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 PDF

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CN115684455A
CN115684455A CN202211345292.3A CN202211345292A CN115684455A CN 115684455 A CN115684455 A CN 115684455A CN 202211345292 A CN202211345292 A CN 202211345292A CN 115684455 A CN115684455 A CN 115684455A
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loofah
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charcoal
identification method
layer chromatography
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张志强
石玉喆
周永康
闫晓鑫
李国鹏
付静
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Beijing Tcmages Pharmaceutical Co Ltd
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Abstract

本发明提供一种丝瓜络及其炮制品的薄层色谱鉴别方法,所述薄层色谱鉴别方法中采用的展开剂包括石油醚、二氯甲烷、乙酸乙酯和甲酸。本发明提供的丝瓜络及其炮制品的薄层色谱鉴别方法,具有准确率高、分离效果佳、色斑清晰的特点,能够鉴别出丝瓜络块、丝瓜络炭不同炮制品,其次能为丝瓜络块到丝瓜络炭炮制过程中化学成分的变化提供研究依据,也能对丝瓜络炭药材、饮片、中间体、配方颗粒的定性鉴别,及对整个工艺过程质量进行控制,保证其质量的均一、稳定、可靠。

Figure 202211345292

The invention provides a thin-layer chromatographic identification method for loofah and processed products thereof. The developer used in the thin-layer chromatographic identification method includes petroleum ether, dichloromethane, ethyl acetate and formic acid. The thin-layer chromatography identification method of loofah and its processed products provided by the invention has the characteristics of high accuracy, good separation effect, and clear stains, and can identify different processed products of loofah block and loofah charcoal, followed by loofah It provides a research basis for the change of chemical composition in the process of processing loofah charcoal from lumps to loofah charcoal. It can also qualitatively identify loofah charcoal medicinal materials, decoction pieces, intermediates, and formula granules, and control the quality of the entire process to ensure uniform quality. ,Stable and reliable.

Figure 202211345292

Description

一种丝瓜络及其炮制品的薄层色谱鉴别方法TLC identification method of loofah and its processed products

技术领域technical field

本发明属于中药分析技术领域,具体涉及一种丝瓜络及其炮制品的薄层色谱鉴别方法。The invention belongs to the technical field of traditional Chinese medicine analysis, and in particular relates to a TLC identification method of loofah and processed products thereof.

背景技术Background technique

丝瓜络为葫芦科植物丝瓜络Luffa cylindrical(L.)Roem.的干燥成熟果实的维管束。具有通经活络、清热化痰、解毒消肿、强心利尿等功效,主治胸胁疼痛、肺热咳痰、闭经、乳汁不通等症。丝瓜络中含有多种丝瓜皂苷、黄酮和酚类、蛋白质、氨基酸类、油脂和无机元素及有机酸类等,药理试验证明其具有降血脂、镇痛镇静等作用,临床效果显著。Luffa is the vascular bundle of the dry ripe fruit of Cucurbitaceae Luffa cylindrical (L.) Roem. It has the effects of dredging the meridian and activating collaterals, clearing heat and reducing phlegm, detoxifying and reducing swelling, strengthening the heart and diuresis, etc. It is mainly used to treat chest and hypochondrium pain, cough due to lung heat, amenorrhea, and milk blockage. Loofah contains a variety of loofah saponins, flavonoids and phenols, proteins, amino acids, oils, inorganic elements and organic acids, etc. Pharmacological tests have proved that it has the effects of lowering blood fat, analgesia and sedation, and has significant clinical effects.

丝瓜络其主要炮制品有丝瓜络(块)、炒丝瓜络、丝瓜络炭三种,其中丝瓜络(块)、丝瓜络炭临床用的较多,但目前均无质量标准,且对丝瓜络(块)、丝瓜络炭两种炮制品差异性的报道较少。由于丝瓜络炭不同剂型的样品是丝瓜络炭药材经一系列工艺制成的,其中一些工艺会破坏原药材的性状特征,故需要一种具现有技术有很好的区分和鉴定能力且操作更加简便、快捷的鉴别方法,以便能够更好地控制质量。Its main processed products of loofah have three kinds of loofah (block), fried loofah and loofah charcoal. Among them, loofah (block) and loofah charcoal are used clinically more, but there is no quality standard at present, and there is no quality standard for loofah. There are few reports on the difference between the two processed products (block) and loofah charcoal. Since the samples of different dosage forms of loofah charcoal are made of loofah charcoal medicinal materials through a series of processes, some of which will destroy the properties and characteristics of the original medicinal materials, it is necessary to have a good ability to distinguish and identify the existing technology and operate Easier and faster identification methods for better quality control.

康阿龙,汤迎爽,孙成荣,张娴,张苏蘅;丝瓜络三种不同炮制品饮片的质量标准研究;陕西中医学院学报,2011,7,34(4)公开丝瓜络三种不同炮制品饮片的质量控制方法包括以下步骤,供试品溶液的制备:取丝瓜络炭2g,剪成小块(长不大于0.5cm),加甲醇50mL,加热回流1h,放冷,滤过,滤液蒸干,残渣加水10mL使溶解,用水饱和正丁醇提取两次,每次20mL,合并正丁醇液,蒸干,残渣加甲醇1mL使溶解,作为供试品溶液。展开剂:三氯甲烷-甲醇-水(28:4:1)10℃以下放置过夜的下层溶液。照薄层色谱法(中国药典2005年版一部附录ⅥB)试验,吸取上述供试品溶液和对照药材溶液各10uL,分别点于同一用0.5%羧甲基纤维素钠溶液为粘合剂制成的硅胶G薄层板上展开,取出,晾干,喷以10%硫酸乙醇溶液液,在105℃加热至斑点显色清晰。置紫外光灯(365nm)下检视,供试品色谱中,在与对照药材色谱相应的位置上,都显相同颜色的斑点。测试结果如图1所示,其中1为丝瓜络炭饮片(YP202105-21-10)3uL;2为丝瓜络炭饮片(YP202105-21-10)5uL。薄层色谱结果分离度差,斑点不清晰,色谱信息少。Kang Along, Tang Yingshuang, Sun Chengrong, Zhang Xian, Zhang Suheng; Study on the quality standards of three different processed loofah slices; Journal of Shaanxi University of Traditional Chinese Medicine, 2011, July, 34 (4) published the quality of three different processed loofah slices Control method comprises the following steps, the preparation of need testing solution: get luffa charcoal 2g, cut into fritters (length is not more than 0.5cm), add methanol 50mL, heat and reflux 1h, let cool, filter, filtrate is evaporated to dryness, residue Add 10 mL of water to dissolve, extract twice with water-saturated n-butanol, 20 mL each time, combine the n-butanol solution, evaporate to dryness, add 1 mL of methanol to the residue to dissolve, and use it as the test solution. Developing solvent: trichloromethane-methanol-water (28:4:1) lower layer solution placed overnight below 10°C. Test according to thin-layer chromatography (Appendix VIB of Chinese Pharmacopoeia in 2005), draw each 10uL of the above-mentioned solution for the test and the solution of the reference medicinal material, and place them on the same spot with 0.5% sodium carboxymethylcellulose solution as the binder. Spread out on a thin-layer silica gel G plate, take it out, dry it in the air, spray it with 10% sulfuric acid ethanol solution, and heat it at 105°C until the spots are clearly colored. Check under the ultraviolet lamp (365nm), in the chromatogram of the test product, on the position corresponding to the chromatogram of the contrast medicinal material, the spots of the same color are all shown. The test results are shown in Figure 1, where 1 is loofah charcoal decoction pieces (YP202105-21-10) 3uL; 2 is loofah charcoal decoction pieces (YP202105-21-10) 5uL. The results of TLC showed poor resolution, unclear spots, and little chromatographic information.

