CN115684455A - Thin-layer chromatography identification method for loofah sponge and processed product thereof - Google Patents
Thin-layer chromatography identification method for loofah sponge and processed product thereof Download PDFInfo
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Abstract
The invention provides a thin-layer chromatography identification method of loofah sponge and a processed product thereof. The thin-layer chromatography identification method of the loofah sponge and the processed products thereof provided by the invention has the characteristics of high accuracy, good separation effect and clear color spots, can identify different processed products of loofah sponge blocks and loofah sponge charcoal, can provide a research basis for the change of chemical components in the processing process from the loofah sponge blocks to the loofah sponge charcoal, can also qualitatively identify the loofah sponge charcoal medicinal materials, decoction pieces, intermediates and formula granules, can control the quality of the whole process, and can ensure the uniformity, stability and reliability of the quality.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a thin-layer chromatography identification method for loofah sponge and a processed product thereof.
Background
The retinervus Luffae fructus is the vascular bundle of dried mature fruit of Luffa cylindrica (L.) Roem. Has effects in dredging meridian passage, clearing away heat, eliminating phlegm, removing toxic materials, relieving swelling, tonifying heart, and promoting urination, and can be used for treating chest and hypochondrium pain, lung heat, expectoration, amenorrhea, and galactostasis. The loofah sponge contains a plurality of loofah saponins, flavones, phenols, proteins, amino acids, grease, inorganic elements, organic acids and the like, and pharmacological tests prove that the loofah sponge has the effects of reducing blood fat, easing pain, calming and the like and has obvious clinical effect.
The main processed products of the loofah sponge comprise three types of loofah sponge (blocks), fried loofah sponge and loofah sponge charcoal, wherein the loofah sponge (blocks) and the loofah sponge charcoal are used in clinic, but at present, the loofah sponge (blocks) and the loofah sponge charcoal have no quality standard, and the difference of the two processed products of the loofah sponge (blocks) and the loofah sponge charcoal is reported less. Because the samples of different formulations of the loofah sponge carbon are prepared from loofah sponge carbon medicinal materials through a series of processes, and some processes can damage the character characteristics of the raw medicinal materials, an identification method which has good distinguishing and identifying capabilities and is simpler and faster to operate in the prior art is needed so as to better control the quality.
KANGALONG, decoction refreshing, grand sung and ZUZHONG; researching the quality standard of three different processed product decoction pieces of loofah sponge; the Shanxi college of traditional Chinese medicine, 2011,7 and 34 (4) disclose quality control methods of three different prepared product decoction pieces of loofah sponge, which comprise the following steps of preparing a test sample solution: taking 2g of Luffa cylindrica Linn charcoal, cutting into small pieces (length is not more than 0.5 cm), adding 50mL of methanol, heating and refluxing for 1h, cooling, filtering, evaporating filtrate to dryness, adding 10mL of water to residue for dissolving, extracting with water saturated n-butanol twice, 20mL each time, combining n-butanol solutions, evaporating to dryness, and adding 1mL of methanol to residue for dissolving to obtain a sample solution. Developing agent: chloroform-methanol-water (28. Performing thin layer chromatography (appendix VI B of the first edition of Chinese pharmacopoeia 2005), sucking 10uL of each of the test solution and the control solution, spreading on a silica gel G thin layer plate with 0.5% sodium carboxymethylcellulose solution as adhesive, taking out, air drying, spraying 10% ethanol sulfate solution, and heating at 105 deg.C until the spots are clearly developed. Inspecting under ultraviolet lamp (365 nm), wherein spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution. The test result is shown in FIG. 1, wherein 1 is retinervus Luffae fructus charcoal decoction pieces (YP 202105-21-10) 3uL;2, 5uL of loofah sponge charcoal decoction pieces (YP 202105-21-10). The thin-layer chromatography result has poor resolution, unclear spots and little chromatographic information.
Therefore, how to provide a thin-layer chromatography identification method for loofah sponge and its processed products becomes a problem to be solved urgently at present.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a thin-layer chromatography identification method for loofah sponge and a processed product thereof. The invention establishes a thin-layer identification method with strong specificity aiming at the loofah sponge and the processed products thereof, and provides experimental basis for the establishment of quality standard and subsequent research. The chromatographic result provided by the invention has moderate separation degree, and the spot clear chromatographic information is rich, stable and reliable.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a thin-layer chromatography identification method for loofah sponge and processed products thereof, wherein developing agents adopted in the thin-layer chromatography identification method comprise petroleum ether, dichloromethane, ethyl acetate and formic acid.
In the invention, the loofah sponge processed product comprises any one of or the combination of at least two of loofah sponge blocks, fried loofah sponge and loofah sponge charcoal.
