A rapid identification method for Alpiniae Oxyphyllae Fructus and salt-processed Alpiniae Oxyphyllae Fructus

Abstract

The present invention discloses a rapid identification method for Alpiniae Oxyphyllae Fructus and salt-processed Alpiniae Oxyphyllae Fructus, including: preparation of test solution of decoction pieces / granules for formula, thin-layer spotting, development and inspection; wherein the test solution is prepared by taking appropriate amounts of Alpiniae Oxyphyllae Fructus, salt-processed Alpiniae Oxyphyllae Fructus decoction pieces or granules for formula, ultrasonically extracting with ethyl acetate, filtering, concentrating the filtrate under reduced pressure to dryness, and dissolving the residue with an appropriate amount of ethyl acetate to obtain the test solution; the developing agent used is petroleum ether - n-hexane - ethyl acetate - glacial acetic acid (7:1:1:0.5); the inspection method is to show under ultraviolet light (365 nm), and the thin-layer plate is a silica gel G plate; the method of the present invention is accurate, rapid and has good reproducibility, and uses the difference in thin-layer chromatography for differentiation, making up for a gap in the field of identification technology of Alpiniae Oxyphyllae Fructus and salt-processed Alpiniae Oxyphyllae Fructus, and can be used as an exclusive identification method for Alpiniae Oxyphyllae Fructus and salt-processed Alpiniae Oxyphyllae Fructus decoction pieces and their preparations such as granules for formula, so as to better control the quality of salt-processed Alpiniae Oxyphyllae Fructus.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine identification, and particularly relates to a rapid identification method for Alpinia oxyphylla Miq. and salt-processed Alpinia oxyphylla Miq. (decoction pieces or formula granules). Background Art

[0002] Alpinia oxyphylla Miq. is the seed of the dried ripe fruit of the plant Alpinia oxyphylla Miq. of the Zingiberaceae family, and salt-processed Alpinia oxyphylla Miq. is the salt-roasted product of Alpinia oxyphylla Miq. Alpinia oxyphylla Miq. presents as irregular oblate seeds or residual petals of a seed mass. The seeds have slightly blunt edges and a diameter of about 3 mm; the surface is grayish-yellow to grayish-brown, with fine wrinkles; it is covered with a light brown membranous aril; it is hard in texture, and the endosperm is milky white. It has a specific aroma, and tastes pungent and slightly bitter. It is mainly produced in Guangdong, Guangxi, Fujian, Hainan and other places, and the currently used Alpinia oxyphylla Miq. is mostly cultivated. It is harvested when the fruit turns from green to red in summer and autumn. Generally, in May to June, when the fruit is light yellow, the seeds are brownish-brown, with a pungent taste, and when the fluff on the pericarp decreases, the fruit stalks are cut and collected. The collected fruit stalks are dried in the sun or dried at a low temperature for use as raw materials. It is pungent and warm. It belongs to the spleen and kidney meridians. It warms the kidney, consolidates essence, reduces urination, warms the spleen, stops diarrhea and controls salivation. It is used for enuresis due to kidney deficiency, frequent urination, spermatorrhea and leukorrhagia, diarrhea due to spleen cold, cold pain in the abdomen, and excessive salivation.

[0003] Processing method of salt-processed Alpinia oxyphylla Miq.: Take Alpinia oxyphylla Miq., mix it evenly with salt water, cover it tightly and let it stand until thoroughly penetrated. Place it in a frying container, heat it with slow fire until the surface is grayish-brown and the aroma escapes, then take it out and let it cool. Mash it before use. For every 100 kg of Alpinia oxyphylla Miq., 2 kg of salt is used. The raw product of Alpinia oxyphylla Miq. is pungent, warm and dry, with a certain degree of dryness. After salt-roasting, it has a moistening effect, guides the drug downward, mainly enters the kidney meridian, can enhance the effects of tonifying the kidney, astringing essence and reducing urination, and is good at tonifying the kidney, reducing urination and consolidating essence. After salt-roasting, the drug and salt synergistically enhance the effect, and the amount of their dissolved substances is higher than that of the corresponding raw product, and the effective components are easily decocted to enhance the curative effect. In order to prevent the misuse and abuse of traditional Chinese medicine preparations containing Alpinia oxyphylla Miq. and salt-processed Alpinia oxyphylla Miq. in clinical practice or research, it is crucial to form a rapid identification method for Alpinia oxyphylla Miq. and salt-processed Alpinia oxyphylla Miq., conduct quality control on salt-processed Alpinia oxyphylla Miq. as a whole, and further improve and perfect the research on the quality standard of salt-processed Alpinia oxyphylla Miq.

