. method as claimed in claim 2 is characterized in that in the described step (1), and the mass ratio of the ganoderma lucidum fruitbody fragment that said 95wt% ethanol and known sample are dry is 33: 1.
9. method as claimed in claim 3 is characterized in that in the described step (1), and the mass ratio of the ganoderma lucidum fruitbody fragment that said 95wt% ethanol and testing sample are dry is 33: 1.
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