In the invention, the volume ratio of the petroleum ether, the dichloromethane, the ethyl acetate and the formic acid is (1.3-1.7): (0.8-1.2): (1.8-2.2): (0.08-0.12);
wherein "1.3-1.7" can be 1.3, 1.4, 1.5, 1.6, 1.7, etc.;
"0.8-1.2" can be 0.8, 0.9, 1, 1.1, 1.2, etc.;
"1.8-2.2" can be 1.8, 1.9, 2, 2.1, 2.2, etc.;
"0.08-0.12" may be 0.08, 0.09, 0.1, 0.11, 0.12, etc.
In the present invention, the petroleum ether has a boiling point of 60 to 90 ℃ (for example, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, 85 ℃, 90 ℃ and the like are possible).
In the invention, the thin layer chromatography identification method specifically comprises the following steps:
and (3) spotting the test sample solution on a silica gel thin-layer plate, developing by adopting a developing agent, spraying a sulfuric acid ethanol solution after the silica gel thin-layer plate is dried, drying, then spraying a vanillin solution, and heating to obtain the positions of all components in the test sample in the thin-layer plate.
In the present invention, the sulfuric acid ethanol solution contains 8 to 12% by mass of sulfuric acid (for example, 8%, 9%, 10%, 11%, 12%, etc.);
preferably, the mass content of vanillin in the vanillin-sulfuric acid solution is 3-7% (e.g. can be 3%, 4%, 5%, 6%, 7%, etc.).
Preferably, the heating temperature is 100-110 ℃ (for example, 100 ℃, 101 ℃, 102 ℃, 103 ℃, 104 ℃,105 ℃, 106 ℃, 107 ℃, 108 ℃, 109 ℃, 110 ℃, etc.).
In the invention, the preparation of the test solution in the thin-layer chromatography identification method comprises the following steps:
(1) Grinding the baked towel gourd and/or the baked towel gourd product, adding water, performing ultrasonic treatment, filtering, and collecting solid;
(2) And (2) extracting the solid obtained in the step (1) by using ethyl acetate, desolventizing the obtained extract, and then performing constant volume by using methanol to obtain a test sample solution.
In the invention, in the step (1), the solid-to-liquid ratio of the towel gourd cauterized and/or towel gourd cauterized processed product to water is 0.5 (15-25) g/mL;
wherein "15-25" can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, etc.
Preferably, in step (1), the power of the ultrasound is 100-500W (for example, 100W, 150W, 200W, 250W, 300W, 350W, 400W, 450W, 500W, etc.), and the time of the ultrasound is 20-40min (for example, 20min, 22min, 24min, 26min, 28min, 30min, 32min, 34min, 36min, 38min, 40min, etc.).
In the invention, the solid-to-liquid ratio of the towel gourd baked and/or towel gourd baked products to ethyl acetate is 0.5 (15-25) g/mL;
wherein "15-25" may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, etc.
Preferably, in step (2), the number of times of extraction is 1 to 3 (e.g., may be 1, 2, 3, etc.).
In the present invention, the amount of spot sample in the thin layer chromatography identification method is 5 to 8. Mu.L (for example, 5. Mu.L, 6. Mu.L, 7. Mu.L, 8. Mu.L, etc.).
Compared with the prior art, the invention has the following beneficial effects:
the thin-layer chromatography identification method of the loofah sponge and the processed products thereof provided by the invention has the characteristics of high accuracy, good separation effect and clear color spots, can identify different processed products of loofah sponge blocks and loofah sponge charcoal, can provide a research basis for the change of chemical components in the processing process from the loofah sponge blocks to the loofah sponge charcoal, can also qualitatively identify the loofah sponge charcoal medicinal materials, decoction pieces, intermediates and formula granules, can control the quality of the whole process, and can ensure the uniformity, stability and reliability of the quality.
Drawings
FIG. 1 is a thin layer chromatogram of a luffa seed charcoal decoction piece in the prior art; wherein 1 is retinervus Luffae fructus charcoal decoction pieces (YP 202105-21-10) 3uL;2, 5uL of loofah sponge charcoal decoction pieces (YP 202105-21-10).
Fig. 2 is a loofah sponge carbon thin-layer chromatogram, wherein 1 is a negative control, 2 is loofah sponge carbon particles, 3 is a loofah sponge carbon intermediate, 4 is loofah sponge carbon decoction pieces, and 5 is a loofah sponge carbon control medicinal material.
FIG. 3 is a thin layer chromatogram of 3 batches of loofah sponge charcoal decoction pieces and 3 batches of loofah sponge blocks, wherein 1, 2 and 3 are 3 batches of loofah sponge charcoal decoction pieces; 4. 5 and 6 are 3 batches of loofah sponge blocks.
