CN113109485A - Method for identifying white cloud ginseng and codonopsis pilosula - Google Patents
Method for identifying white cloud ginseng and codonopsis pilosula Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention relates to the technical field of traditional Chinese medicine identification, in particular to an identification method of white cloud ginseng and codonopsis pilosula, which comprises the following steps of HPLC identification: establishing HPLC characteristic maps of HPLC samples and HPLC reference substances, wherein the HPLC reference substances comprise 9- (2-tetrahydropyran) -8E-alkene-4, 6-diyne-3-nonanol. The scheme solves the technical problem that the prior art can not effectively and accurately identify the white cloud ginseng and the codonopsis pilosula. The establishment of the exclusive identification method of the medicinal material of the codonopsis pilosula belonging to the same family of the dolomitic brevifolia is of great significance for ensuring the safety and the effectiveness of relevant clinical medication.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine identification, in particular to a method for identifying white cloud ginseng and codonopsis pilosula.
Background
The radix Ginseng alba is dried root of Leopard (Campanulaceae) Leopard (Campanumoea javanica BI). Has effects of invigorating spleen, replenishing qi, moistening lung, and promoting fluid production; can be used for treating asthenia, asthenia after illness, palpitation, hyperhidrosis, diarrhea due to spleen deficiency, leukorrhagia, oligogalactia, infantile malnutrition, cough due to lung deficiency, neurasthenia, and traumatic hemorrhage. Baiyun Shen is recorded in Honghe Chinese herbal medicine (1971), also called as "Tudangshen", and is one of the main medicinal materials of the representative medicine "Huli san" series of medicine in traumatology of Yunnan province and also an important guarantee of medicine. The Codonopsis pilosula (Codonopsis pilosula (Franch.) Nannf.) belonging to the same family is a common bulk medicinal material, is low in price, is very similar to the appearance form of the white-cloud ginseng, and has the same chemical components, so that the Codonopsis pilosula and the white-cloud ginseng are easy to be mixed, and the quality and the curative effect of the medicine are seriously influenced, so that the establishment of a special identification and identification method for the Codonopsis pilosula belonging to the same family has important significance for ensuring the safety and the effectiveness of the clinical medication of the sanjieli powder (capsules, powder and tablets). The invention establishes a material-based method for identifying the specificity of the white cloud ginseng and the codonopsis pilosula on the basis of deep chemical component research of the white cloud ginseng and the codonopsis pilosula, and has important practical technical value and economic value.
Disclosure of Invention
The invention aims to provide a method for identifying white cloud ginseng and codonopsis pilosula, and aims to solve the technical problem that the white cloud ginseng and codonopsis pilosula cannot be effectively and accurately identified in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for identifying white cloud ginseng and codonopsis pilosula comprises the following steps of HPLC identification: establishing HPLC characteristic maps of HPLC samples and HPLC reference substances, wherein the HPLC reference substances comprise 9- (2-tetrahydropyran) -8E-alkene-4, 6-diyne-3-nonanol.
The principle and the advantages of the scheme are as follows: through a large number of experimental researches, the inventor finds that 9- (2-tetrahydropyran) -8E-ene-4, 6-diyne-3-nonanol is a special component of white ginseng, and only white ginseng contains the substance in platycodonaceae plants containing lobetyolin (such as platycodon grandiflorum, Chinese lobelia and codonopsis pilosula). Therefore, an HPLC identification method can be established, and the 9- (2-tetrahydropyran) -8E-alkene-4, 6-diyne-3-nonanol is used as a detection index, so that the white Yunnan ginseng and the radix codonopsis can be effectively distinguished. Namely, a 9- (2-tetrahydropyran) -8E-alkene-4, 6-diyne-3-nonanol peak corresponding to the HPLC characteristic spectrum of an HPLC reference substance appears in the HPLC characteristic spectrum of an HPLC sample, and the HPLC sample is from a white yunshen medicinal material; if the peak does not appear, the HPLC sample is from the codonopsis pilosula medicinal material. The inventor discovers that 9- (2-tetrahydropyrane) -8E-alkene-4, 6-diyne-3-nonanol is a special characteristic of the white cloud ginseng for the first time, and identifies and distinguishes the white cloud ginseng and the codonopsis pilosula by utilizing the characteristic. In the scheme, the HPLC method is high performance liquid chromatography.
