CN118130693A - Edible tulip identification method and application of characteristic components thereof - Google Patents

Abstract

The invention discloses a method for identifying edible tulip and application of characteristic components thereof, belonging to the technical field of traditional Chinese medicine identification. The invention discloses an application of a reagent for detecting identification characteristic components of edible tulip in the identification of edible tulip, wherein when the edible tulip is the edible tulip, the characteristic components are tyrosine; when the edible tulip is a hockey stick, the characteristic components are 1, 4-di [4- (glucosyloxy) benzyl ] -2-isobutyl malate and 2-O-glucosyl bletilla glycoside. The characteristic components for identifying the edible tulip are used as index components, so that the edible tulip, the ice hockey and the respective pseudo products thereof can be identified efficiently, and the quality control of the edible tulip is facilitated.

Description

Edible tulip identification method and application of characteristic components thereof

Technical Field

The invention relates to the technical field of traditional Chinese medicine identification, in particular to an edible tulip identification method and application of characteristic components thereof.

Background

The Pseudobulbus Cremastrae seu pleiones is dried pseudobulb of Ericaceae plant CREMASTRA APPENDICULATA (D.don) Makino, herba Duchesneae Indicae Pleione bulbocodioides (Franch.) Rolfe or Yunnan herba Duchesneae Indicae Pleione yunnanensis Rolfe, the former is called Mao Cigu, and the latter is called "puck". Is mainly distributed in regions such as Yunnan, sichuan, guizhou and the like, has the effects of clearing heat and detoxicating, resolving phlegm and resolving masses, and is a common medicinal material in the traditional Chinese medicine anti-tumor prescription.

The edible tulip is a multi-basic medicinal material, and the common-name foreign matter and foreign matter are serious in common-name phenomenon and the variety is disordered because the description of the plant form, the geographical habitat and the like of the edible tulip by the traditional Chinese medicine is slightly different. Although the variety of the edible tulip is gradually clear, the counterfeiting products, mixed products and local study products are still more. In addition, in the current Chinese pharmacopoeia of 2020 edition, the edible tulip contains only two identification items of characters and microscopes, and lacks qualitative and quantitative standards of a system, which also results in good quality and uniformity of edible tulip medicines in the market. Therefore, how to rapidly and effectively identify the edible tulip and the pseudo-product medicinal materials thereof is urgent.

Currently, the quality standards of the edible tulip (unibract) formula granule and the edible tulip (azalea) formula granule are respectively disclosed in Guangdong province and Shanghai city. The thin-layer identification items in the two quality standards respectively adopt the tulip and the hockey puck reference medicinal materials as reference substances, the components with strong specificity are lack as reference substances for reference, the tulip and the hockey puck are respectively very similar to the chemical components of similar varieties, and the thin-layer chromatographic spots are approximately the same, so that the result is easy to be misjudged.

Recently, a scholars also discloses a thin-layer identification method of the ice hockey and the Indian iphigenia bulb, but the method is heavy in distinguishing between the basic sources of the ice hockey and the Indian iphigenia bulb, does not distinguish the pseudo-products of the Indian iphigenia bulb (such as the Indian iphigenia bulb, the cymbidium goeringii, the Indian iphigenia bulb and the like) existing in the market at present, and can be seen that the thin-layer identification of the Indian iphigenia bulb still has the problems of poor specificity, easy misjudgment and the like.

Disclosure of Invention

Aiming at the defect or deficiency that the identification method in the prior art can not effectively distinguish the edible tulip and the pseudo products thereof, the invention provides the application of the reagent for detecting the identification characteristic components of the edible tulip in the identification of the edible tulip, and the adoption of the identification characteristic components of the edible tulip as index components can realize the efficient identification of the edible tulip, the puck and the respective pseudo products thereof, thereby being beneficial to the quality control of the edible tulip.

In one aspect, the invention discloses an application of a reagent for detecting identification characteristic components of edible tulip in identification of edible tulip, wherein when the edible tulip is the edible tulip, the characteristic components are tyrosine; when the edible tulip is a hockey stick, the characteristic components are 1, 4-di [4- (glucosyloxy) benzyl ] -2-isobutyl malate and 2-O-glucosyl bletilla glycoside.

The laboratory finds that the two commodities of the edible tulip, mao Cigu and the puck, have large differences in the aspects of characters and chemical components in early-stage experiments, so that the thin-layer identification method of the edible tulip and the puck is respectively established. In the early stage of the experiment, the index component tyrosine with stronger specificity in Mao Cigu is calibrated through UPLC-Q-TOF-MS/MS. The tyrosine content in Mao Cigu medicinal materials is high and stable, and meanwhile, the components are not detected in the ice hockey, the pseudo product of the rhizoma bletilla, the rhizoma bletillae, the rhizoma pleiones and the rhizoma pleionis or the tyrosine content is low. In addition, the specific index components 1, 4-bis [4- (glucosyloxy) benzyl ] -2-isobutyl malate (militarine) and 2-O-glucosyl bletilla glycoside (dactylorhin A) in the hockey seed are also calibrated, and the two components have obvious differences in the hockey seed and the pseudo-products of the hockey seed, namely, the pachyrhizus, the condyloma verrucosum, the cylindracea, the panhead and the other edible tulip commodity.

Therefore, in the invention, mao Cigu takes tyrosine as a characteristic component, and the puck takes militarine and dactylorhin A as characteristic components, so that respectively corresponding thin layer identification methods with strong specificity can be established.