FIG. 4 is a thin-layer chromatogram of lyophilized powder of retinervus Luffae fructus charcoal obtained by different extraction methods, wherein 1, 2, 3, 4, 5, and 6 correspond to test solutions a, b, c, d, e, and f, respectively.
Fig. 5 is a thin-layer chromatogram of the loofah sponge charcoal lyophilized powder obtained by using developing solvent 1, wherein 1, 2, 3 and 4 correspond to test solution a, d, e and f, respectively.
FIG. 6 is a thin-layer chromatogram of retinervus Luffae fructus charcoal lyophilized powder obtained by using developing agent 2, wherein 1 and 2 correspond to test solution d and e, respectively.
Fig. 7 is a thin-layer chromatogram of the loofah sponge charcoal lyophilized powder obtained by using developing agent 3, wherein 1, 2 and 3 correspond to test solution d, e and f, respectively.
FIG. 8 is a thin-layer chromatogram of the decocted lyophilized powder of retinervus Luffae fructus charcoal with different sample amounts, wherein 1 is blank control, and 2, 3, and 4 are diluted lyophilized powders 202105-21-03 with sample amounts of 5 μ L, 7 μ L, and 10 μ L, respectively; 5. 6 and 7 are standard fried freeze-dried powder 202105-21-09 with sample application amount of 5 μ L, 7 μ L and 10 μ L respectively; 8. 9 and 10 are standard fried freeze-dried powder 202105-21-12 with sample size of 5 μ L, 7 μ L and 10 μ L respectively; 11. 12 and 13 are the sample application amounts of the loofah sponge charcoal control decoction pieces of 1 muL, 3 muL and 5 muL respectively.
FIG. 9 is a thin layer chromatogram of a standard decocted lyophilized powder of retinervus Luffae fructus charcoal obtained by using silica gel thin layer plate provided by Yinlong; wherein, the points are from left to right: stigmasterol, loofah sponge carbon freeze-dried powder 03 batches, loofah sponge carbon freeze-dried powder 09 batches, loofah sponge carbon freeze-dried powder 11 batches and loofah sponge control decoction pieces.
FIG. 10 is a thin layer chromatogram of retinervus Luffae fructus charcoal labeled decoction lyophilized powder obtained by using silica gel thin layer plate provided with sesame 32600; wherein, the points are from left to right: stigmasterol, loofah sponge carbon freeze-dried powder 03 batches, loofah sponge carbon freeze-dried powder 09 batches, loofah sponge carbon freeze-dried powder 11 batches and loofah sponge control decoction pieces.
FIG. 11 is a chromatogram of a lyophilized powder prepared from retinervus Luffae fructus charcoal by silica gel thin layer plate provided by merck; wherein, the points are from left to right: stigmasterol, loofah sponge carbon freeze-dried powder 03 batches, loofah sponge carbon freeze-dried powder 09 batches, loofah sponge carbon freeze-dried powder 11 batches and loofah sponge control decoction pieces.
FIG. 12 is a thin layer chromatogram of a standard-decocted lyophilized powder of retinervus Luffae fructus charcoal obtained at normal temperature and humidity (23 deg.C, 46%); wherein, the points are from left to right: stigmasterol, loofah sponge carbon freeze-dried powder 03 batches, loofah sponge carbon freeze-dried powder 09 batches, loofah sponge carbon freeze-dried powder 11 batches and loofah sponge control decoction pieces.
FIG. 13 is a thin-layer chromatogram of a standard-decocted lyophilized powder of retinervus Luffae fructus charcoal obtained at normal temperature and high humidity (23 deg.C, 82%); wherein, the points are from left to right: stigmasterol, loofah sponge charcoal freeze-dried powder 03 batches, loofah sponge charcoal freeze-dried powder 09 batches, loofah sponge charcoal freeze-dried powder 11 batches and loofah sponge contrast decoction pieces.
FIG. 14 is a thin-layer chromatogram of a lyophilized powder of retinervus Luffae fructus charcoal decocted at normal temperature and low humidity (23 deg.C, 13%); wherein, the points are from left to right: stigmasterol, loofah sponge carbon freeze-dried powder 03 batches, loofah sponge carbon freeze-dried powder 09 batches, loofah sponge carbon freeze-dried powder 11 batches and loofah sponge control decoction pieces.
FIG. 15 is a thin-layer chromatogram of a lyophilized powder of retinervus Luffae fructus charcoal decocted at low temperature and normal humidity (4 deg.C, 46%); wherein, the points are from left to right: stigmasterol, loofah sponge carbon freeze-dried powder 03 batches, loofah sponge carbon freeze-dried powder 09 batches, loofah sponge carbon freeze-dried powder 11 batches and loofah sponge control decoction pieces.