In practical application, the white-cloud ginseng and the codonopsis pilosula have similar functions and indications, but the two medicinal materials cannot be used mutually in clinical application, for example, in a Yunnan province famous prescription preparation 'Huli powder', the white-cloud ginseng in the prescription cannot be replaced by the codonopsis pilosula. In the prior art, an effective method for distinguishing two medicinal materials is not available, related detection indexes are lacked, and the two medicinal materials cannot be effectively distinguished. The codonopsis pilosula is a recorded variety in Chinese pharmacopoeia, and the identification method comprises microscopic identification (cross section characteristics), thin-layer chromatography (TLC method for detecting existence of lobetyolin) and comparison with a codonopsis pilosula reference medicinal material, so that the codonopsis pilosula medicinal material is identified. However, the lobetyolin is not a specific component of codonopsis pilosula, and is contained in other plants of the same family, such as platycodon grandiflorum, Chinese lobelia, and white-cloud ginseng (campanumaea pilosula), so that the codonopsis pilosula and the white-cloud ginseng are difficult to distinguish by a method recorded in the existing Chinese pharmacopoeia.
The white-cloud ginseng is a traditional Chinese medicine, the identification of the white-cloud ginseng (campanumoea pilosula) in the listing of the Chinese pharmacopoeia of 1977 edition is mainly carried out by two modes of appearance shape and physicochemical reaction, and the physicochemical reaction identification is mainly to detect whether the medicinal materials contain saponins and saccharides so as to identify the medicinal materials. The 2003 edition of quality standard of traditional Chinese medicinal materials and national medicinal materials in Guizhou province is added with microscopic identification on the basis of the 1977 edition of Chinese pharmacopoeia. The identification of the white-cloud ginseng recorded in the Chinese medicinal material standard of Yunnan province only comprises a TLC method for comparing and identifying the white-cloud ginseng with a reference medicinal material. However, the calibration of the white Yunnan ginseng reference medicinal material lacks a unified standard, so that the identification method in the Chinese medicinal material standard of Yunnan province cannot be effectively implemented. Because the codonopsis pilosula and the white cloud ginseng have greater similarity in appearance, properties and component content, the methods cannot effectively distinguish the codonopsis pilosula from the white cloud ginseng. Through a large amount of researches on medicinal materials, the inventor finds that 9- (2-tetrahydropyran) -8E-ene-4, 6-diyne-3-nonanol can be used as an index for identifying codonopsis pilosula and white cloud ginseng and is a specific component of the white cloud ginseng. The radix Codonopsis and Caesalpinia crista can be distinguished by detecting whether the sample contains 9- (2-tetrahydropyran) -8E-ene-4, 6-diyne-3-nonanol by HPLC. The technical scheme solves the technical problem which can not be solved in the field for a long time, and fills the technical blank.
In summary, in the prior art, the identification and distinction of the white cloud ginseng and the codonopsis pilosula mainly adopt the characteristics of the medicinal materials, but the identification according to the characteristics has the defect of strong subjectivity, and the characteristics of the medicinal materials are difficult to distinguish after the medicinal materials are pulverized, so that the special detection indexes are lacked, and the powder of the two medicinal materials cannot be effectively distinguished. The invention finds the specific component 9- (2-tetrahydropyran) -8E-ene-4, 6-diyne-3-nonanol of the white cloud ginseng from the chemical components contained in the medicinal materials, can quickly and accurately identify and distinguish the white cloud ginseng and the codonopsis pilosula, and has higher specificity.
Further, HPLC control product also comprises lobetyolin, atractylenolide II and atractylenolide III.
By adopting the technical scheme, under the detection method, atractylenolide II and atractylenolide III can be detected from the codonopsis pilosula, and no atractylenolide is detected from the white cloud ginseng; 9- (2-tetrahydropyran) -8E-ene-4, 6-diyne-3-nonanol can be detected in the white Yunnan ginseng, but is not detected in the radix codonopsis. The method can also be used as one of the exclusive bases for identifying the dolomitic white participating in the codonopsis pilosula. The purpose of detecting the lobetyolin is to detect the lobetyolin in large amount in the plants of Campanulaceae including radix Codonopsis and radix Ginseng alba, further confirm that the detection sample belongs to Campanulaceae, and limit the sample to be detected in the variety containing the lobetyolin in large amount so as to avoid the error of the detection result. Through a large number of compound separation and structure identification experiments, the inventor discovers that the compounds atractylenolide II and atractylenolide III are special components of radix codonopsis, the radix cynanchi atrati does not contain the two components, and the radix codonopsis can be specially identified by utilizing the atractylenolide II and the atractylenolide III.