In some of these embodiments, the authentication is performed by a thin layer authentication method.

In some of these embodiments, the edible tulip identification feature is used to identify edible tulip and cymbidium, round white, light arrowhead, shanlan, autumn flower unibract or wart sheath unibract.

In some of these embodiments, the tyrosine is used to identify mao arrowhead and cymbidium, round white, light arrowhead, mountain orchid, autumn flower unibract, wart sheath unibract or hockey;

the 1, 4-di [4- (glucosyloxy) benzyl ] -2-isobutyl malate and 2-O-glucosyl bletilla glycoside are used for identifying the hockey and autumn flower single garlic blue, wart sheath single garlic blue, cymbidium album, indian kalimeris herb or Indian iphigenia bulb.

On the other hand, the invention discloses a thin-layer identification method of edible tulip, which adopts the following thin-layer identification method of edible tulip and/or puck to identify edible tulip medicinal materials,

The Mao Cigu thin layer identification method comprises the following steps:

(1) Preparation of an identification solution: taking a to-be-identified Mao Cigu medicinal material, adding a methanol aqueous solution with the volume percentage of 75+/-10% as a solvent, performing ultrasonic extraction, and filtering to obtain a filtrate, namely a sample solution;

(2) Thin layer chromatography development: the solution of the sample is absorbed and spotted on a silica gel thin layer plate, and the mixed solvent of n-butanol-glacial acetic acid-water-formic acid with the volume ratio of 4+/-0.4:1+/-0.1:2+/-0.2:0.2+/-0.02 is taken as a developing agent for developing;

(3) And (5) checking: spraying 5+ -2% ninhydrin solution, heating at 105+ -10deg.C until the color of the spot is clear, and inspecting in sunlight, if the spot with the same color in the corresponding position of tyrosine spot in the chromatogram of the sample is detected, judging as genuine;

the ice hockey thin layer identification method comprises the following steps:

(1) Sample solution preparation: taking the quasicrystal medicinal material to be identified, adding an ethanol water solution with the volume percentage of 50+/-10% as a solvent, carrying out ultrasonic extraction, filtering, evaporating filtrate to obtain residues, and adding methanol to dissolve the residues to obtain a sample solution;

(2) Thin layer chromatography development: the solution of the sample is sucked and spotted on a silica gel thin layer plate, and the mixed solvent of n-butanol-ethyl acetate-trichloromethane-methanol-water-glacial acetic acid with the volume ratio of 4+/-0.4:1+/-0.1:2+/-0.2:2+/-0.2:1.5+/-0.15:0.1+/-0.01 is used as a developing agent for developing;

(3) And (5) checking: spraying 10+ -5% sulfuric acid ethanol solution of 5+ -2% vanillin, heating at 105+ -10deg.C until the color of the spots is clear, and inspecting under sunlight, wherein the spots with the same color as the spots of 1, 4-bis [4- (glucosyloxy) benzyl ] -2-isobutyl malate and 2-O-glucosyl bletilla glycoside in the sample chromatogram are judged to be genuine.

The inventor synthesizes the thin layer chromatography behaviors of the edible tulip and the puck based on the research of the characteristic components of the edible tulip, confirms that Mao Cigu thin layer identification and puck Mao Cigu thin layer identification are carried out by the method after experimental finding and trying, not only can the important function of the characteristic components with strong specificity in the thin layer identification be reflected, but also each characteristic and representative spot of Mao Cigu and puck can be comprehensively displayed, and a better thin layer identification effect is obtained.

In some of these embodiments, the Mao Cigu thin layer identification method, the tyrosine spot is obtained by: preparing a reference medicinal material solution by taking a reference medicinal material of Indian iphigenia bulb and referring to a preparation method of a test solution, and taking the reference medicinal material solution, spotting the reference medicinal material solution and the test solution on the same silica gel thin layer plate, and developing and inspecting under the same condition;

And/or taking a tyrosine reference substance, adding methanol to prepare a solution containing 0.1mg of tyrosine per 1mL of the solution as a reference substance solution, taking the reference substance solution, spotting the reference substance solution and the sample solution on the same silica gel thin layer plate, and developing and inspecting under the same condition;

in the ice hockey thin layer identification method, the 1, 4-bis [4- (glucosyloxy) benzyl ] -2-isobutyl malate and 2-O-glucosyl bletilla glycoside spots are obtained by the following method:

Taking a hockey seed reference medicinal material, preparing a reference medicinal material solution by referring to a preparation method of a test sample solution, taking the reference medicinal material solution, spotting the reference medicinal material solution and the test sample solution on the same silica gel thin layer plate, and developing and inspecting under the same condition;

and/or taking 1, 4-di [4- (glucoyloxy) benzyl ] -2-isobutyl malate and 2-O-glucosyl bletilla striata glycoside reference substances, adding methanol to prepare solutions containing 0.1mg of 1, 4-di [4- (glucoyloxy) benzyl ] -2-isobutyl malate or 2-O-glucosyl bletilla striata glycoside per 1mL respectively, taking the reference substance solutions, spotting the reference substance solutions and the sample solutions on the same silica gel thin layer plate, and developing and inspecting under the same conditions.

In some embodiments, the Mao Cigu thin-layer identification method further comprises determining that the sample is a genuine sample, wherein the spot of the sample has the same color as the spot of the Mao Cigu reference drug;

In the ice hockey thin layer identification method, the condition of judging the ice hockey thin layer identification method is that the ice hockey thin layer identification method is a genuine product, and the ice hockey thin layer identification method also comprises spots with the same color as corresponding positions of ice hockey comparison medicinal material chromatographic spots in the chromatogram of the sample to be tested.