FIG. 16 is a chromatogram for the methodological verification of a loofah sponge charcoal labeled decocted lyophilized powder, wherein 1 is blank; 2-16 are 15 batches of standard fried freeze-dried powder batch numbers: 202105-21-01 to 202105-21-15;17 is retinervus Luffae fructus charcoal reference decoction pieces.
FIG. 17 is a thin layer chromatogram of loofah sponge carbon particles with different sample amounts, wherein 1 is a blank control; 2. 3 and 4, the sample volumes of KL19022051 of the loofah sponge carbon particles are respectively 5 muL, 7 muL and 10 muL; 5. 6 and 7 are sample sizes of 5 muL, 7 muL and 10 muL of loofah sponge carbon particles KL19003391 respectively; 8. 9 and 10 are the sample amounts of KL19022051 of loofah sponge carbon particles which are respectively 5 mu L, 7 mu L and 10 mu L; 11. 12 and 13 are the sample application amounts of the loofah sponge charcoal control decoction pieces of 1 mul, 3 mul and 5 mul respectively; the optimal sample amount is determined to be 7 microliters of the sample, and 5 microliters of the reference decoction pieces.
FIG. 18 is a chromatogram of a methodological validation of loofah sponge carbon particles; wherein, 1 is blank control; 2 is luffa vegetable sponge carbon particles KL19022051;3 is loofah sponge carbon particles KL19003391;4 is loofah sponge carbon particles KL19022051;5 is retinervus Luffae fructus charcoal reference decoction pieces.
Detailed Description
The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The sources of the components in the following examples are as follows:
example 1
The embodiment provides a thin-layer chromatography identification method of loofah sponge carbon, which comprises the following steps:
taking 0.5g of loofah sponge charcoal particles, loofah sponge charcoal intermediates and loofah sponge charcoal decoction pieces, grinding, adding 20mL of water, treating for 30min by ultrasound (200W), cooling, shaking and extracting for 2 times by using ethyl acetate, wherein 20mL of ethyl acetate is used each time, combining ethyl acetate solutions, evaporating the ethyl acetate to dryness, and then using methanol to fix the volume to 1mL to obtain a sample solution. Loofah sponge charcoal reference medicinal materials: adding water 100mL into 5g of Siberian snakegourd collaterals charcoal medicinal material, boiling for 30min, spin drying, and making into control medicinal solution by the same method. (note: the loofah sponge charcoal reference medicinal material is not available in the middle school and the market, so the loofah sponge charcoal medicinal material is firstly used as the reference medicinal material). According to a thin-layer chromatography (2015 edition of Chinese pharmacopoeia, general rules of the four parts 0502), absorbing 7 mu L of a test solution (loofah sponge carbon particles, loofah sponge carbon intermediates and loofah sponge carbon reference medicinal materials) and 5 mu L of a loofah sponge carbon decoction piece test solution, respectively dropping the test solution and the test solution on the same silica gel G thin-layer plate, developing the test solution by using petroleum ether (60-90): dichloromethane, ethyl acetate, formic acid (1.5. The thin-layer chromatogram of retinervus Luffae fructus charcoal is shown in 2, wherein 1 is negative control, 2 is retinervus Luffae fructus charcoal granule, 3 is retinervus Luffae fructus charcoal intermediate, 4 is retinervus Luffae fructus charcoal decoction pieces, and 5 is retinervus Luffae fructus charcoal control medicinal material. Fluorescent spots of the same color appear at corresponding positions in the chromatogram of the test sample. As can be seen from fig. 2: the thin-layer chromatography result has moderate separation degree, clear spots and rich chromatographic information, and fluorescent spots with the same color are displayed at corresponding positions in the chromatogram of the test sample.
Example 2
The embodiment provides a thin-layer chromatography identification method of a loofah sponge block, which comprises the following steps:
respectively taking 0.5g of loofah sponge blocks and loofah sponge charcoal decoction pieces, grinding, adding 20mL of water, treating for 30min by ultrasound (400W), cooling, extracting for 2 times by shaking with ethyl acetate, each time with 20mL, combining ethyl acetate solutions, evaporating ethyl acetate to dryness, and then fixing the volume to 1mL by using methanol to obtain a sample solution. Performing a thin-layer chromatography (2015 year edition, general rules of the four parts 0502) test, sucking 7 mu L of loofah sponge block sample solution and 5 mu L of loofah sponge charcoal decoction piece sample solution, respectively spotting on the same silica gel G thin-layer plate, developing by using petroleum ether (60-90): dichloromethane: ethyl acetate: formic acid (1.5: 1: 0.1) as a developing agent, taking out, airing, spraying 10% ethanol sulfate solution, airing, then spraying 5% vanillin sulfate solution, heating at 105 ℃ until spots are clear, and inspecting under sunlight. Fluorescent spots of the same color appear at corresponding positions in the chromatogram of the test sample. As shown in fig. 3, 1, 2 and 3 are 3 batches of loofah sponge charcoal decoction pieces; 4. 5 and 6 are 3 batches of loofah sponge blocks. As can be seen from fig. 3, the thin-layer chromatography result shows that the chromatographic information of the loofah sponge charcoal decoction pieces is different from that of the loofah sponge pieces, and the chromatographic information of the loofah sponge charcoal decoction pieces is rich, and the chemical components of the loofah sponge charcoal decoction pieces and the loofah sponge pieces may be changed in the processing process.