[0004] After traditional Chinese medicine processing, its morphological characteristics change. When the cut herbs are made into formula granules, they lose the morphological characteristics and microscopic features of the cut herbs, and the qualitative and quantitative compositions also change. As a means of physical and chemical identification of traditional Chinese medicine, thin-layer chromatography has the advantages of high efficiency, rapidity, and accuracy compared with other identification techniques such as microscopic identification and fingerprint identification. In the master's thesis "Research on the Improvement of the Quality Standards of Alpiniae Oxyphyllae Fructus and Its Cut Herbs" (Peng Lu, Southwest Jiaotong University), the method of thin-layer chromatography identification of Alpiniae Oxyphyllae Fructus in the Chinese Pharmacopoeia was adopted to prepare the test solutions of Alpiniae Oxyphyllae Fructus and its salt-roasted products in different batches, and a thin-layer chromatography identification method for the active ingredient naringenin in Alpiniae Oxyphyllae Fructus was established. In the thesis, naringenin and the reference herb of Alpiniae Oxyphyllae Fructus were used as references and examined under ultraviolet light (365 nm). In the chromatogram of the test sample, the same fluorescent spots were shown at the positions corresponding to the reference substance of naringenin. Using the thin-layer chromatography identification method of Alpiniae Oxyphyllae Fructus in the pharmacopoeia, it is not possible to effectively distinguish between Alpiniae Oxyphyllae Fructus and salt-roasted Alpiniae Oxyphyllae Fructus cut herbs and their formula granules. At present, no rapid identification technical scheme for Alpiniae Oxyphyllae Fructus and salt-roasted Alpiniae Oxyphyllae Fructus formula granules has been retrieved. In the present invention, ethyl acetate is used as a solvent to extract the volatile oil components in the sample, and a new developing agent condition is established to rapidly identify Alpiniae Oxyphyllae Fructus and salt-roasted Alpiniae Oxyphyllae Fructus formula granules. Moreover, the thin-layer chromatography identification technical scheme adopted in the present invention has not been retrieved in the identification of raw and processed products. In view of the current situation that it is impossible to rapidly distinguish between Alpiniae Oxyphyllae Fructus and salt-roasted Alpiniae Oxyphyllae Fructus formula granules, the present invention provides a highly efficient, rapid, and accurate thin-layer chromatography identification method. Summary of the Invention

[0005] In view of the current situation that there is no simple and rapid detection method to distinguish between Alpiniae Oxyphyllae Fructus cut herbs and salt-roasted Alpiniae Oxyphyllae Fructus cut herbs, and between Alpiniae Oxyphyllae Fructus formula granules and salt-roasted Alpiniae Oxyphyllae Fructus formula granules, the present invention provides a method for identifying Alpiniae Oxyphyllae Fructus and salt-roasted Alpiniae Oxyphyllae Fructus.

[0006] The method of the present invention includes the preparation of the test solution of cut herbs / formula granules, thin-layer spotting, development, and examination. Among them, the test solution is prepared by taking an appropriate amount of Alpiniae Oxyphyllae Fructus, salt-roasted Alpiniae Oxyphyllae Fructus cut herbs or formula granules, ultrasonically extracting with ethyl acetate, filtering, concentrating the filtrate under reduced pressure to dryness, and dissolving the residue with an appropriate amount of ethyl acetate to obtain the test solution; the developing agent used is petroleum ether - n-hexane - ethyl acetate - glacial acetic acid (7:1:1:0.5); the examination method is to show under ultraviolet light (365 nm), and the thin-layer plate is a silica gel G plate.

[0007] The technical solution of the present invention is as follows:

[0008] A rapid identification method for Alpiniae Oxyphyllae Fructus and salt-roasted Alpiniae Oxyphyllae Fructus includes the following steps:

[0009] (1) Preparation of the test solution of cut herbs

[0010] Take the prepared Alpiniae Oxyphyllae Fructus slices and salt-processed Alpiniae Oxyphyllae Fructus slices, add ethyl acetate respectively, extract by ultrasonic wave, filter, concentrate the filtrate under reduced pressure to dryness, and then redissolve with ethyl acetate to obtain the test solution of Alpiniae Oxyphyllae Fructus slices and the test solution of salt-processed Alpiniae Oxyphyllae Fructus slices respectively;

[0011] The prepared Alpiniae Oxyphyllae Fructus slices can be commercially obtained through conventional channels;

[0012] The preparation method of salt-processed Alpiniae Oxyphyllae Fructus slices is as follows: Take an appropriate amount of Alpiniae Oxyphyllae Fructus, mix well with brine, let it stand until thoroughly penetrated, place it in a frying container, heat at 240 °C, fry until the surface is grayish-brown and the aroma escapes, take out, let it cool, and crush it before use; for every 100 kg of Alpiniae Oxyphyllae Fructus, use 2 kg of salt;

[0013] Preferably, the material-liquid ratio of Alpiniae Oxyphyllae Fructus slices to ethyl acetate for extraction is 1:5 - 50, g / mL; preferably, the material-liquid ratio of salt-processed Alpiniae Oxyphyllae Fructus slices to ethyl acetate for extraction is 1:5 - 50, g / mL;

[0014] Preferably, the power of ultrasonic wave is 40 KHz, and the ultrasonic extraction time is 20 - 30 min;