In some of these embodiments, the Mao Cigu thin layer authentication method meets at least one of the following conditions:

(1) The Rf value of the tyrosine spot is 0.58+/-0.02;

(2) The developing agent is a mixed solvent of n-butanol-glacial acetic acid-water-formic acid with the volume ratio of 4:1:2:0.2;

(3) Adding a solvent according to the amount of adding 10+/-5 mL of methanol aqueous solution into each gram Mao Cigu of medicinal materials;

(4) The volume percentage concentration of the methanol aqueous solution is 75+/-10%, preferably 75+/-5%, more preferably 75%;

(5) The power of ultrasonic extraction is 300+/-100 w, the frequency is 50+/-10 KHz, the power is 300w, and the frequency is 50KHz;

(6) The ultrasonic extraction time is 20-40min, preferably 25-35min, more preferably 30min;

(7) The silica gel thin layer plate is a silica gel G thin layer plate;

the ice hockey thin layer identification method meets at least one of the following conditions:

(1) The Rf value of the 1, 4-di [4- (glucosyloxy) benzyl ] -2-isobutyl malate or 2-O-glucosyl bletilla striata glycoside spot is 0.55+/-0.02 and 0.42+/-0.02 respectively;

(2) The developing agent is a mixed solvent of n-butanol-ethyl acetate-chloroform-methanol-water-glacial acetic acid with the volume ratio of 4:1:2:2:1.5:0.1;

(3) Adding a solvent according to the amount of 50+/-10 mL of the ice hockey seed medicinal material per gram;

(4) The volume percentage of the ethanol aqueous solution is 50+/-10%, preferably 50+/-10%, more preferably 50%;

(5) The power of ultrasonic extraction is 300+/-100 w, the frequency is 50+/-10 KHz, the power is 300w, and the frequency is 50KHz;

(6) The ultrasonic extraction time is 20-40min, preferably 25-35min, more preferably 30min;

(7) Dissolving the residue by adding 0.8-0.1.2mL of methanol per gram of the hockey stick medicinal material, preferably 0.9-1.1mL of methanol, more preferably 1.0mL of methanol;

(8) The silica gel thin layer plate is a silica gel G thin layer plate.

On the other hand, the invention also discloses application of the thin-layer identification method for the edible tulip in quality control of the edible tulip.

In some of these embodiments, the method is used to distinguish between Pseudobulbus Cremastrae seu pleiones and pseudobulbus Cremastrae seu pleiones, rhizoma Bletillae, bulbus Iphigeniae Indicae, herba Saxifragae Japonicae, and herba Oenanthes Javanicae;

further, the Mao Cigu thin layer identification method is used for identifying the rhizoma pleionis and the cymbidium sinense, the rhizoma bletillae, the rhizoma pleionis, the rhizoma paridis, the autumn flower unibract, the wart sheath unibract or the hockey;

the thin-layer identification method of the hockey puck is used for identifying the hockey puck and the autumn flower single garlic blue, the wart sheath single garlic blue, the cylindracea, the rhizoma bletillae, the mountain orchid or the Indian iphigenia bulb.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that:

The reagent for detecting the characteristic components of the edible tulip identification is applied to the identification of the edible tulip, tyrosine is used as a reference substance in the edible tulip, 1, 4-bis [4- (glucose oxy) benzyl ] -2-isobutyl malate (militarine, CAS: 58139-23-4) and 2-O-glucosyl bletilla glycoside (dactylorhin A, CAS: 256459-34-4) are used as reference substances in the puck, and respectively corresponding strong-specificity thin-layer identification methods are established. Can realize the high-efficiency identification of the edible tulip and the ice hockey and the respective pseudo products thereof, and is beneficial to the quality control of the edible tulip.

Drawings

FIG. 1 is a thin layer chromatogram of multiple batches of the test solution of Pseudobulbus Cremastrae seu pleiones, the control solution of Mao Cigu, and the control solution of example 1.

FIG. 2 is a thin layer chromatogram of multiple batches of pseudobulb test and control solutions in example 1.

FIG. 3 shows thin-layer chromatograms of ice hockey and its pseudo sample solution, mao Cigu control medicinal material solution and control solution in Mao Cigu thin-layer condition in example 1.

FIG. 4 is a thin layer chromatogram of multiple batches of the hockey puck test solution, control drug solution, and control solution of example 2.

FIG. 5 shows thin layer chromatograms of Mao Cigu and its pseudo sample solution, the pseudo sample solution of the ice hockey, the control medicinal material solution and the control sample solution under the ice hockey thin layer condition in example 2.

FIG. 6 is a thin layer chromatogram (sun view) of comparative example 1.

FIG. 7 is a thin layer chromatogram (sun view) of comparative example 2.

FIG. 8 is a thin layer chromatogram (inspection at 365 nm) of comparative example 2.

FIG. 9 is a thin layer chromatogram (inspection at 365 nm) of comparative example 3.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Instrument, reagent and experimental material

Thin layer imaging system (biostep DD, thin layer chromogenic heater), supelco (silica gel 60 preformed plate).

Reagent: the chemical reagents such as ethyl acetate, n-butanol, chloroform, methanol, ethanol, glacial acetic acid, formic acid and the like are all analytically pure, and the water is purified water.