Because the traditional Chinese medicinal materials are prepared into other dosage forms through a series of processes, the original characters of the traditional Chinese medicinal materials are changed, the identification method provided by the invention can identify different processed products of the loofah sponge block and the loofah sponge charcoal, and can provide a research basis for the change of chemical components in the processing process from the loofah sponge block to the loofah sponge charcoal. The chromatographic result provided by the invention has moderate separation degree, and the spot clear chromatographic information is rich, stable and reliable.
Example 3
Investigation of preparation of test solution
The embodiment provides a thin-layer chromatography identification method of loofah sponge charcoal, which comprises the following steps:
preparation of a test solution:
(a) Taking 0.25g of the luffa pith carbon freeze-dried powder, adding 10mL of methanol, performing ultrasonic treatment for 30min, filtering, and concentrating to 1 mL.
(b) Taking 0.25g of luffa carbon freeze-dried powder, adding 10mL of petroleum ether, performing ultrasonic treatment for 30min, filtering, evaporating to dryness, and fixing the volume to 1mL by using methanol to obtain the luffa carbon freeze-dried powder.
(c) Taking 0.25g of luffa carbon freeze-dried powder, adding 10mL of ethyl acetate, performing ultrasonic treatment for 30min, filtering, and concentrating to 1 mL.
(d) Taking 0.5g of luffa carbon freeze-dried powder, adding 20mL of water, performing ultrasonic treatment for 30min, filtering, extracting with n-butanol for 2 times, combining 20mL of n-butanol layer each time, evaporating to dryness, and diluting with methanol to a constant volume of 1mL to obtain the luffa carbon freeze-dried powder.
(e) Taking 0.5g of the luffa carbon freeze-dried powder, adding 20mL of water, performing ultrasonic treatment for 30min, filtering, extracting with ethyl acetate for 2 times, each time 20mL, combining ethyl acetate layers, evaporating to dryness, and diluting to 1mL with methanol to obtain the luffa carbon freeze-dried powder.
(f) Taking 0.5g of the luffa carbon freeze-dried powder, adding 20mL of methanol, performing ultrasonic treatment for 30min, filtering, evaporating to dryness, adding 20mL of water for dissolving, extracting with ethyl acetate for 2 times, combining 20mL of ethyl acetate layers each time, evaporating to dryness, and diluting to 1mL with methanol to obtain the luffa carbon freeze-dried powder.
Sucking the sample solution mu L, respectively dropping on the same silica gel G thin layer plate, developing with cyclohexane: ethyl acetate (1).
Thin layer chromatogram of lyophilized retinervus Luffae fructus charcoal powder obtained by different extraction methods is shown in FIG. 4, wherein 1, 2, 3, 4, 5, and 6 correspond to test sample solutions a, b, c, d, e, and f, respectively. According to the thin-layer identification result, the thin-layer chromatogram effect of the test solution prepared by b and c is poor.
Example 4
Developer investigation
The embodiment provides a thin-layer chromatography identification method of loofah sponge carbon, which comprises the following steps:
the test solutions were prepared as in example 3.
Developing agent 1: n-hexane, dichloromethane, ethyl acetate (1;
developing agent 2: n-hexane, dichloromethane, ethyl acetate (1;
developing agent 3: petroleum ether 60 to 90: dichloromethane: ethyl acetate: formic acid (1.5.
Sucking 7 μ L of test solution, respectively dropping on the same silica gel G thin layer plate, respectively developing with developing agent 1, 2, and 3, taking out, air drying, spraying 10% sulphuric acid ethanol solution, air drying, spraying 5% vanillin sulphuric acid solution, heating at 105 deg.C to make spots clear, and inspecting in sunlight.
A thin-layer chromatogram of the retinervus Luffae fructus charcoal lyophilized powder obtained by using developer 1 is shown in FIG. 5, wherein 1, 2, 3, and 4 correspond to test solution a, d, e, and f, respectively.
A thin layer chromatogram of the retinervus Luffae fructus charcoal lyophilized powder obtained by using developer 2 is shown in FIG. 6, wherein 1 and 2 correspond to test solution d and e, respectively.