Further, before the HPLC identification step, a TLC identification step is also included: comprises a detection step and an identification detection step; in the detection step, establishing a TLC detection characteristic spectrum of a TLC sample and a detection reference substance, wherein the detection reference substance is lobetyolin; in the identification detection step, establishing TLC identification characteristic maps of a TLC sample and an identification reference substance, wherein the identification reference substance is a mixture formed by atractylenolide II and atractylenolide III.
By adopting the technical scheme, the TLC method is simpler and easier than an HPLC method, and can be used for preliminarily detecting chemical components with higher content, such as lobetyolin, atractylenolide II and atractylenolide III, preliminarily screening a sample to be detected and detecting special substances of codonopsis pilosula. For example, if a specific substance of Codonopsis pilosula is detected in a sample, it can be known that the sample contains a component of Codonopsis pilosula. The HPLC method can find out the slight difference from the trace components, and the TLC identification step and the HPLC identification step are mutually complemented and mutually verified, so that the detection accuracy is improved. In the scheme, the TLC method is thin-layer chromatography.
Comparing the TLC identification characteristic spectrum of the TLC sample with the TLC identification characteristic spectrum of the identification reference substance, and generating spots with the same or similar colors at the same position, namely, indicating that the TLC sample contains lobetyolin, indicating that the TLC sample contains the platycodonaceae plants. Comparing the TLC identification characteristic spectrum of the TLC sample with the TLC identification characteristic spectrum of the identification reference substance, and indicating that the TLC sample contains atractylenolide II and atractylenolide III, wherein the TLC sample contains radix Codonopsis.
Further, the chromatographic conditions for establishing the HPLC characteristic maps of the HPLC sample and the HPLC control are as follows: performing gradient elution by using octadecylsilane chemically bonded silica as a filling agent and a mixture of acetonitrile and water as an eluent; the flow rate was 1ml/min, the column temperature was 30 ℃ and the detection wavelength was 220 nm. The chromatographic conditions can effectively distinguish target components in the codonopsis pilosula and the white cloud ginseng, and the detection and identification of four substance peaks are realized.
Furthermore, the HPLC control contains 180 μ g/ml lobetyolin, 3 μ g/ml 9- (2-tetrahydropyran) -8E-en-4, 6-diyne-3-nonanol, 10 μ g/ml atractylenolide II and 10 μ g/ml atractylenolide III. The HPLC reference substance with the content of the substances can present a more ideal peak pattern after HPLC detection, and is convenient for identification and comparison.
Further, the method for establishing the TLC detection characteristic spectrum comprises the following steps: the TLC sample and the detection reference substance are spotted on a silica gel G thin-layer plate, and a TLC detection characteristic spectrum is obtained by thin-layer chromatography using a mixture consisting of n-butanol, glacial acetic acid and water as a developing agent. The developing solvent consists of n-butyl alcohol, glacial acetic acid and water, so that main components in the TLC sample can be fully developed and separated, and the subsequent identification step can be conveniently carried out.
Further, the method for establishing the TLC identification characteristic spectrum comprises the following steps: the TLC sample and the detection control are spotted on a silica gel G thin-layer plate, and a TLC identification characteristic spectrum is obtained by thin-layer chromatography using a mixture of chloroform and acetone as a developing agent. The developing solvent composed of trichloromethane and acetone can fully develop and separate the main components in the TLC sample, thereby facilitating the subsequent identification steps.
Further, the volume ratio of the n-butyl alcohol to the glacial acetic acid to the water is 7:1: 0.5; the volume ratio of the trichloromethane to the acetone is 10: 1. The developing solvent in the above proportion can obtain the best thin layer chromatography effect.
Further, the preparation method of the identification reference substance comprises the following steps: adding 1mg of lobetyolin into 1ml of methanol to obtain a check and identification reference substance; the preparation method of the identification reference substance comprises the following steps: 1mg of atractylenolide II and 1mg of atractylenolide III per 1ml of methanol are added. The control product with the concentration can form obvious visible spots or fluorescence on the silica gel G thin-layer plate, so that the observation, identification and identification of the medicinal materials are facilitated.