[0015] Preferably, the concentration of the test solution of Alpiniae Oxyphyllae Fructus slices is 1.0 - 3.0 g / mL based on the raw Alpiniae Oxyphyllae Fructus slices; preferably, the concentration of the test solution of salt-processed Alpiniae Oxyphyllae Fructus slices is 1.0 - 3.0 g / mL based on the raw salt-processed Alpiniae Oxyphyllae Fructus slices;

[0016] (2) Preparation of the test solution of formula granules

[0017] Take the Alpiniae Oxyphyllae Fructus formula granules and salt-processed Alpiniae Oxyphyllae Fructus formula granules, add ethyl acetate respectively, extract by ultrasonic wave, filter, concentrate the filtrate under reduced pressure to dryness, and then redissolve with ethyl acetate to obtain the test solution of Alpiniae Oxyphyllae Fructus formula granules and the test solution of salt-processed Alpiniae Oxyphyllae Fructus formula granules respectively;

[0018] The salt-processed Alpiniae Oxyphyllae Fructus formula granules can be commercially obtained through conventional channels;

[0019] The preparation of Alpiniae Oxyphyllae Fructus formula granules is as follows: Take an appropriate amount of Alpiniae Oxyphyllae Fructus slices, decoct with water, collect the volatile oil, prepare the volatile oil β-cyclodextrin inclusion compound, filter the medicinal liquid, concentrate the filtrate into clear paste, dry (or dry and pulverize), add the volatile oil inclusion compound to the dry powder, add an appropriate amount of auxiliary materials, mix well, and granulate to obtain;

[0020] Preferably, the material-liquid ratio of Alpiniae Oxyphyllae Fructus formula granules to ethyl acetate for extraction is 1:10 - 50, g / mL; preferably, the material-liquid ratio of salt-processed Alpiniae Oxyphyllae Fructus formula granules to ethyl acetate for extraction is 1:10 - 50, g / mL;

[0021] Preferably, the power of ultrasonic wave is 40 KHz, and the ultrasonic extraction time is 20 - 30 min;

[0022] The concentration of the test solution of the optimized semen alpiniae oxyphyllae formula granules is 1.2 - 4.5 g / mL based on the raw semen alpiniae oxyphyllae formula granules; the concentration of the test solution of the optimized processed semen alpiniae oxyphyllae formula granules is 1.2 - 4.5 g / mL based on the raw processed semen alpiniae oxyphyllae formula granules;

[0023] (3) Spotting, developing, and inspection

[0024] Spot the test solutions of the semen alpiniae oxyphyllae herbal pieces and the processed semen alpiniae oxyphyllae herbal pieces obtained in step (1) on the same thin-layer plate, use a mixed solution with a volume ratio of petroleum ether / n-hexane / ethyl acetate / glacial acetic acid of 7:1:1:0.5 as the developing agent, develop, take out, dry, and inspect under ultraviolet light (365 nm); compared with the chromatogram of the test solution of the semen alpiniae oxyphyllae herbal pieces, the chromatogram of the test solution of the processed semen alpiniae oxyphyllae herbal pieces shows blue fluorescent spots at an R f value of 0.41 - 0.65, the pink spots at an R f value of 0.25 - 0.40 become significantly brighter, and the magenta spots at an R f value of 0.19 - 0.37 and 0.16 - 0.29 become significantly lighter;

[0025] It is recommended that the spotting volume of both the test solution of the semen alpiniae oxyphyllae herbal pieces and the test solution of the processed semen alpiniae oxyphyllae herbal pieces be 5 - 20 μL;

[0026] Spot the test solutions of the semen alpiniae oxyphyllae formula granules and the processed semen alpiniae oxyphyllae formula granules obtained in step (2) on the same thin-layer plate, use a mixed solution with a volume ratio of petroleum ether / n-hexane / ethyl acetate / glacial acetic acid of 7:1:1:0.5 as the developing agent, develop, take out, dry, and inspect under ultraviolet light (365 nm); compared with the chromatogram of the test solution of the semen alpiniae oxyphyllae formula granules, the chromatogram of the test solution of the processed semen alpiniae oxyphyllae formula granules shows blue fluorescent spots at an R f value of 0.38 - 0.51, and the spots at an R f value of 0.20 - 0.32 and 0.10 - 0.18 become significantly brighter;

[0027] It is recommended that the spotting volume of both the test solution of the semen alpiniae oxyphyllae formula granules and the test solution of the processed semen alpiniae oxyphyllae formula granules be 5 - 20 μL.

[0028] The present invention has the following beneficial effects:

[0029] (1) The present invention uses ethyl acetate to extract the small polar components in the semen alpiniae oxyphyllae herbal pieces and the processed semen alpiniae oxyphyllae herbal pieces, the semen alpiniae oxyphyllae formula granules and the processed semen alpiniae oxyphyllae formula granules, performs thin-layer development and inspection with a developing agent of specific types and ratios, and differentiates using thin-layer chromatography differences, filling a gap in the identification technology field of semen alpiniae oxyphyllae and processed semen alpiniae oxyphyllae, and can be used as an exclusive identification method for semen alpiniae oxyphyllae and processed semen alpiniae oxyphyllae herbal pieces and their formula granule preparations.

