CN113552275A - Identification method of bat grass - Google Patents
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- CN113552275A CN113552275A CN202110707361.XA CN202110707361A CN113552275A CN 113552275 A CN113552275 A CN 113552275A CN 202110707361 A CN202110707361 A CN 202110707361A CN 113552275 A CN113552275 A CN 113552275A
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Abstract
The application provides a bat grass identification method, which is characterized in that a silica gel GF254 thin-layer plate is coated with a mixture of toluene, ethyl formate and methanol in an amount of 5:4:6.5 is developing agent, using 10% sulphuric acid ethanol color developing agent to develop color, dropping sample amount to 7-9 μ l, and performing thin layer identification on the bat grass medicinal material. The method has the advantages of convenient operation, simple equipment, low detection cost, less liquid taking amount, low price of used chemical reagents, no need of special requirements on the temperature and humidity conditions of identification, high identification and analysis speed and capability of being used for daily identification of the bat grass. The identification method is universal for bat grass in different producing areas and different harvesting time, and has the advantages of high separation degree, clear spots and stable Rf value, wherein the Rf value is respectively between 0.4 and 0.45 and between 0.25 and 0.3, and no interference exists; the application lays a theoretical foundation and an experimental basis for distinguishing the bat grass and other plants.
Description
Technical Field
The application relates to the field of medicinal material identification, in particular to a bat grass identification method.
Background
Batwing grass (school name: Christia vespiertioniis (L.f.) Bahn.f.) is a perennial upright herb of the genus Batwing of Leguminosae, which is 60-120 cm high. Also called Rezhou butterfly grass, Feixiang grass, Oenothera flexuosa. Bat Cao is recorded in Chinese plant records, and the whole herb is used for medicine, and is used for treating pulmonary tuberculosis and insect and snake bite; the external application of the leaves is a traumatic fracture setting medicine. Mainly produced in Guangdong, Hainan and Guangxi provinces in China, and mostly grown in grasslands in open fields, shrubbery, roadside and seaside areas.
The root, stem and leaf tissue structure and microscopic characteristics of the bat grass and the paved bat grass are observed by conventional microscopic techniques of Wuxian swallow and the like, and the batwing grass and the paved bat grass contain sheath fiber and calcium oxalate cristobalite and needle crystal and have a conduit with a fringe hole and a threaded conduit. The Liangshuang et al also carried out the ultraviolet-visible spectrum identification research on different medicinal parts of the butterfly grass, and the ultraviolet-visible spectrum curves of different extracting solutions of different medicinal parts of the butterfly grass are considered to have certain difference and obvious characteristics. The Liangshuang et al also researches butterflies in different places produced in Guangxi province by adopting Fourier transform infrared spectroscopy and cluster analysis, and the results show that the butterflies in different places are different and possibly related to external condition factors such as the growth climate conditions, soil conditions, illumination conditions and the like of the butterflies. The two research ideas carried out by Liangshuang et al are to find different parts at the same place, namely to research different parts and different places of the butterfly grass; however, the same idea research is not carried out in different places, namely, the common character of the butterflies in different places is researched, so that the theory and practice which provide the basis for identifying the butterflies and other plants are provided. Wu Xiao Yan et al only studied the commonality of the tissue structure of the bat grass by microscopic technique, and the identification of the whole grass commonality of the bat grass by a thin layer analysis technique is not seen in the prior art.
Disclosure of Invention
Therefore, the present application proposes a bat grass identification method to provide an identification method capable of distinguishing bat grass (whole grass) from other plants.