Further, in the TLC identification step, the TLC sample is prepared by: performing ultrasonic-assisted extraction on the crude powder of the medicinal materials by using methanol as an extraction solvent, and filtering to obtain filtrate to obtain an extracting solution; evaporating the extractive solution to dryness, dissolving in water, and loading onto macroporous adsorbent resin column; eluting the macroporous adsorption resin column with water and then methanol, and collecting methanol eluent; evaporating the methanol eluent to dryness, adding methanol, and uniformly mixing to obtain a TLC sample;
in the HPLC identification step, the HPLC sample is prepared by the following method: performing ultrasonic-assisted extraction on the crude powder of the medicinal materials by using methanol as an extraction solvent, and filtering to obtain filtrate to obtain an extracting solution; evaporating the extracting solution to dryness, then adding methanol, mixing uniformly, and filtering by a microporous filter membrane to obtain an HPLC sample.
By adopting the method, the 9- (2-tetrahydropyran) -8E-alkene-4, 6-diyne-3-nonanol, lobetyolin, atractylenolide II and atractylenolide III in TLC samples and HPLC samples can be fully extracted, and the method is the basis for thin-layer chromatography detection and high performance liquid chromatography detection.
Drawings
FIG. 1 is a TLC identification chart of radix Ginseng alba and radix Codonopsis.
FIG. 2 is a TLC chromatogram for identifying the white cloud ginseng and the radix codonopsis.
FIG. 3 is an HPLC chromatogram of radix Codonopsis identification of radix Codonopsis.
FIG. 4 is an overlay of HPLC chromatogram of ten batches of radix Codonopsis Lanceolatae and radix Codonopsis reference material.
Detailed Description
Examples
TLC method identification (TLC identification step)
1.1 Experimental materials
1.1.1 Experimental drugs
The method comprises the following steps of selecting 10 batches of white Yunnan ginseng medicinal materials from different production places such as Yunnan, Guangxi and the like, wherein the radix codonopsitis medicinal materials are purchased from China food and drug testing research institute: 121057 and 201206, the sources of the experimental materials are shown in Table 1.
Table 1: experimental medicinal material
| Numbering | Origin or place of origin | |
| 1 | Red river of Yunnan province | 20161110 |
| 2 | Red river of Yunnan province | 20131016 |
| 3 | Red river of Yunnan province | 20180101 |
| 4 | Red river of Yunnan province | 20180102 |
| 5 | Red river of Yunnan province | 20161110 |
| 6 | Red river of Yunnan province | 20180103 |
| 7 | Red river of Yunnan province | 20180104 |
| 8 | Puer market in Yunnan province | 20180201 |
| 9 | Wenshan Prefecture, Yunnan Province | 20180202 |
| 10 | Longlin county, Guangxi province | 20180203 |
| 11 | China Institute for food and drug control | Radix Codonopsis reference medicinal material |
1.1.2 Experimental reagents
Methanol; ethanol; trichloromethane; ethyl acetate; n-butanol; glacial acetic acid; n-hexane; all acetone was analytically pure.
1.1.3 control substances
Atractylodes macrocephala lactone II (Kunming Lai chapter medicine science and technology Limited CAS: 73069-14-4), Atractylodes macrocephala lactone III reference substance (Kunming Lai chapter medicine science and technology Limited CAS: 73030-71-4), and Codonopsis pilosula alkyne glycoside reference substance (Kunming Lai chapter medicine science and technology Limited CAS: 136085-37-5)
1.2 Experimental methods
1.2.1 preparation of control solutions
Taking appropriate amount of radix Codonopsis alkyne glycoside, Atractylodis rhizoma lactone II and Atractylodis rhizoma lactone III reference substances, respectively, and adding methanol to obtain 1mg reference substance solution per 1 ml.