因此如何提供一种丝瓜络及其炮制品的薄层色谱鉴别方法,成为目前亟待解决的问题。Therefore how to provide a kind of TLC identification method of luffa and processed product thereof, become the problem demanding prompt solution at present.

发明内容Contents of the invention

针对现有技术的不足,本发明的目的在于提供一种丝瓜络及其炮制品的薄层色谱鉴别方法。本发明针对丝瓜络及其炮制品建立了专属性强的薄层鉴别方法,为其质量标准的建立及后续研究提供实验依据。本发明提供的色谱结果分离度适中,斑点清晰色谱信息丰富、稳定、可靠。Aiming at the deficiencies in the prior art, the object of the present invention is to provide a TLC identification method for loofah and its processed products. The invention establishes a highly specific thin-layer identification method for loofah and its processed products, and provides experimental basis for the establishment of its quality standard and follow-up research. The separation degree of the chromatographic result provided by the invention is moderate, and the spots are clear, and the chromatographic information is rich, stable and reliable.

为达到此发明目的,本发明采用以下技术方案:To achieve this purpose of the invention, the present invention adopts the following technical solutions:

第一方面,本发明提供一种丝瓜络及其炮制品的薄层色谱鉴别方法,所述薄层色谱鉴别方法中采用的展开剂包括石油醚、二氯甲烷、乙酸乙酯和甲酸。In the first aspect, the present invention provides a thin-layer chromatographic identification method for loofah and processed products thereof, wherein the developer used in the thin-layer chromatographic identification method includes petroleum ether, dichloromethane, ethyl acetate and formic acid.

在本发明中,所述丝瓜络的炮制品包括丝瓜络块、炒丝瓜络或丝瓜络炭中的任意一种或至少两种的组合。In the present invention, the processed product of loofah includes any one or a combination of at least two of loofah block, fried loofah or charcoal of loofah.

在本发明中,所述石油醚、二氯甲烷、乙酸乙酯和甲酸的体积比为(1.3-1.7):(0.8-1.2):(1.8-2.2):(0.08-0.12);In the present invention, the volume ratio of the 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);

其中,“1.3-1.7”可以是1.3、1.4、1.5、1.6、1.7等;Among them, "1.3-1.7" can be 1.3, 1.4, 1.5, 1.6, 1.7, etc.;

“0.8-1.2”可以是0.8、0.9、1、1.1、1.2等;"0.8-1.2" can be 0.8, 0.9, 1, 1.1, 1.2, etc.;

“1.8-2.2”可以是1.8、1.9、2、2.1、2.2等;"1.8-2.2" can be 1.8, 1.9, 2, 2.1, 2.2, etc.;

“0.08-0.12”可以是0.08、0.09、0.1、0.11、0.12等。"0.08-0.12" can be 0.08, 0.09, 0.1, 0.11, 0.12, etc.

在本发明中,所述石油醚的沸点为60-90℃(例如可以是60℃、65℃、70℃、75℃、80℃、85℃、90℃等)。In the present invention, the boiling point of the petroleum ether is 60-90°C (for example, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, etc.).

在本发明中,所述薄层色谱鉴别方法具体包括以下步骤:In the present invention, the thin layer chromatography identification method specifically includes the following steps:

将供试品溶液点于硅胶薄层板上,采用展开剂进行展开,硅胶薄层板晾干后,先喷以硫酸乙醇溶液,晾干,然后再喷以香草醛溶液,加热,得到供试品中各组分在薄层板中的位置。Spot the solution of the test product on the silica gel thin-layer plate, and develop it with a developing agent. After the silica gel thin-layer plate is dried, spray the sulfuric acid ethanol solution, dry it, and then spray the vanillin solution and heat to obtain the test product. The position of each component in the product in the thin-layer plate.

在本发明中,所述硫酸乙醇溶液中硫酸的质量含量为8-12%(例如可以是8%、9%、10%、11%、12%等);In the present invention, the mass content of sulfuric acid in the sulfuric acid ethanol solution is 8-12% (such as 8%, 9%, 10%, 11%, 12% etc.);

优选地,所述香草醛硫酸溶液中香草醛的质量含量为3-7%(例如可以是3%、4%、5%、6%、7%等)。Preferably, the mass content of vanillin in the vanillin sulfuric acid solution is 3-7% (for example, it may be 3%, 4%, 5%, 6%, 7%, etc.).

优选地,所述加热的温度为100-110℃(例如可以是100℃、101℃、102℃、103℃、104℃、105℃、106℃、107℃、108℃、109℃、110℃等)。Preferably, the heating temperature is 100-110°C (such as 100°C, 101°C, 102°C, 103°C, 104°C, 105°C, 106°C, 107°C, 108°C, 109°C, 110°C, etc. ).

在本发明中,所述薄层色谱鉴别方法中供试品溶液的配制包括以下步骤:In the present invention, the preparation of need testing solution in the thin layer chromatography identification method comprises the following steps:

(1)取丝瓜烙和/或丝瓜烙炮制品研磨,加水,超声,过滤,收集固体;(1) Grinding loofah roasted and/or loofah roasted processed products, adding water, ultrasonication, filtering, and collecting solids;

(2)将步骤(1)得到的固体采用乙酸乙酯进行萃取,将得到的萃取液进行脱溶,然后采用甲醇进行定容,得到供试品溶液。(2) The solid obtained in step (1) is extracted with ethyl acetate, the obtained extract is subjected to precipitation, and then methanol is used to constant volume to obtain the test solution.

在本发明中,步骤(1)中,所述丝瓜烙和/或丝瓜烙炮制品与水的固液比为0.5:(15-25)g/mL;In the present invention, in step (1), the solid-liquid ratio of the loofah roast and/or loofah roast processed product to water is 0.5: (15-25) g/mL;

其中,“15-25”可以是15、16、17、18、19、20、21、22、23、24、25等。Wherein, "15-25" can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and so on.

优选地,步骤(1)中,所述超声的功率为100-500W(例如可以是100W、150W、200W、250W、300W、350W、400W、450W、500W等),超声的时间为20-40min(例如可以是20min、22min、24min、26min、28min、30min、32min、34min、36min、38min、40min等)。Preferably, in step (1), the power of the ultrasound is 100-500W (such as 100W, 150W, 200W, 250W, 300W, 350W, 400W, 450W, 500W, etc.), and the time of the ultrasound is 20-40min ( For example, it can be 20min, 22min, 24min, 26min, 28min, 30min, 32min, 34min, 36min, 38min, 40min, etc.).

在本发明中,所述丝瓜烙和/或丝瓜烙炮制品与乙酸乙酯的固液比为0.5:(15-25)g/mL;In the present invention, the solid-to-liquid ratio of the loofah roast and/or loofah roasted processed product to ethyl acetate is 0.5: (15-25) g/mL;

其中,“15-25”可以是15、16、17、18、19、20、21、22、23、24、25等。Wherein, "15-25" can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and so on.

优选地,步骤(2)中,所述萃取的次数为1-3次(例如可以是1次、2次、3次等)。Preferably, in step (2), the number of extractions is 1-3 times (for example, it can be 1 time, 2 times, 3 times, etc.).

在本发明中,所述薄层色谱鉴别方法中点样量为5-8μL(例如可以是5μL、6μL、7μL、8μL等)。In the present invention, the sample volume in the TLC identification method is 5-8 μL (for example, 5 μL, 6 μL, 7 μL, 8 μL, etc.).