The technical scheme of the application is realized as follows:
an authentication method of bat grass, the authentication method comprising the steps of:
(1) preparation of reference drug solution: taking a bat grass medicinal material, sequentially carrying out the steps of sieving, adding methanol, ultrasonic treatment and centrifugation, and then taking supernatant as a reference medicinal material solution;
(2) preparation of a test solution: taking a sample to be tested, sequentially carrying out the steps of sieving, adding methanol, ultrasonic treatment and centrifugation, and taking supernate as a test solution;
(3) preparing a blank solvent: the blank solvent is methanol;
(4) respectively dropping the reference medicinal material solution, the test sample solution and the blank solvent with the same dropping amount on the same silica gel GF254 thin-layer plate, developing by a developing agent, and drying the thin-layer plate;
(5) spraying a color developing agent sulfuric acid ethanol on the dried thin layer plate in the step (4), and then heating until spots develop color; and (3) displaying two spots with the same color at corresponding positions of the chromatogram of the test sample and the chromatogram of the reference medicinal material on the thin-layer plate, wherein the Rf values of the two spots are respectively between 0.4-0.45 and 0.25-0.3, so that the sample to be detected is bat grass, otherwise, the sample to be detected is not bat grass.
The further technical scheme is that the sieving in the steps (1) and (2) is a No. 2, No. 3 or No. 4 sieve.
The further technical scheme is that the methanol concentrations in the steps (1), (2) and (3) are the same.
The further technical proposal is that the dosage of the bat grass medicinal material in the step (1) is 0.5-2g, and the dosage of the methanol is 40-60 ml; in the step (2), the dosage of the sample to be detected is 0.5-2g, and the dosage of the methanol is 40-60 ml.
The further technical proposal is that the ultrasonic treatment time is 15-25min, the ultrasonic power is 200-300W, and the ultrasonic frequency is 20-60 kHz.
The further technical proposal is that the rotating speed of the centrifugation is 4000-8000rpm, and the centrifugation time is 2-10 min.
The further technical proposal is that the dropping liquid amount in the step (4) is 7-9 uL.
The further technical scheme is that the volume ratio of the components of the developing solvent in the step (4) is as follows: toluene ethyl formate methanol 5:4: 6.5.
The further technical scheme is that the mass concentration of sulfuric acid in the sulfuric acid ethanol in the step (5) is 8-12%.
The further technical proposal is that the heating temperature in the step (5) is 90-120 ℃.
Compared with the prior art, the beneficial effects of this application are:
(1) the identification method has the advantages of convenient operation, simple equipment, low detection cost, less liquid taking amount, low price of used chemical reagents, no need of making special requirements on the temperature and humidity conditions of identification, high identification and analysis speed and capability of being used for daily identification of the bat grass.
(2) The bat grass identification method determines the thin layer identification condition according to the commonality of the bat grass in different producing areas and different collecting times, so the identification method is universal to the bat grass in different producing areas and different collecting times, and has high separation degree, clear spots and no interference; the invention lays theoretical foundation and experimental basis for distinguishing the bat grass and other plants.
(3) The identification method principle is that under the thin layer identification conditions of the same developing agent, liquid taking amount, color developing agent and the like, the components developed by a sample to be detected and a reference medicinal material are subjected to contrastive analysis, if the position of a thin layer chromatographic spot is the same as the color development, the same medicinal material can be judged, and if not, different medicinal materials are judged.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only preferred embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise.
FIG. 1 is a photograph of a Batwing thin layer chromatography of a developing agent study conducted in example 1 and comparative examples 1-2 of the present application;
FIG. 2 is a slice chromatogram of a bat grass in which the color developing agent of example 1 and comparative example 3 of the present application was studied;
FIG. 3 is a TLC photograph of Batwing grass in which the spot-drip amount of the liquid was investigated in examples 1-2 and comparative examples 4-5 of the present application;
FIG. 4 is a photograph of a Batwing TLC of the present application in which temperature studies were conducted in comparative examples 6-7;
FIG. 5 is a TLC photograph of Batwing grass in comparative examples 8-10 of the present application;
FIG. 6 is a slice chromatogram of bat grass in different production areas and different harvesting times in the experimental example of the application.
Wherein:
1 is a blank solvent, 2 is a reference medicinal material solution, and 3-12 is a test sample solution; wherein 3 and 4 in FIGS. 1-2 and 4-5 are repeated tests of the test solution in the same example or the same comparative example; in FIG. 3, 3 is a sample amount of 3. mu.l of the test solution (comparative example 4), 4 is a sample amount of 5. mu.l of the test solution (comparative example 5), 5 is a sample amount of 7. mu.l of the test solution (example 1), and 6 is a sample amount of 9. mu.l of the test solution (example 2); in fig. 6, 3-12 are the bat grass thin layer chromatograms at different producing areas and different harvesting times.