Experimental materials: mao Cigu control (MCG 9, DNA molecule identified as azalea, supplied by the guangzhou drug test); ice hockey seed control drug 1 (supplied by Shanghai HongYongsheng biotechnology Co., ltd., lot number: 280132-202302); hockey seed control drug 2 (supplied by Sichuan Vickers Biotechnology Co., ltd., lot number: ycwkq 23062903).

Tyrosine control (supplied by the middle inspection department, lot number 140609-201914); 1, 4-bis [4- (glucoyloxy) benzyl ] -2-isobutylmalate control (supplied by Mitsui, lot number 112061-202102); 2-O-glucosyl bletilla striata glycoside reference (supplied by Vickers biotechnology Co., ltd., lot number: wkq22030801, sichuan province).

Mao Cigu samples (lot number: MCG1-MCG 8), puck samples (lot number: DSL1-DSL 6), cymbidium (lot number: TBL1-TBL 8), shanlan (lot number: SL1-SL 3), yuanhe (lot number: YBJ 1), pseudobulbus Cremastrae seu pleiones (lot number: GCG 1), lemongrass (lot number: QH1-QH 2), lemongrass (lot number: YQ 1). The original plant of the sample collected by Guangzhou market medicine inspection is combined with a plant taxonomy method and a molecular biology method to identify the original plant, and the medicinal part pseudobulb is processed according to pharmacopoeia standards and dried to prepare the corresponding medicinal material sample.

Example 1

A thin layer identification method of Pseudobulbus Cremastrae seu pleiones (Mao Cigu).

1. Method of

1.1 Preparation of identification solutions

Taking 1g of each of the medicinal material powder of Indian iphigenia bulb, cymbidium sinense, indian kalimeris herb, indian iphigenia bulb, autumn flower single garlic herb, wart sheath single garlic herb and puck seed, respectively adding 10mL of 75% methanol aqueous solution by volume percent, carrying out ultrasonic treatment (with the power of 300W and the frequency of 50 KHz) for 30 minutes, and filtering to obtain filtrate serving as a sample solution.

1G of rhizoma pleionis control medicinal material powder is prepared, and a control medicinal material solution is prepared by the same method.

And adding 75% methanol into tyrosine reference substance to obtain solution containing 0.1mg per 1mL, and taking the solution as reference substance solution.

1.2 Thin layer chromatography development

Sucking 5 mu L of each of the test solution, the control medicinal solution and the control solution, respectively spotting on the same silica gel G thin layer plate, developing with n-butanol-glacial acetic acid-water-formic acid (4:1:2:0.2) as developing agent, and air drying.

1.3 Inspection of

Spraying 5% ninhydrin ethanol solution, heating at 105deg.C until the color of spots is clear, and inspecting in sunlight.

2. Results

FIG. 1 is a thin layer chromatogram of multiple batches of a test solution of Pseudobulbus Cremastrae seu pleiones, a control solution of Mao Cigu, and a tyrosine control solution; wherein: 1-2. Test solutions of Pseudobulbus Cremastrae seu pleiones (batch number: MCG2, MCG 3); 3. mao Cigu control materials; 4-6. Test solutions of Pseudobulbus Cremastrae seu pleiones (batch number: MCG4, MCG5, MCG 1); 7. tyrosine control.

FIG. 2 is a thin layer chromatogram of multiple batches of pseudobulb of Pseudobulbus Cremastrae seu pleiones test solution and control solution; wherein: 1-8. Sample solution of cymbidium (batch number: TBL1-TBL 8); 9. round white hyacinth bletilla (batch number: YBJ 1); 10. light arrowhead (batch number: GCG 1); 11-12. Sample solutions of the Shanlan (SL 1, SL 3); 13. tyrosine control.

FIG. 3 shows thin layer chromatograms of ice hockey and its pseudo sample solution, mao Cigu control medicinal solution and control solution under thin layer condition of Pseudobulbus Cremastrae seu pleiones; wherein: 1-2. Autumn flower Duganlan (batch number: QH1-QH 2); 3. leucocalyxa (batch number: YQ 1); 4. mao Cigu control materials; 5. a tyrosine control; 6-7 Ice hockey (batch number: DSL1, DSL 4)

From the results, in the chromatography of the test sample of the Indian iphigenia bulb, the spots are clear at the positions corresponding to the chromatography of the tyrosine control, and are basically consistent with the spots of the Mao Cigu control medicinal materials, so that the separation effect is good. No spots corresponding to the tyrosine control were detected in the cymbidium, rhizoma Bletillae, bulbus Iphigeniae Indicae, herba Aristolochiae Mollissimae, and herba Aristolochiae Mollissimae.

In conclusion, a Mao Cigu thin-layer identification method can be established by taking tyrosine as a characteristic component of the tulip with strong specificity, and the tulip thin-layer identification method is determined as follows:

Adding 75% methanol water solution into rhizoma Sagittariae Sagittifoliae powder, performing ultrasonic treatment, and filtering to obtain filtrate as sample solution; preparing a control medicinal material powder of the Indian iphigenia bulb, and preparing a control medicinal material solution by the same method; taking tyrosine reference substance, adding 75% methanol water solution to obtain solution containing 0.1mg per 1mL, and taking the solution as reference substance solution; drawing the sample solution, the reference medicinal material solution and the reference substance solution to be respectively spotted on the same silica gel G thin layer plate, developing by taking n-butanol-glacial acetic acid-water-formic acid (4:1:2:0.2) as developing agents, airing, spraying 5% ninhydrin ethanol solution, heating at 105 ℃ until spots develop clearly, and inspecting under sunlight; spots with the same color appear on the chromatogram of the sample at the positions corresponding to the chromatograms of Mao Cigu reference materials and tyrosine reference materials.