1.2.2 preparation of test solutions (TLC samples)
Taking 1g of crude powder of the medicinal materials, placing the crude powder in a 50ml conical flask with a plug, adding 25ml of methanol, sealing the plug, carrying out ultrasonic extraction for 60 minutes, filtering, evaporating the filtrate to dryness, adding 15ml of water for dissolution, adsorbing by a D101 type macroporous adsorption resin column (the inner diameter is 1.5cm, the column height is 10cm), eluting by 50ml of water, discarding water solution, eluting by 50ml of methanol, collecting the eluent, evaporating to dryness, adding 2ml of methanol into the residue for dissolution to obtain a sample solution for later use.
1.2.3 check-out (check-out detection step)
Taking 2 μ l of radix Codonopsis alkynil reference substance solution and 10 μ l (selected range is 8-10 μ l) of test solution, spreading on the same silica gel G thin layer plate, spreading with n-butanol-glacial acetic acid-water (7:1:0.5) as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, heating at 105 deg.C until the spots are clear, and observing under sunlight.
1.2.4 authentication (authentication detection step)
Taking 2 μ l of atractylenolide II and atractylenolide III reference substance solution, and 10 μ l of test solution (selected range is 8-10 μ l), dropping on the same silica gel G thin layer plate, developing with chloroform-acetone (10:1) as developing agent, taking out, air drying, spraying with 10% sulfuric acid ethanol solution, heating at 105 deg.C until the spots are clear, and observing under ultraviolet light (365 nm).
1.3 results
The control substance and the test solution were detected by TLC conditions of 1.2.3 and 1.2.4, respectively. The results are shown in FIGS. 1 and 2. In fig. 1, the bottom layer numbers represent: 1. 20161110, respectively; 2. 20131016, respectively; 3. 20180101, respectively; 4. 20180102, respectively; 5. 20161110, respectively; 6. mixing radix Ginseng alba and radix Ginseng with reference materials; 7. lobetyolin; 8. 20180103, respectively; 9. 20180104, respectively; 10. 20180201, respectively; 11. 20180202, respectively; 12. 20180203, respectively; 13. radix Codonopsis as reference material. In fig. 2, the bottom layer numbers represent: 1. 20161110, respectively; 2. 20131016, respectively; 3. 20180101, respectively; 4. 20180102, respectively; 5. 20161110, respectively; 6. radix Codonopsis reference material; 7. contrasting atractylenolide II and atractylenolide III; 8. radix Ginseng Indici reference medicinal material; 9. 20180103, respectively; 10. 20180104, respectively; 11. 20180201, respectively; 12. 20180202, respectively; 13. 20180203, respectively; in addition, 1 and 2 in a column represented by 7 in fig. 2 are atractylenolide ii and atractylenolide iii, respectively. As shown in figure 1, the white cloud ginseng medicinal material and the codonopsis pilosula medicinal material have no obvious difference; as shown in FIG. 2, the white cloud ginseng medicinal material has no fluorescent spot with the same color at the position corresponding to the control substance, and the radix Codonopsis control medicinal material has fluorescent spot with the same color at the position corresponding to the control substance.
Detecting radix Ginseng alba under detection condition, wherein spots with same color should be displayed at the same position as TLC detection image of reference substance (radix Codonopsis alkyne glycoside); under the identification condition, the white cloud ginseng medicinal material cannot generate fluorescent spots with the same color at the same positions of the reference substances (atractylenolide II and atractylenolide III), and the codonopsis pilosula medicinal material needs to be detected by the fluorescent spots with the same color. Under the detection method, the atractylenolide II and atractylenolide III contained in the codonopsis pilosula can be detected, but the cynanchum atratum linn cannot be detected, so that the atractylenolide II and atractylenolide III can be used as one of exclusive bases for identifying and identifying the cynanchum atratum linn and the codonopsis pilosula.
HPLC identification (HPLC identification step)
2.1 materials of the experiment
2.1.1 Experimental drugs (see Table 1)
2.1.2 Experimental reagents
Methanol (Merck, chromatography); acetonitrile (Merck, chromatography); methanol (Tianjin, Fengshan chemical reagent science and technology, Inc., analysis); ultrapure water (self-made).
2.1.3 control substances
Atractylodes macrocephala lactone II (Kunming Laishaon medicine science and technology Co., Ltd., CAS: 73069-14-4); atractylodes macrocephala lactone III reference substance (Kunming Laishao medicine science and technology Co., Ltd., CAS: 73030-71-4); radix Codonopsis acetylenic glycoside reference (Kunming Laishaozhang medicine science and technology Co., Ltd., CAS: 136085-37-5); 9- (2-tetrahydropyran) -8E-ene-4, 6-diyne-3-nonanol (self-made).