Detailed Description
For a better understanding of the present technology, specific examples are provided below, which are further described in conjunction with the accompanying drawings.
The No. 2, 3 or 4 sieve mentioned in the application is in R40/3 series according to the standard drug sieve specification of Chinese pharmacopoeia in 2015, the inner diameter of the sieve pore of the No. 2 sieve is 850 mu m +/-29 mu m, and the sieve pore is 25 meshes; the inner diameter of the mesh of the No. 3 sieve is 355 mu m +/-13 mu m, and the mesh number is 50 meshes; the screen hole inner diameter of the No. 4 screen is 250 mu m +/-9.9 mu m, and the mesh number is 65 meshes.
Example 1
An authentication method of bat grass, the authentication method comprising the steps of:
(1) preparation of reference drug solution
Precisely weighing 1g of Hepialus Hainanensis reference medicinal material powder, sieving with No. 2 sieve, placing into 250ml triangular flask with plug, adding 50ml methanol solvent, ultrasonic processing (power 250W, frequency 40kHz) for 20min, centrifuging at 6000rpm for 5min, and collecting supernatant as reference medicinal material solution.
(2) Preparation of test solution
Pulverizing a sample to be tested into powder 1g, sieving with No. 2 sieve, placing in 250ml triangular flask with plug, adding methanol 50ml, performing ultrasonic treatment (power 250W, frequency 40kHz) for 20min, centrifuging at 6000rpm for 5min, and collecting supernatant and filtrate as sample solution.
(3) Development and color development of thin layer chromatography
Sucking the spot liquid amount of 7uL of test solution, reference medicinal material solution and blank solvent, respectively spotting on the same silica gel GF254 thin layer plate, and adding toluene: ethyl formate: developing with methanol (5:4:6.5) as developing agent, taking out, and oven drying. Spraying 10% sulfuric acid ethanol, and heating at 105 deg.C until the spots are clearly developed. In the chromatogram of the test solution, two spots of the same color were observed at the positions corresponding to those of the chromatogram of the control, and the Rf values of the two spots of the test solution 3 were 0.43 and 0.28, and the Rf values of the two spots of the test solution 4 were 0.4 and 0.25 (see FIG. 1). The sample to be tested is proved to be bat grass.
Example 2
An authentication method of bat grass, the authentication method comprising the steps of:
(1) preparation of reference drug solution
Precisely weighing 0.5g of Hepialus Hainanensis reference medicinal material powder, sieving with No. 3 sieve, placing in 250ml triangular flask with plug, adding 40ml of methanol solvent, performing ultrasonic treatment (power 200W, frequency 20kHz) for 15min, centrifuging at 4000rpm for 2min, and collecting supernatant as reference medicinal material solution.
(2) Preparation of test solution
Pulverizing a sample to be tested into 0.5g of powder, sieving with a No. 3 sieve, placing in a 250ml triangular flask with a plug, adding 40ml of methanol, performing ultrasonic treatment (power 200W and frequency 20kHz) for 15min, centrifuging at 4000rpm for 2min, and taking supernatant filtrate as a test solution.
(3) Development and color development of thin layer chromatography
Sucking a sample solution, a reference medicinal material solution and a blank solvent with the dropping liquid amount of 9uL, respectively dropping the sample solution, the reference medicinal material solution and the blank solvent on the same silica gel GF254 thin-layer plate, and adding toluene: ethyl formate: developing with methanol (5:4:6.5) as developing agent, taking out, and oven drying. Spraying 8% sulfuric acid ethanol, and heating at 90 deg.C until the spots are clearly developed. In the chromatogram of the test solution, two spots of the same color appear at the positions corresponding to those of the chromatogram of the control solution, and the Rf values of the two spots of the test solution 6 are 0.45 and 0.3 (see FIG. 3). The sample to be tested is proved to be bat grass.