A thin layer chromatogram of the lyophilized powder of retinervus Luffae fructus charcoal obtained by using developer 3 is shown in FIG. 7, wherein 1, 2, and 3 correspond to test solution d, e, and f, respectively.
As can be seen from comparison of fig. 4 to 7, the developing agent 3 chromatogram results were the best, and therefore, the developing agent 3 (petroleum ether 60 to 90: dichloromethane: ethyl acetate: formic acid (1.5: 1: 0.1)) was used as the developing agent for the loofah sponge charcoal. The detection result is that: spraying 10% ethanol sulfate solution, air drying, spraying 5% vanillin sulfuric acid solution, heating until spots are clear, and inspecting in sunlight.
Example 5
Investigation of different point samples
The embodiment provides a thin-layer chromatography identification method of loofah sponge carbon, which comprises the following steps:
taking 0.5g of the luffa vegetable sponge charcoal target-decocted freeze-dried powder or luffa vegetable sponge charcoal reference decoction piece, grinding, adding 20mL of water, performing ultrasonic treatment for 30min, cooling, extracting with ethyl acetate by shaking for 2 times, each time 20mL, combining ethyl acetate solutions, evaporating ethyl acetate to dryness, and then fixing the volume to 1mL with methanol to obtain a sample solution.
Absorbing 5 muL, 7 muL and 10 muL of the loofah sponge charcoal standard decoction freeze-dried powder sample solution, 1 muL, 3 muL and 5 muL of the loofah sponge charcoal reference decoction piece sample solution, respectively dropping the loofah sponge charcoal reference decoction piece sample solution on the same silica gel G thin layer plate, taking out and drying the loofah sponge charcoal reference decoction piece sample solution by taking petroleum ether (60-90), ethyl acetate, formic acid (1.5.
The thin layer chromatography of the standard-decocted lyophilized powder of retinervus Luffae fructus charcoal with different sample amounts is shown in FIG. 8, wherein 1 is blank control, and 2, 3, and 4 are standard-decocted lyophilized powders 202105-21-03 with sample amounts of 5 μ L, 7 μ L, and 10 μ L respectively; 5. 6 and 7 are standard decoction freeze-dried powder 202105-21-09 with sample application amount of 5 μ L, 7 μ L and 10 μ L respectively; 8. 9 and 10 are standard decoction freeze-dried powder 202105-21-12 with sample application amount of 5 μ L, 7 μ L and 10 μ L respectively; 11. 12 and 13 are the sample application amounts of loofah sponge carbon control decoction pieces of 1 mul, 3 mul and 5 mul respectively; the optimal spot size is determined to be 7 microliters of the test sample and 5 microliters of the control decoction pieces.
Example 6
Investigation of silica gel G thin layer plate by different manufacturers
The embodiment provides a thin-layer chromatography identification method of loofah sponge charcoal, which comprises the following steps:
taking 0.5g of the loofah sponge charcoal target-decocted freeze-dried powder (03, 09 and 11 batches) or the loofah sponge charcoal reference decoction pieces, grinding, adding 20mL of water, carrying out ultrasonic treatment for 30min, cooling, extracting for 2 times by shaking with ethyl acetate, wherein 20mL of the extraction solution is used each time, combining ethyl acetate solutions, evaporating the ethyl acetate to dryness, and then using methanol to fix the volume to 1mL to obtain a sample solution.
Sucking 7 mu L of loofah sponge charcoal standard-decocted freeze-dried powder sample solution, dripping 3 mu L of loofah sponge charcoal reference decoction piece sample solution on the same silica gel G thin-layer plate, spreading by using petroleum ether (60-90): dichloromethane: ethyl acetate: formic acid (1.5.
Fig. 9 is a thin layer chromatogram of loofah sponge charcoal standard-fried lyophilized powder obtained by using a silica gel thin layer plate provided by yinlong, fig. 10 is a thin layer chromatogram of loofah sponge charcoal standard-fried lyophilized powder obtained by using a silica gel thin layer plate provided by cheese 32600, and fig. 11 is a thin layer chromatogram of loofah sponge charcoal standard-fried lyophilized powder obtained by using a silica gel thin layer plate provided by merck; wherein, the points are from left to right: stigmasterol, loofah sponge charcoal freeze-dried powder 03 batches, loofah sponge charcoal freeze-dried powder 09 batches, loofah sponge charcoal freeze-dried powder 11 batches and loofah sponge contrast decoction pieces. As can be seen from FIGS. 9-11, the method of the present invention is suitable for use with silicone thin-layer plates of various manufacturers.
Example 7
Investigation of different humiture
The embodiment provides a thin-layer chromatography identification method of loofah sponge charcoal, which comprises the following steps:
collecting 0.5g of standard decoction lyophilized powder (03, 09, 11 batches) of retinervus Luffae fructus charcoal or retinervus Luffae fructus charcoal reference decoction pieces, grinding, adding 20mL of water, ultrasonically treating for 30min, cooling, extracting with ethyl acetate for 2 times (20 mL each time), mixing ethyl acetate solutions, evaporating ethyl acetate, and diluting to 1mL with methanol to obtain a sample solution.