相对于现有技术,本发明具有以下有益效果:Compared with the prior art, the present invention has the following beneficial effects:

本发明提供的丝瓜络及其炮制品的薄层色谱鉴别方法,具有准确率高、分离效果佳、色斑清晰的特点,能够鉴别出丝瓜络块、丝瓜络炭不同炮制品,其次能为丝瓜络块到丝瓜络炭炮制过程中化学成分的变化提供研究依据,也能对丝瓜络炭药材、饮片、中间体、配方颗粒的定性鉴别,及对整个工艺过程质量进行控制,保证其质量的均一、稳定、可靠。The thin-layer chromatography identification method of loofah and its processed products provided by the invention has the characteristics of high accuracy, good separation effect, and clear stains, and can identify different processed products of loofah block and loofah charcoal, and secondly, loofah It can also provide research basis for the change of chemical composition in the process of processing loofah charcoal from block to loofah charcoal. It can also qualitatively identify loofah charcoal medicinal materials, decoction pieces, intermediates, and formula granules, and control the quality of the entire process to ensure uniform quality. ,Stable and reliable.

附图说明Description of drawings

图1为现有技术中丝瓜络炭饮片的薄层色谱图;其中1为丝瓜络炭饮片(YP202105-21-10)3uL;2为丝瓜络炭饮片(YP202105-21-10)5uL。Fig. 1 is the thin-layer chromatogram of the loofah charcoal decoction pieces in the prior art; wherein 1 is 3uL loofah charcoal decoction pieces (YP202105-21-10); 2 is 5uL loofah charcoal decoction pieces (YP202105-21-10).

图2为丝瓜络炭薄层色谱图,其中,1为阴性对照,2为丝瓜络炭颗粒,3为丝瓜络炭中间体,4为丝瓜络炭饮片,5丝瓜络炭对照药材。Fig. 2 is the thin-layer chromatogram of loofah charcoal, wherein, 1 is negative control, 2 is loofah charcoal particle, 3 is loofah charcoal intermediate, 4 is loofah charcoal decoction piece, 5 loofah charcoal contrast medical material.

图3为3批丝瓜络炭饮片和3批丝瓜络块薄层色谱图,其中,1、2、3为3批丝瓜络炭饮片;4、5、6为3批丝瓜络块。Fig. 3 is 3 batches of loofah charcoal decoction pieces and 3 batches of loofah block thin-layer chromatograms, wherein, 1, 2, 3 are 3 batches of loofah charcoal decoction pieces; 4, 5, 6 are 3 batches of loofah blocks.

图4为采用不同提取方法得到的丝瓜络炭冻干粉薄层色谱图,其中,1、2、3、4、5、6分别对应供试品溶液a、b、c、d、e、f。Fig. 4 is the thin-layer chromatogram of luffa charcoal freeze-dried powder obtained by different extraction methods, wherein, 1, 2, 3, 4, 5, 6 correspond to need testing solution a, b, c, d, e, f respectively .

图5为采用展开剂1得到的丝瓜络炭冻干粉薄层色谱图,其中,1、2、3、4分别对应供试品溶液a、d、e、f。Fig. 5 is the luffa charcoal freeze-dried powder thin-layer chromatogram that adopts developer 1 to obtain, and wherein, 1, 2, 3, 4 correspond to need testing solution a, d, e, f respectively.

图6为采用展开剂2得到的丝瓜络炭冻干粉薄层色谱图,其中,1、2分别对应供试品溶液d、e。Fig. 6 is the luffa charcoal freeze-dried powder thin-layer chromatogram that adopts developing agent 2 to obtain, and wherein, 1,2 correspond to need testing solution d, e respectively.

图7为采用展开剂3得到的丝瓜络炭冻干粉薄层色谱图,其中,1、2、3分别对应供试品溶液d、e、f。Fig. 7 is the thin-layer chromatogram of the luffa charcoal freeze-dried powder that adopts developing agent 3 to obtain, and wherein, 1,2,3 correspond to need testing solution d, e, f respectively.

图8为采用不同点样量的丝瓜络炭标煎冻干粉薄层色谱图,其中,1为空白对照,2、3、4为标煎冻干粉202105-21-03点样量分别为5μL、7μL、10μL;5、6、7为标煎冻干粉202105-21-09点样量分别为5μL、7μL、10μL;8、9、10为标煎冻干粉202105-21-12点样量分别为5μL、7μL、10μL;11、12、13为丝瓜络炭对照饮片点样量分别为1μL、3μL、5μL。Fig. 8 is the thin-layer chromatogram of the luffa charcoal standard decocted freeze-dried powder adopting different sample volumes, wherein, 1 is blank control, 2, 3, 4 is standard decocted freeze-dried powder 202105-21-03 sample volumes are respectively 5μL, 7μL, 10μL; 5, 6, and 7 are the standard fried freeze-dried powder 202105-21-09 The sample volume is 5μL, 7μL, 10μL respectively; 8, 9, 10 are the standard fried freeze-dried powder 202105-21-12 points The sample volumes were 5 μL, 7 μL, and 10 μL; 11, 12, and 13 were loofah charcoal control slices, and the sample volumes were 1 μL, 3 μL, and 5 μL, respectively.

图9为采用银龙提供的硅胶薄层板得到的丝瓜络炭标煎冻干粉薄层色谱图;其中,点从左到右依次为:豆甾醇、丝瓜络炭冻干粉03批、丝瓜络炭冻干粉09批、丝瓜络炭冻干粉11批、丝瓜络对照饮片。Figure 9 is the thin-layer chromatogram of loofah charcoal fried freeze-dried powder obtained by using the silica gel thin-layer plate provided by Yinlong; among them, the points from left to right are: stigmasterol, loofah charcoal freeze-dried powder 03 batches, loofah 09 batches of Luo charcoal freeze-dried powder, 11 batches of loofah charcoal freeze-dried powder, and control slices of loofah.

图10为采用芝罘提供的硅胶薄层板得到的丝瓜络炭标煎冻干粉薄层色谱图;其中,点从左到右依次为:豆甾醇、丝瓜络炭冻干粉03批、丝瓜络炭冻干粉09批、丝瓜络炭冻干粉11批、丝瓜络对照饮片。Figure 10 is the thin-layer chromatogram of loofah charcoal decocted freeze-dried powder obtained by using the silica gel thin-layer plate provided by Zhifu; among them, the points from left to right are: stigmasterol, loofah charcoal freeze-dried powder 03 batches, loofah 09 batches of charcoal freeze-dried powder, 11 batches of loofah charcoal freeze-dried powder, and control slices of loofah.

图11为采用默克提供的硅胶薄层板得到的丝瓜络炭标煎冻干粉薄层色谱图;其中,点从左到右依次为:豆甾醇、丝瓜络炭冻干粉03批、丝瓜络炭冻干粉09批、丝瓜络炭冻干粉11批、丝瓜络对照饮片。Figure 11 is the thin-layer chromatogram of loofah charcoal decocted freeze-dried powder obtained by using the silica gel thin-layer plate provided by Merck; among them, the points from left to right are: stigmasterol, loofah charcoal freeze-dried powder 03 batches, loofah 09 batches of Luo charcoal freeze-dried powder, 11 batches of loofah charcoal freeze-dried powder, and control slices of loofah.

图12为在常温常湿(23℃、46%)得到的丝瓜络炭标煎冻干粉薄层色谱图;其中,点从左到右依次为:豆甾醇、丝瓜络炭冻干粉03批、丝瓜络炭冻干粉09批、丝瓜络炭冻干粉11批、丝瓜络对照饮片。Figure 12 is the thin layer chromatogram of luffa charcoal standard fried freeze-dried powder obtained at normal temperature and humidity (23°C, 46%); wherein, the points are from left to right: stigmasterol, luffa charcoal freeze-dried powder 03 batches , 09 batches of loofah charcoal freeze-dried powder, 11 batches of loofah charcoal freeze-dried powder, loofah control decoction pieces.