Example 2

A thin layer identification method of Pseudobulbus Cremastrae seu pleiones (puck) is provided.

1. Method of

1.1 Preparation of identification solutions

Taking 1g of each of ice hockey, autumn flower single garlic blue, wart sheath single garlic blue, cymbidium sinense, indian iphigenia bulb, round white hyacinth bletilla and mountain orchid medicinal material powder, respectively adding 50mL of 50% ethanol water solution by volume percent, carrying out ultrasonic treatment (with the power of 300W and the frequency of 50 KHz) for 30 minutes, filtering, evaporating filtrate to dryness, and adding 1mL of methanol into residues to dissolve the residues to obtain a sample solution.

Taking 1g of ice hockey seed reference medicinal material powder, and preparing a reference medicinal material solution by the same method.

And adding methanol into 1, 4-di [4- (glucosyloxy) benzyl ] -2-isobutyl malate reference substance and 2-O-glucosyl bletilla glycoside reference substance to prepare solutions containing 1mg/mL respectively as reference substance solutions.

1.2 Thin layer chromatography development

Sucking 4 μl of each of the sample solution, the control medicinal material solution and the control solution, respectively spotting on the same silica gel G thin layer plate, spreading with n-butanol-ethyl acetate-chloroform-methanol-water-glacial acetic acid (4:1:2:2:1.5:0.1) as developing agent, and air drying.

1.3 Inspection of

Spraying 10% sulfuric acid ethanol solution of 5% vanillin, heating at 105deg.C until the color of spots is clear, and inspecting in sunlight.

2. Results

FIG. 4 is a thin layer chromatogram of multiple batches of hockey subject solution, control drug solution, and control solution; wherein: 1-3. The puck sample solution (batch number: DSL1-DSL 3); 4. the hockey seed is used for controlling the medicinal material 1;5. the hockey seed is used for controlling the medicinal material 2;6.1,4-bis [4- (glucoyloxy) benzyl ] -2-isobutyl malate control; 7.2-O-glucosyl bletilla striata glycoside reference substance; 8-10. The puck sample solution (batch number: DSL4-DSL 6).

FIG. 5 is a thin layer chromatogram of Mao Cigu and its pseudo sample solution, and pseudo sample solution, control medicinal solution, and control solution of hockey seed under thin layer condition;

Wherein: 1-2. Autumn flower Duganlan (batch number: QH1-QH 2); 3. leucocalyxa (batch number: YQ 1); 4. cylinder flange (batch number: TBL 4); 5. round white hyacinth bletilla (batch number: YBJ 1); 6. mountain blue (lot: SL 3); 7. mao Cigu control materials; 8. the hockey seed is used for controlling the medicinal material 1;9. the hockey seed is used for controlling the medicinal material 2;10.1,4-bis [4- (glucoyloxy) benzyl ] -2-isobutyl malate control; 2-O-glucosyl bletilla striata glycoside reference substance.

As can be seen from the results, the corresponding spots of 1, 4-bis [4- (glucoyloxy) benzyl ] -2-isobutyl malate and 2-O-glucosyl-bletilla glycoside were detected for the puck samples. Whereas the working controls of autumn flower single garlic blue, wart sheath single garlic blue, mao Cigu pseudo-product mountain blue and Indian iphigenia bulb did not detect spots corresponding to 1, 4-bis [4- (glucosyloxy) benzyl ] -2-isobutyl malate and 2-O-glucosyl bletilla glycoside; spots corresponding to 2-O-glucosyl bletilla striata glycoside were not detected for cymbidium and bletilla striata.

In summary, the method for identifying the thin layer of the ice hockey ball can be established by taking 1, 4-di [4- (glucosyloxy) benzyl ] -2-isobutyl malate and 2-O-glucosyl bletilla glycoside as characteristic components of the ice hockey ball with strong specificity, and the method for identifying the thin layer of the ice hockey ball is determined as follows:

Taking ice hockey herb powder, carrying out ultrasonic treatment on the ice hockey herb powder by using an ethanol water solution with the volume percentage of 50 percent, filtering, evaporating filtrate to dryness, and adding 1mL of methanol into residues to dissolve the residues to be used as a sample solution; preparing a control medicinal material powder of the hockey puck, and preparing a control medicinal material solution by the same method; taking 1, 4-di [4- (glucosyloxy) benzyl ] -2-isobutyl malate reference substance and 2-O-glucosyl bletilla striata glycoside reference substance, respectively adding methanol to prepare solutions containing 1mg/mL respectively, and taking the solutions as reference substance solutions; drawing the sample solution, the reference medicinal material solution and the reference substance solution to be respectively spotted on the same silica gel G thin layer plate, developing with n-butanol-ethyl acetate-chloroform-methanol-water-glacial acetic acid (4:1:2:2:1.5:0.1) as developing agent, air drying, spraying 5% vanillin-10% sulfuric acid ethanol solution, heating at 105deg.C until the spot color is clear, and inspecting in sunlight; in the chromatogram of the sample, spots with the same color appear at the positions corresponding to the chromatograms of the hockey seed control medicine, the 1, 4-di [4- (glucosyloxy) benzyl ] -2-isobutyl malate control and the 2-O-glucosyl bletilla glycoside control.