2.2 Experimental methods
2.2.1 control substance solution preparation (HPLC control)
Respectively taking radix Codonopsis alkyne glycoside, 9- (2-tetrahydropyrane) -8E-ene-4, 6-diyne-3-nonanol, atractylenolide II and atractylenolide III reference substances and a proper amount, precisely weighing, adding methanol to prepare reference substance solutions with the concentrations of the radix Codonopsis alkyne glycoside, the 9- (2-tetrahydropyrane) -8E-ene-4, 6-diyne-3-nonanol, the atractylenolide II and the atractylenolide III of 180 mu g/ml, 3 mu g/ml, 10 mu g/ml and 10 mu g/ml respectively, and filtering with a 0.22 mu m microporous filter membrane to obtain the medicine.
2.2.2 preparation of test solutions (HPLC samples)
Taking 1g of crude powder of the medicinal materials, precisely weighing, adding 25ml of methanol, carrying out ultrasonic treatment for 60min, filtering, and concentrating to dryness. Dissolving the residue with methanol, diluting to 5ml volumetric flask, and filtering with microporous membrane (0.22 μm).
2.2.3 assay
Precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into high performance liquid chromatograph, and measuring. The chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent, acetonitrile-water is used as an eluent, and gradient elution is performed according to the specification in the following table (table 2); flow rate: 1 ml/min; column temperature: 30 ℃; detection wavelength: 220 nm.
Table 2: gradient elution chart
2.2.4 results of measurement
Radix Codonopsis alkyne glycoside and 9- (2-tetrahydropyran) -8E-ene-4, 6-diyne-3-nonanol are detected in 10 batches of radix Picrasmae medicinal materials at the same time, and atractylenolide II and atractylenolide III are not detected; radix Codonopsis reference material is detected by radix Codonopsis alkyne glycoside, atractylenolide II and atractylenolide III, and is not detected by 9- (2-tetrahydropyran) -8E-ene-4, 6-diyne-3-nonanol, as shown in figure 3 and figure 4. As shown in figure 3, radix Codonopsis Lanceolatae medicinal material has radix Codonopsis alkyne glycoside and 9- (2-tetrahydropyran) -8E-ene-4, 6-diyne-3-nonanol detected at the same time, and has no Atractylodes macrocephala lactone II and Atractylodes macrocephala lactone III detected; radix Codonopsis has radix Codonopsis alkyne glycoside, atractylenolide II and atractylenolide III detected, and no 9- (2-tetrahydropyran) -8E-ene-4, 6-diyne-3-nonanol detected. As shown in figure 4, different batches of the white Yunnan ginseng medicinal material have radix Codonopsis alkyne glycoside and 9- (2-tetrahydropyrane) -8E-alkene-4, 6-diyne-3-nonanol detected at the same time, and have no atractylenolide II and atractylenolide III detected. Thus realizing the identification and the differentiation of the white cloud ginseng and the codonopsis pilosula. The sample case represented by the right-hand number in fig. 4 is: s1, comparison products; s2, radix Codonopsis control; s3, 20161110; s4, 20131016; s5, 20180101; s6, 20180102; s7, 20161110; s8, 20180103; s9, 20180104; s10, 20180201; s11, 20180202; s12, 20180203.
The lobetyolin can be detected in HPLC chromatogram; the two medicinal materials are different in that: the radix Codonopsis has a chromatographic peak at retention time corresponding to the reference substances Atractylodes lactone II and Atractylodes lactone III, and the ultraviolet spectrogram is checked to be consistent with the reference substances; the white Yunnan ginseng can detect chromatographic peak on the retention time corresponding to the reference substance 9- (2-tetrahydropyran) -8E-alkene-4, 6-diyne-3-nonanol, and check the ultraviolet spectrogram, which should be consistent with the reference substance. Under the detection method, atractylenolide II and atractylenolide III can be detected in the codonopsis pilosula medicinal material, and are not detected in the white cloud ginseng; 9- (2-tetrahydropyran) -8E-ene-4, 6-diyne-3-nonanol can be detected in the white Yunnan ginseng, but is not detected in the radix codonopsis. The method can also be used as one of the exclusive bases for identifying the dolomitic white participating in the codonopsis pilosula.