[0030] (2) The present invention is accurate, fast, and has good reproducibility, providing a method for quickly identifying Alpiniae Oxyphyllae Fructus and salt-processed Alpiniae Oxyphyllae Fructus. Description of the Drawings

[0031] Figure 1 : TLC chromatograms of the Alpiniae Oxyphyllae Fructus decoction pieces and Alpiniae Oxyphyllae Fructus formula granules obtained in Example 3, (1 - TLC chromatogram of Alpiniae Oxyphyllae Fructus decoction pieces, 2 - TLC chromatogram of Alpiniae Oxyphyllae Fructus formula granules).

[0032] Figure 2 : TLC chromatograms of the salt-processed Alpiniae Oxyphyllae Fructus decoction pieces and salt-processed Alpiniae Oxyphyllae Fructus formula granules obtained in Example 3, (1 - TLC chromatogram of salt-processed Alpiniae Oxyphyllae Fructus decoction pieces, 2 - TLC chromatogram of salt-processed Alpiniae Oxyphyllae Fructus formula granules).

[0033] Figure 3 : TLC chromatograms of the Alpiniae Oxyphyllae Fructus decoction pieces and salt-processed Alpiniae Oxyphyllae Fructus decoction pieces obtained in Example 3, (1 - TLC chromatogram of Alpiniae Oxyphyllae Fructus decoction pieces, 2 - TLC chromatogram of salt-processed Alpiniae Oxyphyllae Fructus decoction pieces).

[0034] Figure 4 : TLC chromatograms of the Alpiniae Oxyphyllae Fructus formula granules and salt-processed Alpiniae Oxyphyllae Fructus formula granules obtained in Example 3, (1 - TLC chromatogram of Alpiniae Oxyphyllae Fructus formula granules, 2 - TLC chromatogram of salt-processed Alpiniae Oxyphyllae Fructus formula granules).

[0035] Figure 5 : TLC repeatability chromatograms of three batches of Alpiniae Oxyphyllae Fructus formula granules and salt-processed Alpiniae Oxyphyllae Fructus formula granules from different production areas and batches obtained in Example 5, (in each figure, numbers 1, 2, 3 - TLC chromatograms of Alpiniae Oxyphyllae Fructus formula granules with batch numbers 20210901, 20211101, and 20220601 from Yulin, Guangxi; 4, 5, 6 - TLC chromatograms of salt-processed Alpiniae Oxyphyllae Fructus formula granules with batch numbers YYZR-Y191003 from Lingshan, Guangxi, YYZR-Y191007 from Lucheng, Guangxi, and YYZR-Y191008 from Wenshan, Yunnan).

[0036] Figure 6 : TLC chromatograms of three batches of Alpiniae Oxyphyllae Fructus formula granules and salt-processed Alpiniae Oxyphyllae Fructus formula granules from different production areas and batches obtained in Example 5 under different temperature and humidity conditions, (in sequence: 10 ± 2°C, 20 ± 5%, 10 ± 2°C, 80 ± 5%, 40 ± 2°C, 20 ± 5%, 40 ± 2°C, 80 ± 5%; in each figure, numbers 1, 2, 3 - TLC chromatograms of Alpiniae Oxyphyllae Fructus formula granules with batch numbers 20210901, 20211101, and 20220601 from Yulin, Guangxi; 4, 5, 6 - TLC chromatograms of salt-processed Alpiniae Oxyphyllae Fructus formula granules with batch numbers YYZR-Y191003 from Lingshan, Guangxi, YYZR-Y191007 from Lucheng, Guangxi, and YYZR-Y191008 from Wenshan, Yunnan).

[0037] Figure 7: TLC durability thin layer chromatograms of three batches of Alpiniae Oxyphyllae Fructus formula granules and salted Alpiniae Oxyphyllae Fructus formula granules from different producing areas obtained in Example 5, (from top to bottom: Qingdao Ocean Chemical silica gel G thin layer plate, Merck silica gel G thin layer plate; in each figure, numbers 1, 2, 3 - thin layer chromatograms of Alpiniae Oxyphyllae Fructus formula granules with batch numbers 20210901, 20211101, 20220601 from Yulin, Guangxi; 4, 5, 6 - thin layer chromatograms of salted Alpiniae Oxyphyllae Fructus formula granules with batch numbers YYZR - Y191003 from Lingshan, Guangxi, YYZR - Y191007 from Luchuan, Guangxi, and YYZR - Y191008 from Wenshan, Yunnan). Detailed implementation manners

[0038] The present invention will be further described below through specific examples, but the protection scope of the present invention is not limited thereto.

[0039] In the following examples, the instruments involved include: SQP electronic balance (Sartorius Scientific Instruments Co., Ltd.), KQ - 250DB ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), rotary evaporator (R210 + V700, Buchi, Switzerland).