图13为在常温高湿(23℃、82%)得到的丝瓜络炭标煎冻干粉薄层色谱图;其中,点从左到右依次为:豆甾醇、丝瓜络炭冻干粉03批、丝瓜络炭冻干粉09批、丝瓜络炭冻干粉11批、丝瓜络对照饮片。Figure 13 is the thin-layer chromatogram of the luffa charcoal standard fried freeze-dried powder obtained at normal temperature and high humidity (23°C, 82%); wherein, the points are from left to right: stigmasterol, luffa charcoal freeze-dried powder 03 batches , 09 batches of loofah charcoal freeze-dried powder, 11 batches of loofah charcoal freeze-dried powder, loofah control decoction pieces.

图14为在常温低湿(23℃、13%)得到的丝瓜络炭标煎冻干粉薄层色谱图;其中,点从左到右依次为:豆甾醇、丝瓜络炭冻干粉03批、丝瓜络炭冻干粉09批、丝瓜络炭冻干粉11批、、丝瓜络对照饮片。Fig. 14 is the thin-layer chromatogram of the luffa charcoal standard fried freeze-dried powder obtained at normal temperature and low humidity (23°C, 13%); wherein, the points from left to right are: stigmasterol, luffa charcoal freeze-dried powder 03 batches, Loofah charcoal freeze-dried powder 09 batches, loofah charcoal freeze-dried powder 11 batches, loofah control decoction pieces.

图15为在低温常湿(4℃、46%)得到的丝瓜络炭标煎冻干粉薄层色谱图;其中,点从左到右依次为:豆甾醇、丝瓜络炭冻干粉03批、丝瓜络炭冻干粉09批、丝瓜络炭冻干粉11批、丝瓜络对照饮片。Fig. 15 is the thin-layer chromatogram of the luffa charcoal standard decocted freeze-dried powder obtained at low temperature and normal humidity (4°C, 46%); wherein, the points are from left to right: stigmasterol, luffa charcoal freeze-dried powder 03 batches , 09 batches of loofah charcoal freeze-dried powder, 11 batches of loofah charcoal freeze-dried powder, loofah control decoction pieces.

图16为丝瓜络炭标煎冻干粉方法学验证色谱图,其中,1为空白;2-16为15批标煎冻干粉批号:202105-21-01~202105-21-15;17为丝瓜络炭对照饮片。Figure 16 is the methodological verification chromatogram of loofah charcoal standard fried freeze-dried powder, wherein, 1 is blank; 2-16 is 15 batches of standard fried freeze-dried powder. Loofah charcoal control drink pieces.

图17为采用不同点样量的丝瓜络炭颗粒薄层色谱图,其中,1为空白对照;2、3、4为丝瓜络炭颗粒KL19022051点样量分别为5μL、7μL、10μL;5、6、7为丝瓜络炭颗粒KL19003391点样量分别为5μL、7μL、10μL;8、9、10为丝瓜络炭颗粒KL19022051点样量分别为5μL、7μL、10μL;11、12、13为丝瓜络炭对照饮片点样量分别为1μL、3μL、5μL;确定最佳点样量为供试品7微升,对照饮片5微升。Figure 17 is the thin-layer chromatogram of luffa charcoal particles with different sample volumes, wherein, 1 is a blank control; 2, 3, and 4 are loofah charcoal particles KL19022051 sample volumes are 5 μL, 7 μL, and 10 μL respectively; 5, 6 , 7 are loofah charcoal particles KL19003391, the sample volumes are 5 μL, 7 μL, and 10 μL; 8, 9, and 10 are loofah charcoal particles KL19022051 sample volumes are 5 μL, 7 μL, and 10 μL; 11, 12, and 13 are loofah charcoal The sampling volumes of the control decoction pieces were 1 μL, 3 μL, and 5 μL respectively; the optimal sampling volume was determined to be 7 μl for the test sample and 5 μl for the control decoction pieces.

图18为丝瓜络炭颗粒方法学验证色谱图;其中,1为空白对照;2为丝瓜络炭颗粒KL19022051;3为丝瓜络炭颗粒KL19003391;4为丝瓜络炭颗粒KL19022051;5为丝瓜络炭对照饮片。Figure 18 is the methodological verification chromatogram of loofah charcoal particles; among them, 1 is blank control; 2 is loofah charcoal particles KL19022051; 3 is loofah charcoal particles KL19003391; 4 is loofah charcoal particles KL19022051; 5 is loofah charcoal control Pieces.

具体实施方式Detailed ways

下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了,所述实施例仅仅是帮助理解本发明,不应视为对本发明的具体限制。The technical solutions of the present invention will be further described below through specific embodiments. It should be clear to those skilled in the art that the embodiments are only for helping to understand the present invention, and should not be regarded as specific limitations on the present invention.

以下实施例中各组分的来源如下所示:The source of each component in the following examples is as follows:

Figure BDA0003916988020000071
Figure BDA0003916988020000071

实施例1Example 1

本实施例提供一种丝瓜络炭的薄层色谱鉴别方法,所述丝瓜络炭的薄层色谱鉴别方法包括以下步骤:The present embodiment provides a kind of TLC identification method of loofah charcoal, the TLC identification method of described loofah charcoal comprises the following steps:

取丝瓜络炭颗粒、丝瓜络炭中间体、丝瓜络炭饮片0.5g,研细,加水20mL,超声(200W)处理30min,放冷,用乙酸乙酯振摇提取2次,每次20mL,合并乙酸乙酯液,将乙酸乙酯蒸干,然后用甲醇定容至1mL,作为供试品溶液。丝瓜络炭对照药材:取丝瓜络炭药材5g加100mL水,加热煮沸30min,旋干,同法制成对照药材溶液。(注:中检院及市面暂无丝瓜络炭对照药材,故先以丝瓜络炭药材作为对照药材)。照薄层色谱法(中国药典2015年版四部通则0502)试验,吸取供试品(丝瓜络炭颗粒、丝瓜络炭中间体、丝瓜络炭对照药材)溶液7μL,丝瓜络炭饮片供试品溶液5μL,分别点于同一硅胶G薄层板上,以石油醚(60-90):二氯甲烷:乙酸乙酯:甲酸(1.5:1:2:0.1)为展开剂,展开,取出,晾干,先喷以10%的硫酸乙醇溶液,晾干,再喷以5%的香草醛硫酸溶液,105℃加热至斑点清晰,日光下检视。丝瓜络炭薄层色谱图如2所示,其中,1为阴性对照,2为丝瓜络炭颗粒,3为丝瓜络炭中间体,4为丝瓜络炭饮片,5丝瓜络炭对照药材。供试品色谱中相应的位置上,显相同颜色的荧光斑点。从图2可以看出:薄层色谱结果分离度适中,斑点清晰,色谱信息丰富,供试品色谱中相应的位置上,显相同颜色的荧光斑点。Take loofah charcoal granules, loofah charcoal intermediate, loofah charcoal decoction pieces 0.5g, grind finely, add 20mL of water, treat with ultrasonic (200W) for 30min, let cool, shake and extract twice with ethyl acetate, 20mL each time, combine Ethyl acetate solution, evaporate the ethyl acetate to dryness, then dilute to 1mL with methanol, as the test solution. Loofah charcoal reference medicinal material: Take 5 g of loofah charcoal medicinal material and add 100 mL of water, heat to boil for 30 minutes, spin dry, and prepare the reference medicinal material solution in the same way. (Note: There is no loofah charcoal reference medicinal material in the National Inspection Institute and the market, so the loofah charcoal medicinal material is used as the reference medicinal material first). According to thin-layer chromatography (Chinese Pharmacopoeia 2015 edition four general rules 0502) test, absorb 7 μ L of the solution of the test product (loofah charcoal particle, loofah charcoal intermediate, loofah charcoal reference medicinal material), and 5 μ L of the test product solution of loofah charcoal decoction pieces , respectively point on the same silica gel G thin-layer plate, using petroleum ether (60-90): dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) as the developer, develop, take out, and dry in the air. First spray with 10% sulfuric acid ethanol solution, dry it, then spray with 5% vanillin sulfuric acid solution, heat at 105°C until the spots are clear, and inspect under sunlight. The thin-layer chromatogram of loofah charcoal is shown in 2, wherein, 1 is negative control, 2 is loofah charcoal particles, 3 is loofah charcoal intermediate, 4 is loofah charcoal decoction pieces, and 5 is loofah charcoal control medicinal material. At the corresponding position in the chromatogram of the test product, fluorescent spots of the same color are displayed. It can be seen from Figure 2 that the thin-layer chromatography results have moderate separation, clear spots, and rich chromatographic information. On the corresponding position in the chromatogram of the test product, there are fluorescent spots of the same color.