3. Summary of the invention
FIG. 1 shows that lobetyolin can be detected in TLC chromatogram of radix Codonopsis and radix Codonopsis; meanwhile, the TLC test of the white cloud ginseng medicinal material in the figure 2 cannot detect the fluorescent spots which are consistent with the reference substances of atractylenolide II and atractylenolide III; thus being identified as the white cloud ginseng. FIG. 1 shows that lobetyolin can be detected in TLC chromatogram of radix Codonopsis and radix Codonopsis; meanwhile, in the picture 2, the codonopsis pilosula medicinal material needs to be detected by fluorescent spots of atractylenolide II and atractylenolide III which are consistent with reference substances. So as to be identified as the codonopsis pilosula medicinal material.
FIG. 4 shows that peaks consistent with the retention time of lobetyolin reference substance should be detected in HPLC chromatogram of radix Codonopsis and radix Codonopsis; meanwhile, the Yunnan ginseng medicinal material must have the chromatographic peak that is consistent with the retention time of the reference substance 9- (2-tetrahydropyran) -8E-alkene-4, 6-diyne-3-nonanol and the ultraviolet spectrum to detect at the same time, must not have the chromatographic peak that is consistent with the retention time of the reference substances atractylenolide II and atractylenolide III and the ultraviolet spectrum to detect; thus, the ginseng is identified as the white cloud ginseng medicinal material. FIG. 4 shows that peaks consistent with the retention time of lobetyolin reference substance should be detected in HPLC chromatogram of radix Codonopsis and radix Codonopsis; meanwhile, the codonopsis pilosula medicinal material also needs to simultaneously detect atractylenolide II and atractylenolide III; thus, the radix codonopsitis is identified as the radix codonopsitis medicinal material.
The differences between the white cloud ginseng and the codonopsis pilosula are as follows: TLC identification, wherein the radix Codonopsis contains fluorescent spots with different colors from those of the radix Codonopsis Lanceolatae; in HPLC identification, TLC identification results are verified, and meanwhile, trace components 9- (2-tetrahydropyran) -8E-ene-4, 6-diyne-3-nonanol are found in the white Yunnan ginseng and are not detected in the radix codonopsis.
Comparative example
The relative content of 9- (2-tetrahydropyrane) -8E-ene-4, 6-diyne-3-nonanol in the cloudberry is small, the relative content difference of different producing areas/batches is large, and whether sufficient 9- (2-tetrahydropyrane) -8E-ene-4, 6-diyne-3-nonanol can be extracted from medicinal materials in HPLC detection is the key for successful detection and reduction of false negative of the cloudberry. In the HPLC detection, the method for processing the medicinal materials is researched, and different HPLC samples are obtained by trying, and the method comprises the following specific steps:
(1) the HPLC sample was obtained by taking the crude powder (from 20161110), extracting with 80% methanol solution, and following the rest of the procedure in reference to 2.2.2 test solution preparation.
(2) The HPLC sample was obtained by taking the crude powder (from 20161110), extracting with 50% methanol solution, and following the rest of the procedure in reference to 2.2.2 test solution preparation.
(3) The HPLC sample was obtained by taking the crude powder (from 20161110), extracting with ethanol, and performing the rest steps, see 2.2.2 for sample solution preparation.
And (3) carrying out HPLC detection on the HPLC samples obtained in the steps (1), (2) and (3), wherein the HPLC characteristic spectrum of the obtained HPLC sample does not show a peak at the 9- (2-tetrahydropyran) -8E-ene-4, 6-diyne-3-nonanol peak of an HPLC control. This indicates that none of the methods (1), (2) and (3) described above can effectively extract the key component 9- (2-tetrahydropyran) -8E-en-4, 6-diyne-3-nonanol from the sample.
The foregoing is merely an example of the present invention and common general knowledge in the art of designing and/or characterizing particular aspects and/or features is not described in any greater detail herein. It should be noted that, for those skilled in the art, without departing from the technical solution of the present invention, several variations and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (10)
1. A method for identifying white cloud ginseng and codonopsis pilosula is characterized by comprising the following steps: comprises the following steps of HPLC identification: establishing HPLC characteristic maps of HPLC samples and HPLC reference substances, wherein the HPLC reference substances comprise 9- (2-tetrahydropyran) -8E-alkene-4, 6-diyne-3-nonanol.