[0040] In the following examples, the reagents involved include: petroleum ether (Shanghai Titan Scientific Co., Ltd., analytical pure), n - hexane (Shanghai Macklin Biochemical Co., Ltd., analytical pure), ethyl acetate (Xilong Science Co., Ltd., analytical pure), glacial acetic acid (Sinopharm Chemical Reagent Co., Ltd., analytical pure).

[0041] Example 1

[0042] (1) Preparation of test solution of crude drug

[0043] Take 1.0 g each of Alpiniae Oxyphyllae Fructus crude drug and salted Alpiniae Oxyphyllae Fructus crude drug, add 5 ml of ethyl acetate respectively, ultrasonically extract for 20 min, filter, concentrate the filtrate to dryness under reduced pressure (30 - 50 °C), and redissolve the residue with 1 ml of ethyl acetate to obtain the test solutions of Alpiniae Oxyphyllae Fructus crude drug and salted Alpiniae Oxyphyllae Fructus crude drug respectively.

[0044] (2) Preparation of test solution of formula granule

[0045] Take 1.2 g each of Alpiniae Oxyphyllae Fructus formula granule and salted Alpiniae Oxyphyllae Fructus formula granule, add 60 ml of ethyl acetate respectively, ultrasonically extract for 20 min, filter, concentrate the filtrate to dryness under reduced pressure (30 - 50 °C), and redissolve the residue with 1 ml of ethyl acetate to obtain the test solutions of Alpiniae Oxyphyllae Fructus formula granule and salted Alpiniae Oxyphyllae Fructus formula granule respectively.

[0046] (3) Spotting, developing, and inspection

[0047] Absorb 20 μL of the test solution of the cut crude drug and 20 μL of the test solution of the formula granules, spot them on the same silica gel G thin-layer plate of Yantai Jiangyou Silica Gel Development Co., Ltd., use a mixed solution with a volume ratio of petroleum ether: n-hexane: ethyl acetate: glacial acetic acid of 7:1:1:0.5 as the developing agent, develop, take out, dry, and examine under ultraviolet light (365 nm).

[0048] Compared with the chromatogram of the test solution of the crude drug of Alpiniae Oxyphyllae Fructus, the chromatogram of the test solution of the salt-processed crude drug of Alpiniae Oxyphyllae Fructus shows a blue fluorescent spot at an R f value of 0.59, the pink spot at an R f value of 0.39 becomes significantly brighter, and the magenta spots at R f values of 0.36 and 0.29 become significantly lighter.

[0049] Compared with the chromatogram of the test solution of the formula granules of Alpiniae Oxyphyllae Fructus, the chromatogram of the test solution of the salt-processed formula granules of Alpiniae Oxyphyllae Fructus shows a blue fluorescent spot at an R f value of 0.48, and the spots at R f values of 0.28 and 0.18 become significantly brighter.

[0050] Example 2

[0051] (1) Preparation of the test solution of the cut crude drug

[0052] Take 1.5 g each of the cut crude drug of Alpiniae Oxyphyllae Fructus and the salt-processed cut crude drug of Alpiniae Oxyphyllae Fructus, add 75 ml of ethyl acetate respectively, extract by ultrasonic for 10 min, filter, concentrate the filtrate to dryness under reduced pressure (30 - 50 °C), and redissolve the residue with 1 ml of ethyl acetate to obtain the test solution of the cut crude drug of Alpiniae Oxyphyllae Fructus and the test solution of the salt-processed cut crude drug of Alpiniae Oxyphyllae Fructus respectively.

[0053] (2) Preparation of the test solution of the formula granules

[0054] Take 3.0 g each of the formula granules of Alpiniae Oxyphyllae Fructus and the salt-processed formula granules of Alpiniae Oxyphyllae Fructus, add 50 ml of ethyl acetate respectively, extract by ultrasonic for 10 min, filter, concentrate the filtrate to dryness under reduced pressure (30 - 50 °C), and redissolve the residue with 1 ml of ethyl acetate to obtain the test solution of the formula granules of Alpiniae Oxyphyllae Fructus and the test solution of the salt-processed formula granules of Alpiniae Oxyphyllae Fructus respectively.

[0055] (3) Spotting, developing, and examination

[0056] Absorb 15 μL of the test solution of the cut crude drug and 15 μL of the test solution of the formula granules, spot them on the same silica gel G thin-layer plate of Yantai Jiangyou Silica Gel Development Co., Ltd., use a mixed solution with a volume ratio of petroleum ether: n-hexane: ethyl acetate: glacial acetic acid of 7:1:1:0.5 as the developing agent, develop, take out, dry, and examine under ultraviolet light (365 nm).

[0057] Compared with the chromatogram of the test solution of the cut crude drug of Alpiniae Oxyphyllae Fructus, the chromatogram of the test solution of the salt-processed cut crude drug of Alpiniae Oxyphyllae Fructus shows at an R fA blue fluorescent spot appears at a value of 0.59, R f The pink spot at the value of 0.38 becomes significantly brighter, R f The rose-red spots at values ​​of 0.37 and 0.29 become significantly lighter.