实施例2Example 2

本实施例提供一种丝瓜络块的薄层色谱鉴别方法,所述丝瓜络块的薄层色谱鉴别方法包括以下步骤:The present embodiment provides a kind of TLC identification method of loofah block, the TLC identification method of described loofah block comprises the following steps:

分别取丝瓜络块、丝瓜络炭饮片0.5g,研细,加水20mL,超声(400W)处理30min,放冷,用乙酸乙酯振摇提取2次,每次20mL,合并乙酸乙酯液,将乙酸乙酯蒸干,然后用甲醇定容至1mL,作为供试品溶液。照薄层色谱法(中国药典2015年版四部通则0502)试验,吸取取丝瓜络块供试品溶液7μL,丝瓜络炭饮片供试品溶液5μL,分别点于同一硅胶G薄层板上,以石油醚(60~90):二氯甲烷:乙酸乙酯:甲酸(1.5:1:2:0.1)为展开剂,展开,取出,晾干,先喷以10%的硫酸乙醇溶液,晾干,再喷以5%的香草醛硫酸溶液,105℃加热至斑点清晰,日光下检视。供试品色谱中相应的位置上,显相同颜色的荧光斑点。如下图3所示,1、2、3为3批丝瓜络炭饮片;4、5、6为3批丝瓜络块。由图3可知,薄层色谱结果显示丝瓜络炭饮片与丝瓜络块的色谱信息有差异,前者色谱信息较丰富,可能是炮制过程中两者化学成分发生了变化。Take 0.5g loofah block and loofah charcoal decoction slices respectively, grind them finely, add 20mL of water, treat with ultrasonic (400W) for 30min, let it cool, shake and extract twice with ethyl acetate, 20mL each time, combine the ethyl acetate solution, and Ethyl acetate was evaporated to dryness, and then dilute to 1mL with methanol as the test solution. Test according to the thin-layer chromatography (Chinese Pharmacopoeia 2015 edition four general rules 0502), draw loofah block test solution 7 μ L, loofah charcoal slices test solution 5 μ L, point respectively on the same silica gel G thin-layer plate, with petroleum Ether (60~90): dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) as developing agent, develop, take out, dry in the air, first spray with 10% sulfuric acid ethanol solution, dry in the air, then Spray with 5% vanillin sulfuric acid solution, heat at 105°C until the spots are clear, and inspect under sunlight. At the corresponding position in the chromatogram of the test product, fluorescent spots of the same color are displayed. As shown in Figure 3 below, 1, 2, and 3 are 3 batches of loofah charcoal decoction pieces; 4, 5, and 6 are 3 batches of loofah cubes. It can be seen from Figure 3 that the TLC results show that the chromatographic information of the loofah charcoal slices and the loofah block are different, and the chromatographic information of the former is richer, which may be due to changes in the chemical composition of the two during the processing.

由于中药材通过一系列的工艺制成其他剂型,其原有的性状已发生变化,故本发明提供的鉴别方法能够鉴别出丝瓜络块、丝瓜络炭不同炮制品,而且能为丝瓜络块到丝瓜络炭炮制过程中化学成分的变化提供研究依据。本发明提供的色谱结果分离度适中,斑点清晰色谱信息丰富、稳定、可靠。Because Chinese medicinal materials are made into other dosage forms through a series of processes, their original properties have changed, so the identification method provided by the invention can identify different processed products of loofah block and luffa charcoal, and can be used for loofah block to The change of chemical composition in the process of loofah charcoal processing provides a research basis. The separation degree of the chromatographic result provided by the invention is moderate, and the spots are clear, and the chromatographic information is rich, stable and reliable.

实施例3Example 3

供试品溶液的制备考察Preparation and investigation of the test solution

本实施例提供一种丝瓜络炭的薄层色谱鉴别方法,所述丝瓜络炭的薄层色谱鉴别方法包括以下步骤:The present embodiment provides a kind of TLC identification method of loofah charcoal, the TLC identification method of described loofah charcoal comprises the following steps:

供试品溶液的制备:Preparation of the test solution:

(a)取丝瓜络炭冻干粉0.25g,加10mL甲醇,超声30min,过滤,浓缩至1mL即得。(a) Take 0.25 g of loofah charcoal freeze-dried powder, add 10 mL of methanol, ultrasonicate for 30 min, filter, and concentrate to 1 mL.

(b)取丝瓜络炭冻干粉0.25g,加10mL石油醚,超声30min,过滤,蒸干,用甲醇定容至1mL即得。(b) Take 0.25g of loofah charcoal freeze-dried powder, add 10mL of petroleum ether, sonicate for 30min, filter, evaporate to dryness, and dilute to 1mL with methanol.

(c)取丝瓜络炭冻干粉0.25g,加10mL乙酸乙酯,超声30min,过滤,浓缩至1mL即得。(c) Take 0.25 g of loofah charcoal freeze-dried powder, add 10 mL of ethyl acetate, sonicate for 30 min, filter, and concentrate to 1 mL.

(d)取丝瓜络炭冻干粉0.5g,加20mL水,超声30min,过滤,正丁醇萃取2次,每次20mL合并正丁醇层,蒸干,用甲醇定容至1mL即得。(d) Take 0.5g of loofah charcoal freeze-dried powder, add 20mL of water, sonicate for 30min, filter, extract with n-butanol twice, 20mL each time, combine the n-butanol layer, evaporate to dryness, and dilute to 1mL with methanol.

(e)取丝瓜络炭冻干粉0.5g,加20mL水,超声30min,过滤,乙酸乙酯萃取2次,每次20mL,合并乙酸乙酯层,蒸干,用甲醇定容至1mL即得。(e) Take 0.5g of loofah charcoal freeze-dried powder, add 20mL of water, sonicate for 30min, filter, extract with ethyl acetate twice, 20mL each time, combine the ethyl acetate layers, evaporate to dryness, and dilute to 1mL with methanol. .

(f)取丝瓜络炭冻干粉0.5g,加20mL甲醇,超声30min,过滤,蒸干,加20mL水溶解,用乙酸乙酯萃取2次,每次20mL合并乙酸乙酯层,蒸干,用甲醇定容至1mL即得。(f) Take 0.5g of loofah charcoal freeze-dried powder, add 20mL of methanol, ultrasonicate for 30min, filter, evaporate to dryness, add 20mL of water to dissolve, extract twice with ethyl acetate, each 20mL combined ethyl acetate layer, evaporate to dryness, Dilute to 1mL with methanol.

吸取供试品溶液μL,分别点于同一硅胶G薄层板上,以环己烷:乙酸乙酯(1:2)为展开剂,展开,取出,晾干,先喷以10%的硫酸乙醇溶液,晾干,在喷以5%的香草醛硫酸溶液,105℃加热至斑点清晰,日光下检视。Take μL of the test solution, respectively spot on the same silica gel G thin-layer plate, use cyclohexane: ethyl acetate (1:2) as developing agent, develop, take out, dry in the air, spray with 10% ethanol sulfate first The solution was dried in the air, sprayed with 5% vanillin sulfuric acid solution, heated at 105°C until the spots were clear, and inspected under sunlight.