Example 3
An authentication method of bat grass, the authentication method comprising the steps of:
(1) preparation of reference drug solution
Precisely weighing 2g of Hepialus Hainanensis reference medicinal material powder, sieving with No. 4 sieve, placing in 250ml triangular flask with plug, adding 60ml methanol solvent, ultrasonic processing (power 300W, frequency 60kHz) for 25min, centrifuging at 8000rpm for 10min, and collecting supernatant as reference medicinal material solution.
(2) Preparation of test solution
Pulverizing sample to be tested into powder 2g, sieving with No. 4 sieve, placing in 250ml triangular flask with plug, adding methanol 60ml, ultrasonic processing (power 300W, frequency 60kHz) for 25min, centrifuging at 8000rpm for 10min, and collecting supernatant and filtrate as sample solution.
(3) Development and color development of thin layer chromatography
Sucking the sample solution, the reference medicinal material solution and the blank solvent with the dropping liquid amount of 8uL, respectively dropping the sample solution, the reference medicinal material solution and the blank solvent on the same silica gel GF254 thin-layer plate, and adding toluene: ethyl formate: developing with methanol (5:4:6.5) as developing agent, taking out, and oven drying. Spraying 12% sulfuric acid ethanol, and heating at 120 deg.C until the spots are clearly developed. In the chromatogram of the test solution, two spots of the same color appear at the positions corresponding to those of the chromatogram of the control solution, and the Rf values of the two spots of the test solution are 0.44 and 0.27. The sample to be tested is proved to be bat grass.
Comparative example 1
The developing solvent was ethyl acetate, methanol, glacial acetic acid, and water 15:1:1:2, and the other steps were the same as in example 1.
Comparative example 2
The developing solvent is dichloromethane-methane 20:1, and other steps are the same as example 1.
The results of the developer study conducted by example 1 and comparative examples 1-2 show (see fig. 1): developing agent in the examples: the separation effect of the toluene, ethyl formate and methanol (5:4:6.5) on the bat herb materials is good, the separation degree is high, and spots are clear, so that a system of the toluene, ethyl formate and methanol (5:4:6.5) is adopted as a developing agent for identifying the bat herb materials by thin-layer chromatography.
Comparative example 3
The developer is 10% aluminum trichloride ethanol, and other steps are the same as example 1. The results show (see FIG. 2) that the 10% ethanol sulfate developed clear spots.
Comparative example 4
The liquid dropping amount of the sample solution, the reference medicinal material solution and the blank solvent is 3uL, and the sample solution, the reference medicinal material solution and the blank solvent are respectively dropped on the same silica gel GF254 thin-layer plate. The other steps are the same as in example 1.
Comparative example 5
The liquid dropping amount of the sample solution, the reference medicinal material solution and the blank solvent is 5uL, and the sample solution, the reference medicinal material solution and the blank solvent are respectively dropped on the same silica gel GF254 thin-layer plate. The other steps are the same as in example 1.
The dot blot amount study for identification of bat grass was conducted by examples 1-2 and comparative examples 4-5 (see fig. 3), and the results show that: spot size of 7-9. mu.l, spot clarity was better.
Comparative example 6
A method for identifying bat grass is carried out at 4 ℃, and other steps are the same as example 1.
Comparative example 7
A method for identifying bat grass is carried out at 25 ℃, and other steps are the same as example 1.
A temperature study of batwing identification was conducted by comparative examples 6-7 (see fig. 4), and the results showed: the temperature condition has little influence on the thin layer spreading effect, so the temperature for identifying the bat grass thin layer chromatography is not required.
Comparative example 8
A method for identifying bat grass is carried out under the condition of 32% of humidity, and other steps are the same as those of the example 1.
Comparative example 9
A method for identifying bat grass is carried out under the condition of 68% of humidity, and other steps are the same as those of the example 1.
Comparative example 10
A method for identifying bat grass is carried out under the condition of humidity of 80%, and other steps are the same as those of the example 1.
Humidity studies of batwing identification were conducted by comparative examples 8-10 (see fig. 5), and the results showed: the humidity condition has little influence on the thin layer spreading effect, so the humidity identified by the bat grass thin layer chromatography is not required.