[0058] Compared with the chromatogram of the Alpinia oxyphylla formula granules, the chromatogram of the salt Alpinia oxyphylla formula granules was f A blue fluorescent spot appears at a value of 0.47, R f The spots become significantly brighter at values ​​of 0.26 and 0.17.

[0059] Example 3

[0060] (1) Preparation of sample solution

[0061] Take 2.0 g of Alpinia oxyphylla slices and salt Alpinia oxyphylla slices, add 20 ml of ethyl acetate respectively, extract by ultrasonic for 30 min, filter, concentrate the filtrate to dryness (30-50°C) under reduced pressure, and re-dissolve the residue with 1 ml of ethyl acetate to obtain Alpinia oxyphylla slice test solution and salt Alpinia oxyphylla slice test solution respectively.

[0062] (2) Preparation of test solution of formulated granules

[0063] Take 2.0g of Alpinia oxyphylla formula granules and salt Alpinia oxyphylla formula granules, add 30ml of ethyl acetate respectively, extract by ultrasonic for 30min, filter, concentrate the filtrate to dryness (30-50℃) under reduced pressure, and dissolve the residue with 1ml of ethyl acetate to obtain the test solution of Alpinia oxyphylla formula granules and salt Alpinia oxyphylla formula granules, respectively.

[0064] (3) Click, expand, and inspect

[0065] 10 μL of the test solution of the decoction pieces and 10 μL of the test solution of the formulated granules were spotted on the same silica gel G thin layer plate produced by Yantai Jiangyou Silica Gel Development Co., Ltd., and a mixed solution of petroleum ether: n-hexane: ethyl acetate: glacial acetic acid in a volume ratio of 7:1:1:0.5 was used as the developing agent. The plate was developed, taken out, dried, and examined under ultraviolet light (365 nm).

[0066] Compared with the chromatogram of the Alpinia oxyphylla decoction piece test sample, the chromatogram of the salt Alpinia oxyphylla decoction piece test sample is f A blue fluorescent spot appears at a value of 0.62, R f The pink spot at the value of 0.38 becomes significantly brighter, R f The rose-red spots at values ​​of 0.34 and 0.28 become significantly lighter.

[0067] Compared with the chromatogram of the Alpinia oxyphylla formula granules, the chromatogram of the salt Alpinia oxyphylla formula granules was f A blue fluorescent spot appears at a value of 0.46, R fThe spots become significantly brighter at values ​​of 0.29 and 0.18.

[0068] Example 4

[0069] (1) Preparation of sample solution

[0070] Take 3.0 g of Alpinia oxyphylla slices and salt Alpinia oxyphylla slices, add 25 ml of ethyl acetate respectively, extract by ultrasonic for 35 min, filter, concentrate the filtrate to dryness (30-50°C) under reduced pressure, and re-dissolve the residue with 1 ml of ethyl acetate to obtain Alpinia oxyphylla slice test solution and salt Alpinia oxyphylla slice test solution respectively.

[0071] (2) Preparation of test solution of formulated granules

[0072] Take 4.5 g of Alpinia oxyphylla formula granules and salt Alpinia oxyphylla formula granules, add 45 ml of ethyl acetate respectively, extract by ultrasonic for 35 min, filter, concentrate the filtrate to dryness (30-50°C) under reduced pressure, and dissolve the residue with 1 ml of ethyl acetate to obtain the test solution of Alpinia oxyphylla formula granules and the test solution of salt Alpinia oxyphylla formula granules, respectively.

[0073] (3) Click, expand, and inspect

[0074] 5 μL of the test solution of the decoction pieces and 5 μL of the test solution of the formulated granules were spotted on the same silica gel G thin layer plate produced by Yantai Jiangyou Silica Gel Development Co., Ltd., and a mixed solution of petroleum ether: n-hexane: ethyl acetate: glacial acetic acid in a volume ratio of 7:1:1:0.5 was used as the developing agent. The plate was developed, taken out, dried, and examined under ultraviolet light (365 nm).

[0075] Compared with the chromatogram of the Alpinia oxyphylla decoction piece test sample, the chromatogram of the salt Alpinia oxyphylla decoction piece test sample is f A blue fluorescent spot appears at a value of 0.56, R f The pink spot at the value of 0.37 becomes significantly brighter, R f The rose-red spots at values ​​of 0.34 and 0.28 become significantly lighter.

[0076] Compared with the chromatogram of the Alpinia oxyphylla formula granules, the chromatogram of the salt Alpinia oxyphylla formula granules was f A blue fluorescent spot appears at a value of 0.49, R f The spots become significantly brighter at values ​​of 0.26 and 0.18.