采用不同提取方法得到的丝瓜络炭冻干粉的薄层色谱图如图4所示,其中,1、2、3、4、5、6分别对应供试品溶液a、b、c、d、e、f。根据薄层鉴别结果,得知采用b和c制备的供试品溶液,薄层色谱图效果差。The thin-layer chromatograms of the luffa charcoal freeze-dried powder obtained by different extraction methods are shown in Figure 4, wherein, 1, 2, 3, 4, 5, 6 correspond to need testing solution a, b, c, d, respectively. e, f. According to the TLC identification results, it is learned that the TLC effect of the test solution prepared by b and c is poor.

实施例4Example 4

展开剂考察Developing agent inspection

本实施例提供一种丝瓜络炭的薄层色谱鉴别方法,所述丝瓜络炭的薄层色谱鉴别方法包括以下步骤:The present embodiment provides a kind of TLC identification method of loofah charcoal, the TLC identification method of described loofah charcoal comprises the following steps:

供试品溶液的制备同实施例3。The preparation of need testing solution is with embodiment 3.

展开剂1:正己烷:二氯甲烷:乙酸乙酯(1:1:2);Developing agent 1: n-hexane: dichloromethane: ethyl acetate (1:1:2);

展开剂2:正己烷:二氯甲烷:乙酸乙酯(1:1:2.5);Developing agent 2: n-hexane: dichloromethane: ethyl acetate (1:1:2.5);

展开剂3:石油醚60~90:二氯甲烷:乙酸乙酯:甲酸(1.5:1:2:0.1)。Developing agent 3: petroleum ether 60-90: dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1).

吸取供试品溶液7μL,分别点于同一硅胶G薄层板上,分别采用展开剂1、2、3进行展开,取出,晾干,先喷以10%的硫酸乙醇溶液,晾干,在喷以5%的香草醛硫酸溶液,105℃加热至斑点清晰,日光下检视。Take 7 μL of the test solution, spot it on the same silica gel G thin-layer plate, develop it with developer 1, 2, and 3 respectively, take it out, dry it in the air, spray it with 10% sulfuric acid ethanol solution, dry it in the spray Use 5% vanillin sulfuric acid solution, heat at 105°C until the spots are clear, and inspect under sunlight.

采用展开剂1得到的丝瓜络炭冻干粉薄层色谱图如图5所示,其中,1、2、3、4分别对应供试品溶液a、d、e、f。The thin-layer chromatogram of the luffa charcoal freeze-dried powder obtained by developing agent 1 is shown in Figure 5, wherein, 1, 2, 3, 4 correspond to need testing solution a, d, e, f respectively.

采用展开剂2得到的丝瓜络炭冻干粉薄层色谱图如图6所示,其中,1、2分别对应供试品溶液d、e。The thin-layer chromatogram of the luffa charcoal freeze-dried powder obtained by developing agent 2 is shown in Figure 6, wherein 1 and 2 correspond to the testing solution d and e respectively.

采用展开剂3得到的丝瓜络炭冻干粉薄层色谱图如图7所示,其中,1、2、3分别对应供试品溶液d、e、f。The thin-layer chromatogram of the luffa charcoal freeze-dried powder that adopts developing agent 3 to obtain is shown in Figure 7, and wherein, 1, 2, 3 correspond to need testing solution d, e, f respectively.

通过对比图4-图7可知,展开剂3色谱图结果最佳,故以展开剂3(石油醚60~90:二氯甲烷:乙酸乙酯:甲酸(1.5:1:2:0.1))为丝瓜络炭的展开剂。检视为:先喷以10%硫酸乙醇溶液,晾干,再喷以5%香草醛硫酸溶液,加热至斑点清晰,日光下检视。By comparing Figure 4-Figure 7, it can be seen that the chromatogram results of developer 3 are the best, so developer 3 (petroleum ether 60-90: dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1)) is used as the Spreading agent for loofah charcoal. Inspection method: first spray with 10% sulfuric acid ethanol solution, dry it, then spray with 5% vanillin sulfuric acid solution, heat until the spots are clear, and inspect under sunlight.

实施例5Example 5

不同点样量考察Inspection of different sample volumes

本实施例提供一种丝瓜络炭的薄层色谱鉴别方法,所述丝瓜络炭的薄层色谱鉴别方法包括以下步骤:The present embodiment provides a kind of TLC identification method of loofah charcoal, the TLC identification method of described loofah charcoal comprises the following steps:

取丝瓜络炭标煎冻干粉或丝瓜络炭对照饮片0.5g,研细,加水20mL,超声处理30min,放冷,用乙酸乙酯振摇提取2次,每次20mL,合并乙酸乙酯液,将乙酸乙酯蒸干,然后用甲醇定容至1mL,作为供试品溶液。Take loofah charcoal decoction freeze-dried powder or 0.5g of loofah charcoal control decoction slices, grind finely, add 20mL of water, ultrasonically treat for 30min, let cool, shake and extract twice with ethyl acetate, 20mL each time, combine ethyl acetate solution , Evaporate ethyl acetate to dryness, and then dilute to 1 mL with methanol as the test solution.

吸取丝瓜络炭标煎冻干粉供试品溶液5μL、7μL、10μL,丝瓜络炭对照饮片供试品溶液1μL、3μL、5μL,分别点于同一硅胶G薄层板上,以石油醚(60-90):二氯甲烷:乙酸乙酯:甲酸(1.5:1:2:0.1)为展开剂,展开,取出,晾干,先喷以10%的硫酸乙醇溶液,晾干,再在喷以5%的香草醛硫酸溶液,105℃加热至斑点清晰,日光下检视。Take 5 μL, 7 μL, and 10 μL of the test solution of loofah charcoal decocted and freeze-dried powder, and 1 μL, 3 μL, and 5 μL of the test solution of loofah charcoal control decoction pieces. -90): dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) as developing agent, develop, take out, dry, first spray with 10% sulfuric acid ethanol solution, dry, then spray with 5% vanillin sulfuric acid solution, heated at 105°C until the spots are clear, and inspected under sunlight.

采用不同点样量的丝瓜络炭标煎冻干粉薄层色谱如图8所示,其中,1为空白对照,2、3、4为标煎冻干粉202105-21-03点样量分别为5μL、7μL、10μL;5、6、7为标煎冻干粉202105-21-09点样量分别为5μL、7μL、10μL;8、9、10为标煎冻干粉202105-21-12点样量分别为5μL、7μL、10μL;11、12、13为丝瓜络炭对照饮片点样量分别为1μL、3μL、5μL;确定最佳点样量为供试品7微升,对照饮片5微升。The thin-layer chromatogram of loofah charcoal standard decocted freeze-dried powder with different sample volumes is shown in Figure 8, wherein, 1 is a blank control, and 2, 3, and 4 are standard decocted freeze-dried powders. 5μL, 7μL, 10μL; 5, 6, 7 are the standard decocted freeze-dried powder 202105-21-09 The sampling volume is 5μL, 7μL, 10μL respectively; 8, 9, 10 are the standard fried freeze-dried powder 202105-21-12 The sample volumes were 5 μL, 7 μL, and 10 μL; 11, 12, and 13 were loofah charcoal control slices, and the sample volumes were 1 μL, 3 μL, and 5 μL; the optimal sample volume was determined to be 7 μl for the test sample and 5 μL for the control slices. microliter.

实施例6Example 6

硅胶G薄层板不同厂家考察Inspection of different manufacturers of silica gel G thin-layer board

本实施例提供一种丝瓜络炭的薄层色谱鉴别方法,所述丝瓜络炭的薄层色谱鉴别方法包括以下步骤:The present embodiment provides a kind of TLC identification method of loofah charcoal, the TLC identification method of described loofah charcoal comprises the following steps:

取丝瓜络炭标煎冻干粉(03、09、11批)或丝瓜络炭对照饮片0.5g,研细,加水20mL,超声处理30min,放冷,用乙酸乙酯振摇提取2次,每次20mL,合并乙酸乙酯液,将乙酸乙酯蒸干,然后用甲醇定容至1mL,作为供试品溶液。Take loofah charcoal standard fried freeze-dried powder (03, 09, 11 batches) or loofah charcoal control decoction pieces 0.5g, grind finely, add 20mL of water, ultrasonicate for 30min, let cool, shake and extract with ethyl acetate twice, each 20 mL at a time, combined ethyl acetate solution, evaporated ethyl acetate to dryness, and then dilute to 1 mL with methanol, as the test solution.