Respectively detecting at different temperatures and humidities, sucking 7 mu L of loofah sponge charcoal standard decoction freeze-dried powder sample solution, respectively dropping 5 mu L of loofah sponge charcoal control decoction piece sample solution on the same silica gel G thin-layer plate, respectively spreading by using petroleum ether (60-90): dichloromethane: ethyl acetate: formic acid (1.5.
Fig. 12 is a thin-layer chromatogram of a retinervus luffae fructus boiled freeze-dried powder obtained at normal temperature and normal humidity (23 ℃, 46%), fig. 13 is a thin-layer chromatogram of a retinervus luffae fructus boiled freeze-dried powder obtained at normal temperature and high humidity (23 ℃, 82%), fig. 14 is a thin-layer chromatogram of a retinervus luffae fructus boiled freeze-dried powder obtained at normal temperature and low humidity (23 ℃, 13%), and fig. 15 is a thin-layer chromatogram of a retinervus luffae fructus boiled freeze-dried powder obtained at low temperature and normal humidity (4 ℃, 46%); wherein, the points are from left to right: stigmasterol, loofah sponge charcoal freeze-dried powder 03 batches, loofah sponge charcoal freeze-dried powder 09 batches, loofah sponge charcoal freeze-dried powder 11 batches and loofah sponge contrast decoction pieces. As can be seen from fig. 12-15, the method provided by the present invention is applicable to various temperatures and humidities.
Example 8
Methodology validation
The embodiment provides a thin-layer chromatography identification method of loofah sponge carbon, which comprises the following steps:
collecting 0.5g of standard decoction lyophilized powder (202105-21-01-202105-21-15) of retinervus Luffae fructus or retinervus Luffae fructus control decoction pieces, grinding, adding 20mL of water, performing ultrasonic treatment for 30min, cooling, extracting with ethyl acetate for 2 times (20 mL each time), mixing ethyl acetate solutions, evaporating ethyl acetate, and diluting to 1mL with methanol to obtain a sample solution.
Absorbing 7 mu L of loofah sponge charcoal target-decocted freeze-dried powder sample solution and 5 mu L of loofah sponge charcoal control decoction piece sample solution under normal temperature and normal humidity (23 ℃ and 46%), respectively dropping on the same silica gel G thin-layer plate, spreading by using petroleum ether (60-90): dichloromethane: ethyl acetate: formic acid (1.5: 0.1) as a developing agent, taking out, airing, spraying 10% ethanol sulfate solution, airing, then spraying 5% vanillin sulfate solution, heating at 105 ℃ until spots are clear, and inspecting under sunlight.
The chromatogram of the standard decocted lyophilized powder of retinervus Luffae fructus charcoal methodology is shown in FIG. 16, wherein 1 is blank; 2-16 are 15 batches of standard fried freeze-dried powder batch numbers: 202105-21-01 to 202105-21-15;17 is retinervus Luffae fructus charcoal reference decoction pieces.
Example 9
Investigation of different dot sizes
The embodiment provides a thin-layer chromatography identification method of loofah sponge carbon, which comprises the following steps:
taking 0.5g of loofah sponge carbon particles or loofah sponge carbon reference decoction pieces, grinding, adding 20mL of water, carrying out ultrasonic treatment for 30min, cooling, extracting for 2 times by shaking with ethyl acetate, each time with 20mL, combining ethyl acetate solutions, evaporating the ethyl acetate to dryness, and then fixing the volume to 1mL with methanol to obtain a sample solution.
Absorbing 5 muL, 7 muL and 10 muL of the loofah sponge charcoal particle test solution, dispensing 1 muL, 3 muL and 5 muL of the loofah sponge charcoal control decoction piece test solution on the same silica gel G thin-layer plate respectively, taking out and drying by using petroleum ether (60-90): dichloromethane: ethyl acetate: formic acid (1.5.
The thin-layer chromatogram of the loofah sponge carbon particles with different sample amounts is shown in fig. 17, wherein 1 is blank control, and 2, 3 and 4 are loofah sponge carbon particles with KL19022051 sample amounts of 5 μ L, 7 μ L and 10 μ L respectively; 5. 6 and 7, the sample application amount of the loofah sponge carbon particles KL19003391 is 5 muL, 7 muL and 10 muL respectively; 8. 9 and 10 are that the sample volumes of the loofah sponge carbon particles KL19022051 are respectively 5 muL, 7 muL and 10 muL; 11. 12 and 13 are the sample application amounts of the loofah sponge charcoal control decoction pieces of 1 mul, 3 mul and 5 mul respectively; the optimal sample amount is determined to be 7 microliters of the sample, and 5 microliters of the reference decoction pieces.