2. The method for identifying white cloud ginseng and codonopsis pilosula according to claim 1, wherein the identification method comprises the following steps: HPLC control substance also comprises lobetyolin, atractylenolide II and atractylenolide III.
3. The method for identifying white cloud ginseng and codonopsis pilosula according to claim 2, wherein the identification method comprises the following steps: before the HPLC identification step, a TLC identification step is also included: comprises a detection step and an identification detection step; in the detection step, establishing a TLC detection characteristic spectrum of a TLC sample and a detection reference substance, wherein the detection reference substance is lobetyolin; in the identification detection step, establishing TLC identification characteristic maps of a TLC sample and an identification reference substance, wherein the identification reference substance is a mixture formed by atractylenolide II and atractylenolide III.
4. The method for identifying white cloud ginseng and codonopsis pilosula according to claim 3, wherein the identification method comprises the following steps: the chromatographic conditions for establishing HPLC characteristic maps of the HPLC sample and the HPLC control are as follows: performing gradient elution by using octadecylsilane chemically bonded silica as a filling agent and a mixture of acetonitrile and water as an eluent; the flow rate was 1ml/min, the column temperature was 30 ℃ and the detection wavelength was 220 nm.
5. The method for identifying white cloud ginseng and codonopsis pilosula according to claim 4, wherein the identification method comprises the following steps: the HPLC control contains 180 μ g/ml lobetyolin, 3 μ g/ml 9- (2-tetrahydropyran) -8E-en-4, 6-diyne-3-nonanol, 10 μ g/ml atractylenolide II and 10 μ g/ml atractylenolide III.
6. The method for identifying white cloud ginseng and codonopsis pilosula according to claim 5, wherein the identification method comprises the following steps: the method for establishing the TLC detection characteristic spectrum comprises the following steps: the TLC sample and the detection reference substance are spotted on a silica gel G thin-layer plate, and a TLC detection characteristic spectrum is obtained by thin-layer chromatography using a mixture consisting of n-butanol, glacial acetic acid and water as a developing agent.
7. The method for identifying white cloud ginseng and codonopsis pilosula according to claim 6, wherein the identification method comprises the following steps: the method for establishing the TLC identification characteristic spectrum comprises the following steps: the TLC sample and the detection control are spotted on a silica gel G thin-layer plate, and a TLC identification characteristic spectrum is obtained by thin-layer chromatography using a mixture of chloroform and acetone as a developing agent.
8. The method for identifying white cloud ginseng and codonopsis pilosula according to claim 7, wherein the identification method comprises the following steps: the volume ratio of the n-butyl alcohol to the glacial acetic acid to the water is 7:1: 0.5; the volume ratio of the trichloromethane to the acetone is 10: 1.
9. The method for identifying white cloud ginseng and codonopsis pilosula according to claim 8, wherein the identification method comprises the following steps: the preparation method of the identification reference substance comprises the following steps: adding 1mg of lobetyolin into 1ml of methanol to obtain a check and identification reference substance; the preparation method of the identification reference substance comprises the following steps: 1mg of atractylenolide II and 1mg of atractylenolide III per 1ml of methanol are added.
10. The method for identifying white cloud ginseng and codonopsis pilosula according to claim 9, wherein the identification method comprises the following steps: in the TLC identification step, the TLC sample was prepared as follows: performing ultrasonic-assisted extraction on the crude powder of the medicinal materials by using methanol as an extraction solvent, and filtering to obtain filtrate to obtain an extracting solution; evaporating the extractive solution to dryness, dissolving in water, and loading onto macroporous adsorbent resin column; eluting the macroporous adsorption resin column with water and then methanol, and collecting methanol eluent; evaporating the methanol eluent to dryness, adding methanol, and uniformly mixing to obtain a TLC sample;
in the HPLC identification step, the HPLC sample is prepared by the following method: performing ultrasonic-assisted extraction on the crude powder of the medicinal materials by using methanol as an extraction solvent, and filtering to obtain filtrate to obtain an extracting solution; evaporating the extracting solution to dryness, then adding methanol, mixing uniformly, and filtering by a microporous filter membrane to obtain an HPLC sample.
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