吸取丝瓜络炭标煎冻干粉供试品溶液7μL,丝瓜络炭对照饮片供试品溶液3μL,分别点于同一硅胶G薄层板上,以石油醚(60-90):二氯甲烷:乙酸乙酯:甲酸(1.5:1:2:0.1)为展开剂,展开,取出,晾干,先喷以10%的硫酸乙醇溶液,晾干,再在喷以5%的香草醛硫酸溶液,105℃加热至斑点清晰,日光下检视。Absorb loofah charcoal decocted and freeze-dried powder test solution 7 μL, loofah charcoal control decoction pieces test solution 3 μL, respectively, on the same silica gel G thin-layer plate, with petroleum ether (60-90): dichloromethane: Ethyl acetate: formic acid (1.5:1:2:0.1) as developing agent, develop, take out, and dry, first spray with 10% sulfuric acid ethanol solution, dry, then spray with 5% vanillin sulfuric acid solution, Heat at 105°C until the spots are clear, and inspect under sunlight.

图9为采用银龙提供的硅胶薄层板得到的丝瓜络炭标煎冻干粉薄层色谱图,图10为采用芝罘提供的硅胶薄层板得到的丝瓜络炭标煎冻干粉薄层色谱图,图11为采用默克提供的硅胶薄层板得到的丝瓜络炭标煎冻干粉薄层色谱图;其中,点从左到右依次为:豆甾醇、丝瓜络炭冻干粉03批、丝瓜络炭冻干粉09批、丝瓜络炭冻干粉11批、丝瓜络对照饮片。从图9-图11可知,本发明提供的方法适用于各个厂家的硅胶薄层板。Figure 9 is the thin layer chromatogram of loofah charcoal decocted freeze-dried powder obtained by using the silica gel thin layer plate provided by Yinlong, and Figure 10 is the thin layer of loofah charcoal decocted freeze-dried powder obtained by using the silica gel thin layer plate provided by Zhifu Chromatogram, Figure 11 is the thin layer chromatogram of loofah charcoal decocted freeze-dried powder obtained by using the silica gel thin-layer plate provided by Merck; among them, the points from left to right are: stigmasterol, loofah charcoal freeze-dried powder 03 09 batches of loofah charcoal freeze-dried powder, 11 batches of loofah charcoal freeze-dried powder, loofah control decoction pieces. It can be seen from Fig. 9-Fig. 11 that the method provided by the present invention is applicable to the silica gel thin-layer boards of various manufacturers.

实施例7Example 7

不同温湿度考察Investigation of different temperature and humidity

本实施例提供一种丝瓜络炭的薄层色谱鉴别方法,所述丝瓜络炭的薄层色谱鉴别方法包括以下步骤:The present embodiment provides a kind of TLC identification method of loofah charcoal, the TLC identification method of described loofah charcoal comprises the following steps:

取丝瓜络炭标煎冻干粉(03、09、11批)或丝瓜络炭对照饮片0.5g,研细,加水20mL,超声处理30min,放冷,用乙酸乙酯振摇提取2次,每次20mL,合并乙酸乙酯液,将乙酸乙酯蒸干,然后用甲醇定容至1mL,作为供试品溶液。Take loofah charcoal standard fried freeze-dried powder (03, 09, 11 batches) or loofah charcoal control decoction pieces 0.5g, grind finely, add 20mL of water, ultrasonicate for 30min, let cool, shake and extract with ethyl acetate twice, each 20 mL at a time, combined ethyl acetate solution, evaporated ethyl acetate to dryness, and then dilute to 1 mL with methanol, as the test solution.

分别在不同温湿度下进行检测,吸取丝瓜络炭标煎冻干粉供试品溶液7μL,丝瓜络炭对照饮片供试品溶液5μL,分别点于同一硅胶G薄层板上,以石油醚(60-90):二氯甲烷:乙酸乙酯:甲酸(1.5:1:2:0.1)为展开剂,展开,取出,晾干,先喷以10%的硫酸乙醇溶液,晾干,再在喷以5%的香草醛硫酸溶液,105℃加热至斑点清晰,日光下检视。Tested at different temperature and humidity respectively, absorb 7 μL of the test solution of loofah charcoal decoction and freeze-dried powder, and 5 μL of the test solution of loofah charcoal control decoction pieces, respectively spot on the same silica gel G thin-layer plate, and use petroleum ether ( 60-90): dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) as the developer, unfold, take out, and dry, first spray with 10% sulfuric acid ethanol solution, dry, and then spray Use 5% vanillin sulfuric acid solution, heat at 105°C until the spots are clear, and inspect under sunlight.

图12为在常温常湿(23℃、46%)得到的丝瓜络炭标煎冻干粉薄层色谱图,图13为在常温高湿(23℃、82%)得到的丝瓜络炭标煎冻干粉薄层色谱图,图14为在常温低湿(23℃、13%)得到的丝瓜络炭标煎冻干粉薄层色谱图,图15为在低温常湿(4℃、46%)得到的丝瓜络炭标煎冻干薄层色谱图;其中,点从左到右依次为:豆甾醇、丝瓜络炭冻干粉03批、丝瓜络炭冻干粉09批、丝瓜络炭冻干粉11批、丝瓜络对照饮片。从图12-图15可知,本发明提供的方法适用于各种温湿度。Figure 12 is the TLC of the luffa charcoal decocted freeze-dried powder obtained at normal temperature and humidity (23°C, 46%), and Figure 13 is the luffa charcoal decoction obtained at normal temperature and humidity (23°C, 82%) The thin-layer chromatogram of freeze-dried powder, Fig. 14 is the thin-layer chromatogram of fried freeze-dried powder obtained at normal temperature and low humidity (23°C, 13%), and Fig. 15 is at low temperature and low humidity (4°C, 46%) The obtained loofah charcoal standard fried and freeze-dried thin-layer chromatogram; among them, the points from left to right are: stigmasterol, loofah charcoal freeze-dried powder 03 batches, loofah charcoal freeze-dried powder 09 batches, loofah charcoal freeze-dried Powder 11 batches, loofah control decoction pieces. It can be seen from Fig. 12-Fig. 15 that the method provided by the present invention is applicable to various temperatures and humidity.

实施例8Example 8

方法学验证Methodology Validation

本实施例提供一种丝瓜络炭的薄层色谱鉴别方法,所述丝瓜络炭的薄层色谱鉴别方法包括以下步骤:The present embodiment provides a kind of TLC identification method of loofah charcoal, the TLC identification method of described loofah charcoal comprises the following steps:

取丝瓜络炭标煎冻干粉(202105-21-01~202105-21-15)或丝瓜络炭对照饮片0.5g,研细,加水20mL,超声处理30min,放冷,用乙酸乙酯振摇提取2次,每次20mL,合并乙酸乙酯液,将乙酸乙酯蒸干,然后用甲醇定容至1mL,作为供试品溶液。Take loofah charcoal decoction freeze-dried powder (202105-21-01~202105-21-15) or loofah charcoal reference slice 0.5g, grind finely, add 20mL of water, sonicate for 30min, let cool, shake with ethyl acetate Extract 2 times, 20mL each time, combine the ethyl acetate solution, evaporate the ethyl acetate to dryness, and then dilute to 1mL with methanol as the test solution.