Comparative example 1

The comparative example uses other known thin layer identification methods to identify the tulip bulb.

1. Method of

Taking 0.4g of each of the Indian iphigenia bulb, the Indian kalimeris herb and the cymbidium herb powder, respectively adding 30mL of ethyl acetate, heating and refluxing for 30min, filtering, evaporating filtrate to dryness, and adding 1mL of ethyl acetate into residues to dissolve the residues to obtain a sample solution; the preparation method comprises preparing control medicinal material solution by the same method with 0.4g of rhizoma Sagittariae Sagittifoliae control medicinal material powder. According to thin layer chromatography (China pharmacopoeia general rule 0502), 20 μl of each of the above two solutions is absorbed and respectively spotted on the same silica gel G thin layer plate, toluene-ethyl acetate (5:2) is used as developing agent, and the mixture is developed, taken out, dried, sprayed with 5% vanillin sulfuric acid solution, heated at 105deg.C until the color of the spots is clear, and inspected under sunlight (see FIG. 6).

2. Results

FIG. 6 is a thin layer chromatogram of this comparative example; wherein: 1-3. Test solution of Pseudobulbus Cremastrae seu pleiones (batch number: MCG1-MCG 3); 4. mao Cigu control materials; 5-6. Iphigenia Indici sample solution (batch number: MCG4-MCG 5); 7-9. Sample solution of Shanlan (batch number: SL1-SL 3); 10. and the cylinder valve blue (batch number is TBL 6).

As can be seen from the results, mao Cigu is basically identical to the thin-layer chromatography spots of the pseudo-panda, and is almost identical to the spots of the pseudo-panda, and misjudgment is easy to occur although the panda has one more red spot. Under this condition, mao Cigu cannot be distinguished from the pseudo-shanlan and the cylindracea.

Comparative example 2

The present comparative example uses other known thin layer identification methods to identify the puck.

1. Method of

Taking 3g of each of ice hockey, autumn flower single garlic blue, wart sheath single garlic blue, cymbidium sinense, indian iphigenia bulb, round white hyacinth bletilla and mountain orchid medicinal material powder, respectively adding 50mL of water, heating and refluxing for 30 minutes, filtering, evaporating the filtrate to dryness, adding 30mL of chloroform into the residue with a proper amount of concentrated ammonia solution to moisten the residue, heating and refluxing for 30 minutes, cooling, filtering, evaporating the filtrate to dryness, and adding 1mL of methanol into the residue to dissolve the residue to serve as a sample solution; and 3g of a tulip control medicinal material and a hockey puck control medicinal material powder are respectively prepared, and a control medicinal material solution is prepared by the same method. According to thin layer chromatography (general rule 0502), the above solutions are respectively absorbed by 15 μl and respectively spotted on the same silica gel G thin layer plate, chloroform-acetone-methanol-concentrated ammonia test solution (7:2:0.2:0.1) is used as developing agent, and then developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105deg.C until the color of spots is clear, and respectively placed under sunlight and ultraviolet lamp (365 nm) for inspection (see FIG. 7-8).

2. Results

FIG. 7 is a thin layer chromatogram of the present comparative example as viewed under sunlight; wherein: 1. mao Cigu control materials; 2-4. Test solution of Pseudobulbus Cremastrae seu pleiones (batch number: MCG1-MCG 3); 5-6. Sample solutions of the Shanlan (batch number: SL1, SL 3); 7. round white hyacinth bletilla (lot number YBJ 1); 8-9. Sample solutions of cymbidium (batch number: TBL1, TBL 6); 10. the hockey seed is used for controlling the medicinal material 1;11-13, puck sample solution (batch number: DSL1-DSL2, DSL 4); 14-15. Autumn flower Duganlan (batch number: QH1-QH 2); 16. leucocalyxa (batch number: YQ 1).

FIG. 8 is a thin layer chromatogram of this comparative example at 365 nm; wherein: 1. mao Cigu control materials; 2-4. Test solution of Pseudobulbus Cremastrae seu pleiones (batch number: MCG1-MCG 3); 5-6. Sample solutions of the Shanlan (batch number: SL1, SL 3); 7. round white hyacinth bletilla (lot number YBJ 1); 8-9. Sample solutions of cymbidium (batch number: TBL1, TBL 6); 10. the hockey seed is used for controlling the medicinal material 1;11-13, puck sample solution (batch number: DSL1-DSL2, DSL 4); 14-15. Autumn flower Duganlan (batch number: QH1-QH 2); 16. leucocalyxa (batch number: YQ 1).

As can be seen from the results, the thin-layer chromatography shows that under the 365nm of sunlight and ultraviolet light, the color spectrum spots of the hockey seed medicinal material are basically consistent with those of the pseudo-product of the autumn flower single garlic blue, the wart sheath single garlic blue, the round white and the cymbidium, and can not be distinguished. In addition, mao Cigu and the pseudo-mountain orchid have fewer spots and have no obvious difference and cannot be distinguished under the method. Under this condition, it is not possible to distinguish the hockey puck from the pseudo-autumn flower unibract, the wart sheath unibract, the round white and the cymbidium.

Comparative example 3

The comparative example uses other known thin layer identification methods to identify Pseudobulbus Cremastrae seu pleiones.