[0077] Example 5

[0078] Based on Example 3, this example conducts the following methodological investigation on the thin layer chromatography identification process, as follows:

[0079] (1) Repeatability study

[0080] Prepare three batches of test solutions of semen alpiniae oxyphyllae formula granules and salted semen alpiniae oxyphyllae formula granules respectively. Pipette 10 μL of the test solution and spot it on the same silica gel G thin layer plate of Yantai Jiangyou Silica Gel Development Co., Ltd. Use a mixed solution with a volume ratio of petroleum ether: n-hexane: ethyl acetate: glacial acetic acid of 7:1:1:0.5 as the developing agent, develop, take out, dry, and examine under ultraviolet light (365 nm).

[0081] Compared with the chromatogram of the test solution of semen alpiniae oxyphyllae formula granules, the chromatogram of the test solution of salted semen alpiniae oxyphyllae formula granules shows blue fluorescent spots at an R f value of 0.42 - 0.44, and the spots at R f values of 0.24 - 0.26 and 0.15 - 0.17 become significantly brighter.

[0082] (2) Investigation of temperature and humidity durability

[0083] Prepare three batches of test solutions of semen alpiniae oxyphyllae formula granules and salted semen alpiniae oxyphyllae formula granules respectively. Fix the relative temperature at 10°C and 40°C respectively, and at different humidities (20%, 80%). Pipette 10 μL of the test solution and spot it on the same silica gel G thin layer plate of Yantai Jiangyou Silica Gel Development Co., Ltd. Use a mixed solution with a volume ratio of petroleum ether: n-hexane: ethyl acetate: glacial acetic acid of 7:1:1:0.5 as the developing agent, develop, take out, dry, and examine under ultraviolet light (365 nm).

[0084] Compared with the chromatogram of the test solution of semen alpiniae oxyphyllae formula granules, the chromatogram of the test solution of salted semen alpiniae oxyphyllae formula granules shows blue fluorescent spots at an R f value of 0.42 - 0.49, and the spots at R f values of 0.21 - 0.29 and 0.10 - 0.17 become significantly brighter.

[0085] The results show that: under different temperature conditions, the purpose of rapid identification can be achieved, indicating that the method has good temperature and humidity durability.

[0086] (3) Durability of thin layer plate

[0087] Prepare three batches of test solutions of semen alpiniae oxyphyllae formula granules and salted semen alpiniae oxyphyllae formula granules respectively. Pipette 10 μL of the test solution and spot it on the same silica gel G thin layer plate of Qingdao Ocean Chemical Industry. Use a mixed solution with a volume ratio of petroleum ether: n-hexane: ethyl acetate: glacial acetic acid of 7:1:1:0.5 as the developing agent, develop, take out, dry, and examine under ultraviolet light (365 nm).

[0088] Compared with the chromatogram of the test solution of semen alpiniae oxyphyllae formula granules, the chromatogram of the test solution of salted semen alpiniae oxyphyllae formula granules shows blue fluorescent spots at an R f value of 0.51, and at R fThe spots at values of 0.32 and 0.16 become significantly brighter.

[0089] Prepare three different batches of test solutions of semen alpiniae oxyphyllae formula granules and salt-processed semen alpiniae oxyphyllae formula granules respectively. Pipette 10 μL of the test solution and spot it on the same Merck silica gel G thin-layer plate. Use a mixed solution with a volume ratio of petroleum ether:n-hexane:ethyl acetate:glacial acetic acid of 7:1:1:0.5 as the developing agent. Develop, take out, and dry. Examine under ultraviolet light (365 nm).

[0090] Compared with the test chromatogram of semen alpiniae oxyphyllae formula granules, the test chromatogram of salt-processed semen alpiniae oxyphyllae formula granules shows a blue fluorescent spot at an R f value of 0.38, and the spots at R f values of 0.20 and 0.12 become significantly brighter.

[0091] The results show that under different thin-layer plate conditions, the purpose of rapid identification can be achieved, indicating that the thin-layer plate of this method has good durability.