Example 10
Methodology validation
The embodiment provides a thin-layer chromatography identification method of loofah sponge carbon, which comprises the following steps:
taking 0.5g of loofah sponge charcoal particles (KL 19022051, KL19003391, KL 19022051) or loofah sponge charcoal control decoction pieces, grinding, adding 20mL of water, performing ultrasonic treatment for 30min, cooling, extracting with ethyl acetate for 2 times, 20mL each time, combining ethyl acetate solutions, evaporating ethyl acetate to dryness, and then adding methanol to a constant volume of 1mL to obtain a sample solution.
Absorbing 7 mu L of loofah sponge carbon particle test solution and 5 mu L of loofah sponge carbon control decoction piece test solution under normal temperature and normal humidity (23 ℃ and 46%), respectively dropping on the same silica gel G thin layer plate, taking out and airing by using petroleum ether (60-90), ethyl acetate, formic acid (1.5.
The chromatogram of the loofah sponge carbon particle methodology is shown in fig. 18, wherein 1 is blank control; 2 is loofah sponge carbon particles KL19022051;3 is loofah sponge carbon particles KL19003391;4 is loofah sponge carbon particles KL19022051; and 5, loofah sponge charcoal reference decoction pieces.
The applicant states that the present invention is illustrated by the above examples of the process of the present invention, but the present invention is not limited to the above process steps, i.e. it is not meant that the present invention must rely on the above process steps to be carried out. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.
Claims (10)
1. A thin-layer chromatography identification method for loofah sponge and its processed products is characterized in that developing agents adopted in the thin-layer chromatography identification method comprise petroleum ether, dichloromethane, ethyl acetate and formic acid.
2. The thin-layer chromatography identification method of loofah sponge and the processed products thereof according to claim 1, wherein the processed products of loofah sponge comprise any one or a combination of at least two of loofah sponge blocks, fried loofah sponge and loofah sponge charcoal.
3. The thin layer chromatography identification method of vegetable sponge and its processed product according to claim 1 or 2, characterized in that the volume ratio of petroleum ether, dichloromethane, ethyl acetate and formic acid is (1.3-1.7): (0.8-1.2): (1.8-2.2): (0.08-0.12).
4. The thin-layer chromatography identification method of loofah sponge and processed products thereof according to any one of claims 1-3, characterized in that the boiling point of the petroleum ether is 60-90 ℃.
5. The thin-layer chromatography identification method of loofah sponge and processed products thereof according to any one of claims 1 to 4, characterized in that the thin-layer chromatography identification method specifically comprises the following steps:
and (2) spotting the test sample solution on a silica gel thin-layer plate, developing by adopting a developing agent, spraying a sulfuric acid ethanol solution after the silica gel thin-layer plate is dried, drying, spraying a vanillin solution, and heating to obtain the positions of the components in the test sample in the thin-layer plate.
6. The thin-layer chromatography identification method for the loofah sponge and the processed products thereof according to claim 5, characterized in that the mass content of sulfuric acid in the sulfuric acid ethanol solution is 8-12%;
preferably, the mass content of vanillin in the vanillin sulfuric acid solution is 3-7%;
preferably, the heating temperature is 100-110 ℃.
7. The thin-layer chromatography identification method of loofah sponge and processed products thereof according to claim 5 or 6, characterized in that the preparation of the test solution in the thin-layer chromatography identification method comprises the following steps:
(1) Grinding the baked towel gourd and/or the baked towel gourd product, adding water, performing ultrasonic treatment, filtering, and collecting solid;
(2) And (2) extracting the solid obtained in the step (1) by using ethyl acetate, desolventizing the obtained extract, and then performing constant volume by using methanol to obtain a test sample solution.
8. The thin-layer chromatography identification method for the loofah sponge and the processed products thereof according to claim 7, characterized in that in the step (1), the solid-to-liquid ratio of the loofah baked and/or the processed products of loofah baked to water is 0.5 (15-25) g/mL;
preferably, in the step (1), the power of the ultrasound is 100-500W, and the time of the ultrasound is 20-40min.
9. The thin-layer chromatography identification method for the loofah sponge and the processed products thereof according to claim 7 or 8, characterized in that the solid-to-liquid ratio of the loofah baked and/or the processed products of loofah baked to ethyl acetate is 0.5 (15-25) g/mL;
preferably, in the step (2), the number of times of extraction is 1 to 3.
10. The thin-layer chromatography identification method of loofah sponge and its processed products according to any one of claims 1 to 9, characterized in that the spot size is 5 to 8 μ L in the thin-layer chromatography identification method.
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