1. Method of

Taking 0.5g of each of the ice hockey, the autumn flower single garlic blue, the wart sheath single garlic blue, the cymbidium sinense, the Indian iphigenia bulb, the round white hyacinth and the mountain orchid medicinal material powder, respectively adding 2mL of ethanol water solution with the volume percentage of 70%, carrying out ultrasonic extraction for 10min, and filtering to obtain a sample solution; the control medicinal material solution is prepared by the same method with 0.5g of the control medicinal material powder of Indian iphigenia bulb and the control medicinal material powder of the puck. According to the thin layer chromatography (general rule 0502), the above solutions were sucked into 4. Mu.L each and respectively spotted on the same silica gel G thin layer plate, petroleum ether-ethyl acetate-glacial acetic acid (25:5:1) was used as developing agent, developed, taken out, dried in the air, sprayed with 10% sulfuric acid ethanol solution, heated at 105℃for 3 minutes to develop clear spots, and then examined under an ultraviolet lamp (365 nm) (see FIG. 9).

2. Results

FIG. 9 is a thin layer chromatogram of this comparative example at 365 nm; wherein: 1. mao Cigu control materials; 2-4. Test solution of Pseudobulbus Cremastrae seu pleiones (batch number: MCG1-MCG 3); 5-6. Sample solutions of the Shanlan (batch number: SL1, SL 3); 7. round white (lot number YBJ 1); 8-9. Sample solutions of cymbidium (batch number: TBL1, TBL 6); 10. the hockey seed is used for controlling the medicinal material 1;11-13, puck sample solution (batch number: DSL1-DSL2, DSL 4); 14-15. Autumn flower Duganlan (batch number: QH1-QH 2);

As can be seen from the results, the thin-layer chromatography shows that under the 365nm ultraviolet lamp, the Rf value of the chromatogram is 0.14-0.21, and the sample solutions of the hockey puck medicinal material and the pseudo-product of the autumn flower single garlic blue, the round white and the cymbidium are all provided with yellow-green fluorescent spots, so that the hockey puck and the pseudo-product of the autumn flower single garlic blue, the round white and the cymbidium can not be distinguished. Meanwhile, mao Cigu is basically consistent with the pseudo-mountain orchid chromatographic spots, and cannot be distinguished.

Claims (10)

1. Application of a reagent for detecting characteristic components of edible tulip in the identification of edible tulip, wherein when the edible tulip is the edible tulip, the characteristic components are tyrosine; when the edible tulip is a hockey stick, the characteristic components are 1, 4-di [4- (glucosyloxy) benzyl ] -2-isobutyl malate and 2-O-glucosyl bletilla glycoside.

2. The use according to claim 1, wherein said authentication is by means of thin layer authentication.

3. The use according to claim 1, wherein the edible tulip identification characteristic is used for identifying edible tulip and pseudo-cymbidium goeringii, bletilla striata, phophia indica, shanlan, autumn-flower-unibract, or verruca-unibract.

4. Use according to claim 3, wherein the tyrosine is used to identify tulip and cymbidium, bletilla striata, tulip, shanlan, autumn-flower unibract, condyloma-like unibract or hockey;

the 1, 4-di [4- (glucosyloxy) benzyl ] -2-isobutyl malate and 2-O-glucosyl bletilla glycoside are used for identifying the hockey and autumn flower single garlic blue, wart sheath single garlic blue, cymbidium album, indian kalimeris herb or Indian iphigenia bulb.

5. A thin-layer identification method for edible tulip is characterized in that the thin-layer identification method for edible tulip to be identified is carried out by adopting the following thin-layer identification method for edible tulip and/or puck,

The Mao Cigu thin layer identification method comprises the following steps:

(1) Preparation of an identification solution: taking a to-be-identified Mao Cigu medicinal material, adding a methanol aqueous solution with the volume percentage of 75+/-10% as a solvent, performing ultrasonic extraction, and filtering to obtain a filtrate, namely a sample solution;

(2) Thin layer chromatography development: the solution of the sample is absorbed and spotted on a silica gel thin layer plate, and the mixed solvent of n-butanol-glacial acetic acid-water-formic acid with the volume ratio of 4+/-0.4:1+/-0.1:2+/-0.2:0.2+/-0.02 is taken as a developing agent for developing;

(3) And (5) checking: spraying 5+ -2% ninhydrin solution, heating at 105+ -10deg.C until the color of the spot is clear, and inspecting in sunlight, if the spot with the same color in the corresponding position of tyrosine spot in the chromatogram of the sample is detected, judging as genuine;

the ice hockey thin layer identification method comprises the following steps:

(1) Sample solution preparation: taking the quasicrystal medicinal material to be identified, adding an ethanol water solution with the volume percentage of 50+/-10% as a solvent, carrying out ultrasonic extraction, filtering, evaporating filtrate to obtain residues, and adding methanol to dissolve the residues to obtain a sample solution;

(2) Thin layer chromatography development: the solution of the sample is sucked and spotted on a silica gel thin layer plate, and the mixed solvent of n-butanol-ethyl acetate-trichloromethane-methanol-water-glacial acetic acid with the volume ratio of 4+/-0.4:1+/-0.1:2+/-0.2:2+/-0.2:1.5+/-0.15:0.1+/-0.01 is used as a developing agent for developing;

(3) And (5) checking: spraying 10+ -5% sulfuric acid ethanol solution of 5+ -2% vanillin, heating at 105+ -10deg.C until the color of the spots is clear, and inspecting under sunlight, wherein the spots with the same color as the spots of 1, 4-bis [4- (glucosyloxy) benzyl ] -2-isobutyl malate and 2-O-glucosyl bletilla glycoside in the sample chromatogram are judged to be genuine.