常温常湿下(23℃、46%),吸取丝瓜络炭标煎冻干粉供试品溶液7μL,丝瓜络炭对照饮片供试品溶液5μL,分别点于同一硅胶G薄层板上,以石油醚(60-90):二氯甲烷:乙酸乙酯:甲酸(1.5:1:2:0.1)为展开剂,展开,取出,晾干,先喷以10%的硫酸乙醇溶液,晾干,再在喷以5%的香草醛硫酸溶液,105℃加热至斑点清晰,日光下检视。Under normal temperature and humidity (23°C, 46%), absorb 7 μL of the test solution of loofah charcoal decoction and freeze-dried powder, and 5 μL of the test solution of loofah charcoal control decoction pieces, and place them on the same silica gel G thin-layer plate respectively. Petroleum ether (60-90): dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) as the developing agent, develop, take out, and dry in the air, first spray with 10% sulfuric acid ethanol solution, dry in the air, Then spray with 5% vanillin sulfuric acid solution, heat at 105°C until the spots are clear, and inspect under sunlight.

丝瓜络炭标煎冻干粉方法学验证色谱图如图16所示,其中,1为空白;2-16为15批标煎冻干粉批号:202105-21-01~202105-21-15;17为丝瓜络炭对照饮片。The methodological verification chromatogram of loofah charcoal standard fried freeze-dried powder is shown in Figure 16, where 1 is blank; 2-16 is 15 batches of standard fried freeze-dried powder Batch number: 202105-21-01~202105-21-15; 17 is loofah charcoal contrast decoction pieces.

实施例9Example 9

不同点样量考察Inspection of different sample volumes

本实施例提供一种丝瓜络炭的薄层色谱鉴别方法,所述丝瓜络炭的薄层色谱鉴别方法包括以下步骤:The present embodiment provides a kind of TLC identification method of loofah charcoal, the TLC identification method of described loofah charcoal comprises the following steps:

取丝瓜络炭颗粒或丝瓜络炭对照饮片0.5g,研细,加水20mL,超声处理30min,放冷,用乙酸乙酯振摇提取2次,每次20mL,合并乙酸乙酯液,将乙酸乙酯蒸干,然后用甲醇定容至1mL,作为供试品溶液。Take loofah charcoal granules or loofah charcoal control slices 0.5g, grind finely, add 20mL of water, ultrasonically treat for 30min, let cool, shake and extract twice with ethyl acetate, 20mL each time, combine the ethyl acetate solution, and dilute ethyl acetate The ester was evaporated to dryness, and then dilute to 1mL with methanol as the test solution.

吸取丝瓜络炭颗粒供试品溶液5μL、7μL、10μL,丝瓜络炭对照饮片供试品溶液1μL、3μL、5μL,分别点于同一硅胶G薄层板上,以石油醚(60-90):二氯甲烷:乙酸乙酯:甲酸(1.5:1:2:0.1)为展开剂,展开,取出,晾干,先喷以10%的硫酸乙醇溶液,晾干,再在喷以5%的香草醛硫酸溶液,105℃加热至斑点清晰,日光下检视。Take 5 μL, 7 μL, and 10 μL of the test solution of loofah charcoal granules, and 1 μL, 3 μL, and 5 μL of loofah charcoal control decoction pieces, respectively, and place them on the same silica gel G thin-layer plate, with petroleum ether (60-90): Dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) as the developing agent, develop, take out, and dry, first spray with 10% sulfuric acid ethanol solution, dry, and then spray with 5% vanilla Aldehyde sulfuric acid solution, heated at 105°C until the spots are clear, and inspected under sunlight.

采用不同点样量的丝瓜络炭颗粒薄层色谱图如图17所示,其中,1为空白对照,2、3、4为丝瓜络炭颗粒KL19022051点样量分别为5μL、7μL、10μL;5、6、7为丝瓜络炭颗粒KL19003391点样量分别为5μL、7μL、10μL;8、9、10为丝瓜络炭颗粒KL19022051点样量分别为5μL、7μL、10μL;11、12、13为丝瓜络炭对照饮片点样量分别为1μL、3μL、5μL;确定最佳点样量为供试品7微升,对照饮片5微升。The thin-layer chromatograms of loofah charcoal particles with different sample volumes are shown in Figure 17, wherein, 1 is a blank control, 2, 3, and 4 are loofah charcoal particles KL19022051 sample volumes are 5 μL, 7 μL, and 10 μL respectively; 5 , 6, and 7 are loofah charcoal particles KL19003391 with sample volumes of 5 μL, 7 μL, and 10 μL; 8, 9, and 10 are loofah charcoal particles KL19022051 with sample volumes of 5 μL, 7 μL, and 10 μL; 11, 12, and 13 are loofah The sampling volumes of Luotan control decoction pieces were 1 μL, 3 μL, and 5 μL respectively; the optimal sampling volume was determined to be 7 μl for the test product and 5 μl for the control decoction pieces.

实施例10Example 10

方法学验证Methodology Validation

本实施例提供一种丝瓜络炭的薄层色谱鉴别方法,所述丝瓜络炭的薄层色谱鉴别方法包括以下步骤:The present embodiment provides a kind of TLC identification method of loofah charcoal, the TLC identification method of described loofah charcoal comprises the following steps:

取丝瓜络炭颗粒(KL19022051、KL19003391、KL19022051)或丝瓜络炭对照饮片0.5g,研细,加水20mL,超声处理30min,放冷,用乙酸乙酯振摇提取2次,每次20mL,合并乙酸乙酯液,将乙酸乙酯蒸干,然后用甲醇定容至1mL,作为供试品溶液。Take loofah charcoal granules (KL19022051, KL19003391, KL19022051) or loofah charcoal control slices 0.5g, grind finely, add 20mL of water, ultrasonically treat for 30min, let cool, shake and extract twice with ethyl acetate, 20mL each time, combine acetic acid Ethyl solution, evaporate the ethyl acetate to dryness, then dilute to 1mL with methanol, as the test solution.

常温常湿下(23℃、46%),吸取丝瓜络炭颗粒供试品溶液7μL,丝瓜络炭对照饮片供试品溶液5μL,分别点于同一硅胶G薄层板上,以石油醚(60-90):二氯甲烷:乙酸乙酯:甲酸(1.5:1:2:0.1)为展开剂,展开,取出,晾干,先喷以10%的硫酸乙醇溶液,晾干,再在喷以5%的香草醛硫酸溶液,105℃加热至斑点清晰,日光下检视。Under normal temperature and humidity (23°C, 46%), absorb 7 μL of the test solution of loofah charcoal particles and 5 μL of the test solution of loofah charcoal control decoction pieces, respectively spot on the same silica gel G thin-layer plate, and add petroleum ether (60 -90): dichloromethane: ethyl acetate: formic acid (1.5:1:2:0.1) as developing agent, develop, take out, dry, first spray with 10% sulfuric acid ethanol solution, dry, then spray with 5% vanillin sulfuric acid solution, heated at 105°C until the spots are clear, and inspected under sunlight.

丝瓜络炭颗粒方法学验证色谱图如图18所示,其中,1为空白对照;2为丝瓜络炭颗粒KL19022051;3为丝瓜络炭颗粒KL19003391;4为丝瓜络炭颗粒KL19022051;5为丝瓜络炭对照饮片。The method verification chromatogram of loofah charcoal granules is shown in Figure 18, in which, 1 is the blank control; 2 is loofah charcoal granules KL19022051; 3 is loofah charcoal granules KL19003391; 4 is loofah charcoal granules KL19022051; 5 is loofah Charcoal control drink pieces.

申请人声明,本发明通过上述实施例来说明本发明的工艺方法,但本发明并不局限于上述工艺步骤,即不意味着本发明必须依赖上述工艺步骤才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明所选用原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。The applicant declares that the present invention illustrates the process method of the present invention through the above examples, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of the selected raw materials in the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of 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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