Claims

1. A rapid identification method for Fructus Alpiniae Oxyphyllae and salt-processed Fructus Alpiniae Oxyphyllae, characterized in that, It includes the following steps: (1) Preparation of the test solution of the cut crude drug Take the cut crude drug of Alpiniae Oxyphyllae Fructus and the salt-processed cut crude drug of Alpiniae Oxyphyllae Fructus, add ethyl acetate respectively, extract by ultrasonic wave, filter, concentrate the filtrate under reduced pressure to dryness, and then redissolve with ethyl acetate to obtain the test solution of the cut crude drug of Alpiniae Oxyphyllae Fructus and the test solution of the salt-processed cut crude drug of Alpiniae Oxyphyllae Fructus respectively; (2) Preparation of the test solution of the formula granules Take the formula granules of Alpiniae Oxyphyllae Fructus and the salt-processed formula granules of Alpiniae Oxyphyllae Fructus, add ethyl acetate respectively, extract by ultrasonic wave, filter, concentrate the filtrate under reduced pressure to dryness, and then redissolve with ethyl acetate to obtain the test solution of the formula granules of Alpiniae Oxyphyllae Fructus and the test solution of the salt-processed formula granules of Alpiniae Oxyphyllae Fructus respectively; (3) Spotting, developing, and inspection Spot the test solution of the prepared Alpiniae Oxyphyllae Fructus slices and the test solution of the salt-processed Alpiniae Oxyphyllae Fructus slices obtained in step (1) on the same thin-layer plate. Use a mixed solution with a volume ratio of petroleum ether / n-hexane / ethyl acetate / glacial acetic acid of 7:1:1:0.5 as the developing agent. Develop, take out, and air dry. Examine under ultraviolet light. Compared with the chromatogram of the test solution of the Alpiniae Oxyphyllae Fructus slices, the chromatogram of the test solution of the salt-processed Alpiniae Oxyphyllae Fructus slices shows blue fluorescent spots at an R f value of 0.41 - 0.65, the pink spots at an R f value of 0.25 - 0.40 become significantly brighter, and the magenta spots at an R f value of 0.19 - 0.37 and 0.16 - 0.29 become significantly lighter; Spot the test solution of the prepared Alpiniae Oxyphyllae Fructus formula granules and the test solution of the salt-processed Alpiniae Oxyphyllae Fructus formula granules obtained in step (2) on the same thin-layer plate. Use a mixed solution with a volume ratio of petroleum ether / n-hexane / ethyl acetate / glacial acetic acid of 7:1:1:0.5 as the developing agent, develop, take out, dry, and examine under ultraviolet light; compared with the chromatogram of the test sample of the Alpiniae Oxyphyllae Fructus formula granules, the chromatogram of the test sample of the salt-processed Alpiniae Oxyphyllae Fructus formula granules shows blue fluorescent spots at an R f value of 0.38 - 0.51, and the spots at an R f value of 0.20 - 0.32 and 0.10 - 0.18 become significantly brighter.

2. The rapid identification method of Alpiniae Oxyphyllae Fructus and salt-processed Alpiniae Oxyphyllae Fructus according to claim 1, characterized in that, In step (1), the material-liquid ratio of the cut crude drug of Alpiniae Oxyphyllae Fructus to ethyl acetate for extraction is 1:5 - 50, g / mL; the material-liquid ratio of the salt-processed cut crude drug of Alpiniae Oxyphyllae Fructus to ethyl acetate for extraction is 1:5 - 50, g / mL.

3. The rapid identification method of Alpiniae Oxyphyllae Fructus and salt-processed Alpiniae Oxyphyllae Fructus according to claim 1, characterized in that, In step (1), the concentration of the test solution of the cut crude drug of Alpiniae Oxyphyllae Fructus is 1.0 - 3.0 g / mL calculated based on the raw cut crude drug of Alpiniae Oxyphyllae Fructus; the concentration of the test solution of the salt-processed cut crude drug of Alpiniae Oxyphyllae Fructus is 1.0 - 3.0 g / mL calculated based on the raw salt-processed cut crude drug of Alpiniae Oxyphyllae Fructus.

4. The rapid identification method of Alpiniae Oxyphyllae Fructus and salt-processed Alpiniae Oxyphyllae Fructus according to claim 1, characterized in that, In step (2), the material-liquid ratio of the formula granules of Alpiniae Oxyphyllae Fructus to ethyl acetate for extraction is 1:10 - 50, g / mL; the material-liquid ratio of the salt-processed formula granules of Alpiniae Oxyphyllae Fructus to ethyl acetate for extraction is 1:10 - 50, g / mL.

5. The rapid identification method of Alpiniae Oxyphyllae Fructus and salt-processed Alpiniae Oxyphyllae Fructus according to claim 1, characterized in that, In step (2), the concentration of the test solution of the formula granules of Alpiniae Oxyphyllae Fructus is 1.2 - 4.5 g / mL calculated based on the raw formula granules of Alpiniae Oxyphyllae Fructus; the concentration of the test solution of the salt-processed formula granules of Alpiniae Oxyphyllae Fructus is 1.2 - 4.5 g / mL calculated based on the raw salt-processed formula granules of Alpiniae Oxyphyllae Fructus.

6. The rapid identification method of Alpiniae Oxyphyllae Fructus and salt-processed Alpiniae Oxyphyllae Fructus according to claim 1, characterized in that, In step (1) or step (2), the power of ultrasonic wave is 40 KHz, and the time for ultrasonic extraction is 20 - 30 min.

7. The rapid identification method of Alpiniae Oxyphyllae Fructus and salt-processed Alpiniae Oxyphyllae Fructus according to claim 1, characterized in that, In step (3), the spotting volume of the test solution of the cut crude drug of Alpiniae Oxyphyllae Fructus and the test solution of the salt-processed cut crude drug of Alpiniae Oxyphyllae Fructus is both 5 - 20 μL; the spotting volume of the test solution of the formula granules of Alpiniae Oxyphyllae Fructus and the test solution of the salt-processed formula granules of Alpiniae Oxyphyllae Fructus is both 5 - 20 μL.

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