6. The method for thin-layer identification of edible tulip as claimed in claim 5, wherein the Mao Cigu thin-layer identification method is characterized in that the tyrosine spots are obtained by the following method: preparing a reference medicinal material solution by taking a reference medicinal material of Indian iphigenia bulb and referring to a preparation method of a test solution, and taking the reference medicinal material solution, spotting the reference medicinal material solution and the test solution on the same silica gel thin layer plate, and developing and inspecting under the same condition;

and/or taking a tyrosine reference substance, adding 75% methanol aqueous solution by volume percent to prepare a solution containing 0.1mg tyrosine per 1mL as a reference substance solution, taking the reference substance solution, spotting the reference substance solution and the sample solution on the same silica gel thin layer plate, and developing and inspecting under the same condition;

in the ice hockey thin layer identification method, the 1, 4-bis [4- (glucosyloxy) benzyl ] -2-isobutyl malate and 2-O-glucosyl bletilla glycoside spots are obtained by the following method:

Taking a hockey seed reference medicinal material, preparing a reference medicinal material solution by referring to a preparation method of a test sample solution, taking the reference medicinal material solution, spotting the reference medicinal material solution and the test sample solution on the same silica gel thin layer plate, and developing and inspecting under the same condition;

and/or taking 1, 4-di [4- (glucoyloxy) benzyl ] -2-isobutyl malate and 2-O-glucosyl bletilla striata glycoside reference substances, adding methanol to prepare solutions containing 0.1mg of 1, 4-di [4- (glucoyloxy) benzyl ] -2-isobutyl malate or 2-O-glucosyl bletilla striata glycoside per 1mL respectively, taking the reference substance solutions, spotting the reference substance solutions and the sample solutions on the same silica gel thin layer plate, and developing and inspecting under the same conditions.

7. The thin-layer identification method of edible tulip as claimed in claim 6, wherein the condition for judging as genuine product in the thin-layer identification method of Mao Cigu further comprises spots of the same color in the sample chromatogram corresponding to the Mao Cigu reference medicinal material chromatogram spots;

In the ice hockey thin layer identification method, the condition of judging the ice hockey thin layer identification method is that the ice hockey thin layer identification method is a genuine product, and the ice hockey thin layer identification method also comprises spots with the same color as corresponding positions of ice hockey comparison medicinal material chromatographic spots in the chromatogram of the sample to be tested.

8. The method of claim 5, wherein the Mao Cigu thin layer identification method meets at least one of the following conditions:

(1) The Rf value of the tyrosine spot is 0.58+/-0.02;

(2) The developing agent is a mixed solvent of n-butanol-glacial acetic acid-water-formic acid with the volume ratio of 4:1:2:0.2;

(3) Adding a solvent according to the amount of adding 10+/-5 mL of methanol aqueous solution into each gram Mao Cigu of medicinal materials;

(4) The volume percentage concentration of the methanol aqueous solution is 75+/-10%, preferably 75+/-5%, more preferably 75%;

(5) The power of ultrasonic extraction is 300+/-100 w, the frequency is 50+/-10 KHz, the power is 300w, and the frequency is 50KHz;

(6) The ultrasonic extraction time is 20-40min, preferably 25-35min, more preferably 30min;

(7) The silica gel thin layer plate is a silica gel G thin layer plate;

the ice hockey thin layer identification method meets at least one of the following conditions:

(1) Rf values of the 1, 4-bis [4- (glucosyloxy) benzyl ] -2-isobutyl malate and 2-O-glucosyl bletilla striata spots are respectively 0.55+/-0.02 and 0.42+/-0.02;

(2) The developing agent is a mixed solvent of n-butanol-ethyl acetate-chloroform-methanol-water-glacial acetic acid with the volume ratio of 4:1:2:2:1.5:0.1;

(3) Adding a solvent according to the amount of 50+/-10 mL of solvent added into each gram of the hockey medicinal material;

(4) The volume percentage of the ethanol aqueous solution is 50+/-10%, preferably 50+/-10%, more preferably 50%;

(5) The power of ultrasonic extraction is 300+/-100 w, the frequency is 50+/-10 KHz, the power is 300w, and the frequency is 50KHz;

(6) The ultrasonic extraction time is 20-40min, preferably 25-35min, more preferably 30min;

(7) Dissolving the residue by adding 0.8-1.2mL of methanol per gram of the hockey stick medicinal material, preferably 0.9-1.1mL of methanol, more preferably 1.0mL of methanol;

(8) The silica gel thin layer plate is a silica gel G thin layer plate.

9. Use of the thin-layer identification method of edible tulip according to any one of claims 5-8 for quality control of edible tulip.

10. The use according to claim 9 for distinguishing between edible tulip and cymbidium, round white, edible tulip, shanlan, autumn-flower cymbidium or condyloma-cymbidium;

further, the Mao Cigu thin layer identification method is used for identifying the rhizoma pleionis and the cymbidium sinense, the rhizoma bletillae, the rhizoma pleionis, the rhizoma paridis, the autumn flower unibract, the wart sheath unibract or the hockey;

the thin-layer identification method of the hockey puck is used for identifying the hockey puck and the autumn flower single garlic blue, the wart sheath single garlic blue, the cylindracea, the rhizoma bletillae, the mountain orchid or the Indian iphigenia bulb.