Examples of the experiments
By adopting the bat grass identification method in the embodiment 1 of the application, the bat grass medicinal materials in different batches are identified, and the result shows that (see fig. 6): two spots with the same color are displayed at the corresponding positions of the chromatogram of the reference medicinal material, and the Rf values of the spots are respectively between 0.4 and 0.45 and 0.25 to 0.3; the identification method of the bat grass can be used for identifying the bat grass thin layer in different producing areas or different harvesting times, and has the advantages of high separation degree, clear spots and no interference.
TABLE 1 Batwing herb samples of different batches
| Numbering | Producing area | Time of harvest | |
| 1 | Blank solvent | —— | —— |
| 2 | Oriental (contrast medicinal material) | 2020.05 | 0.43,0.27 |
| 3 | Oriental | 2020.08 | 0.45,0.3 |
| 4 | Oriental | 2020.08 | 0.44,0.29 |
| 5 | Oriental | 2020.08 | 0.43,0.28 |
| 6 | Le Dong | 2020.09 | 0.42,0.28 |
| 7 | Le Dong | 2020.09 | 0.42,0.27 |
| 8 | Changjiang river | 2020.09 | 0.41,0.27 |
| 9 | Changjiang river | 2020.09 | 0.41,0.26 |
| 10 | Changjiang river | 2020.08 | 0.41,0.25 |
| 11 | Changjiang river | 2020.08 | 0.4,0.25 |
| 12 | Oriental | 2020.08 | 0.4,0.25 |
The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (10)
1. A bat grass identification method is characterized in that: the authentication method comprises the following steps:
(1) preparation of reference drug solution: taking a bat grass medicinal material, sequentially carrying out the steps of sieving, adding methanol, ultrasonic treatment and centrifugation, and then taking supernatant as a reference medicinal material solution;
(2) preparation of a test solution: taking a sample to be tested, sequentially carrying out the steps of sieving, adding methanol, ultrasonic treatment and centrifugation, and taking supernate as a test solution;
(3) preparing a blank solvent: the blank solvent is methanol;
(4) respectively dropping the reference medicinal material solution, the test sample solution and the blank solvent with the same dropping amount on the same silica gel GF254 thin-layer plate, developing by a developing agent, and drying the thin-layer plate;
(5) spraying a color developing agent sulfuric acid ethanol on the dried thin layer plate in the step (4), and then heating until spots develop color; and (3) displaying two spots with the same color at corresponding positions of the chromatogram of the test sample and the chromatogram of the reference medicinal material on the thin-layer plate, wherein the Rf values of the two spots are respectively between 0.4-0.45 and 0.25-0.3, so that the sample to be detected is bat grass, otherwise, the sample to be detected is not bat grass.
2. The method of authenticating batcao as claimed in claim 1, wherein: the sieving in the steps (1) and (2) is a No. 2, No. 3 or No. 4 sieve.
3. The method of authenticating batcao as claimed in claim 1, wherein: the methanol concentrations in steps (1), (2) and (3) are the same.
4. The method of authenticating batcao as claimed in claim 1, wherein: the using amount of the bat grass medicinal material in the step (1) is 0.5-2g, and the using amount of the methanol is 40-60 ml; in the step (2), the dosage of the sample to be detected is 0.5-2g, and the dosage of the methanol is 40-60 ml.
5. The method of authenticating batcao as claimed in claim 1, wherein: the ultrasonic treatment time is 15-25min, the ultrasonic power is 200-300W, and the ultrasonic frequency is 20-60 kHz.
6. The method of authenticating batcao as claimed in claim 1, wherein: the rotating speed of the centrifugation is 4000-.
7. The method of authenticating batcao as claimed in claim 1, wherein: the amount of the liquid dropping in the step (4) is 7-9 uL.
8. The method of authenticating batcao as claimed in claim 1, wherein: the volume ratio of the components of the developing agent in the step (4) is as follows: toluene ethyl formate methanol 5:4: 6.5.
9. The method of authenticating batcao as claimed in claim 1, wherein: the mass concentration of sulfuric acid in the sulfuric acid ethanol in the step (5) is 8-12%.
10. The method of authenticating batcao as claimed in claim 1, wherein: the heating temperature in the step (5) is 90-120 ℃.
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