技术领域Technical Field
本发明属于纳米材料的制备及应用技术领域，具体涉及柠檬酸系列碳点及其在影响草酸钙晶体结晶方面的应用。The invention belongs to the technical field of preparation and application of nano materials, and specifically relates to citric acid series carbon dots and their application in influencing the crystallization of calcium oxalate crystals.
背景技术Background technique
结石病因发病率高，且治疗后易复发(十年复发率高达50％)，难以防治，已成为威胁人类健康的一种常见多发病。草酸钙是泌尿系结石中常见组分，约占结石病病因的70％。其中，一水草酸钙(COM)、二水草酸钙(COD)和三水草酸钙(COT)是草酸钙晶体的主要形式。在这三种晶型中，COM的热力学稳定性最高，且COM晶体表面带正电荷，与带负电荷的受损伤肾上皮细胞黏附能力最强，致病性也最强；相比之下，表面电荷近中性的COD晶体容易随尿液排出体外；而热稳性较差的COT则在肾结石中较少见。因此除了直接抑制COM结晶之外，将COM转化成COD也有利于降低尿结石形成风险。The cause of stones has a high incidence rate and is prone to recurrence after treatment (the recurrence rate is as high as 50% in ten years). It is difficult to prevent and treat, and has become a common and frequently occurring disease that threatens human health. Calcium oxalate is a common component of urinary stones, accounting for about 70% of the causes of stone disease. Among them, calcium oxalate monohydrate (COM), calcium oxalate dihydrate (COD) and calcium oxalate trihydrate (COT) are the main forms of calcium oxalate crystals. Among these three crystal forms, COM has the highest thermodynamic stability, and the surface of COM crystals is positively charged, with the strongest adhesion ability to negatively charged damaged renal epithelial cells, and the strongest pathogenicity; in contrast, COD crystals with a near-neutral surface charge are easily excreted from the body with urine; while COT with poor thermal stability is less common in kidney stones. Therefore, in addition to directly inhibiting COM crystallization, converting COM into COD is also beneficial to reduce the risk of urinary stone formation.
曾有美国休斯敦大学于2016年在Nature第536卷7617期446–450页发表了一篇关于羟基柠檬酸影响草酸钙晶体结晶的研究，该文献中提到羟基柠檬酸能通过吸附在草酸钙结晶体表面明显抑制其晶体生长，同时还能破坏草酸钙中的离子化学键，从而破坏晶体内部结构的稳定性，使其溶解。此外还有报道采用小分子有机酸及其衍生物在控制草酸钙结晶过程的应用。例如申请号为CN201911304432.0的发明专利公开了一种小分子酸影响草酸钙结晶的应用方法，即通过没食子酸及其衍生物来控制草酸钙的结晶过程。但是以上报道的两种小分子有机酸及其衍生物在控制草酸钙结晶过程中，小分子酸的稳定性差，难以被代谢，生物相容性差，且难以进入细胞，这些缺点限制了其在人体内抑制草酸钙结晶的实际应用。In 2016, the University of Houston in the United States published a study on the effect of hydroxycitric acid on the crystallization of calcium oxalate crystals in Nature, Vol. 536, No. 7617, pp. 446-450. The document mentioned that hydroxycitric acid can significantly inhibit the growth of calcium oxalate crystals by adsorbing on the surface of calcium oxalate crystals, and can also destroy the ionic chemical bonds in calcium oxalate, thereby destroying the stability of the internal structure of the crystals and dissolving them. In addition, there are reports on the use of small molecule organic acids and their derivatives in controlling the crystallization process of calcium oxalate. For example, the invention patent with application number CN201911304432.0 discloses an application method for small molecule acids to affect the crystallization of calcium oxalate, that is, to control the crystallization process of calcium oxalate by gallic acid and its derivatives. However, in the process of controlling the crystallization of calcium oxalate, the two small molecule organic acids and their derivatives reported above have poor stability, are difficult to be metabolized, have poor biocompatibility, and are difficult to enter cells. These shortcomings limit their practical application in inhibiting the crystallization of calcium oxalate in the human body.
碳点是一类尺寸小于10纳米的碳纳米粒子，具有石墨化的碳核和由有机表面基团(如羧基、羟基和羰基等)组成的无定型外壳，这种独特的结构和组成赋予碳点良好的稳定性、水溶性以及生物相容性。同时碳点可以通过合成原料和方法的选择来设计其结构，本发明通过设计合成出了表面富含羟基与羧基官能团的柠檬酸系列碳点，该碳点在保有小分子有机酸(柠檬酸)抑制草酸钙结晶的影响效果同时还具有碳点內禀的高亮度荧光等优良性质。因此，柠檬酸系列碳点在草酸钙结石治疗方面展现出了巨大的应用潜力。Carbon dots are a type of carbon nanoparticles with a size of less than 10 nanometers, with a graphitized carbon core and an amorphous shell composed of organic surface groups (such as carboxyl, hydroxyl and carbonyl groups, etc.). This unique structure and composition give carbon dots good stability, water solubility and biocompatibility. At the same time, the structure of carbon dots can be designed by selecting synthetic raw materials and methods. The present invention synthesized a series of citric acid carbon dots with hydroxyl and carboxyl functional groups on the surface. The carbon dots retain the effect of small molecule organic acids (citric acid) in inhibiting the crystallization of calcium oxalate while also having the excellent properties of carbon dots such as high brightness fluorescence. Therefore, the citric acid series of carbon dots show great application potential in the treatment of calcium oxalate stones.
发明内容Summary of the invention
针对上述现有技术存在的问题，本发明提供一种具有水溶性好、稳定性好、有良好的生物相容性、水溶液pH值偏中性的柠檬酸系列碳点，及其在影响草酸钙晶体结晶方面的应用也即在钙结晶抑制方面的应用，使得其可期望用于钙结石的预防和治疗。In view of the problems existing in the above-mentioned prior art, the present invention provides a citric acid series carbon dots with good water solubility, good stability, good biocompatibility, and a neutral pH value of the aqueous solution, and its application in affecting the crystallization of calcium oxalate crystals, that is, in inhibiting calcium crystallization, so that it can be expected to be used for the prevention and treatment of calcium stones.
为了实现上述目的，本发明提供了一种稳定性好的柠檬酸系列碳点，该类碳点可以提高草酸钙晶体的亲水性，抑制草酸钙晶体的生长，促进一水草酸钙向二水草酸钙转化；同时该类碳点表面具有丰富的羧基和羟基官能团，可与游离的钙离子结合生成可溶性配合物，降低尿液中草酸钙晶体的过饱和度。In order to achieve the above-mentioned purpose, the present invention provides a citric acid series carbon dots with good stability, which can improve the hydrophilicity of calcium oxalate crystals, inhibit the growth of calcium oxalate crystals, and promote the conversion of calcium oxalate monohydrate to calcium oxalate dihydrate; at the same time, the surface of this type of carbon dots has abundant carboxyl and hydroxyl functional groups, which can combine with free calcium ions to form soluble complexes, thereby reducing the supersaturation of calcium oxalate crystals in urine.
本发明所述的柠檬酸系列碳点包括羟基柠檬酸碳点，柠檬酸钾碳点，柠檬酸碳点等等，其稳定性好、有良好的生物相容性、水溶液pH值偏中性，其表面含有丰富的羧基和羟基官能团。The citric acid series carbon dots of the present invention include hydroxycitric acid carbon dots, potassium citrate carbon dots, citric acid carbon dots, etc., which have good stability, good biocompatibility, neutral pH value of aqueous solution, and rich carboxyl and hydroxyl functional groups on the surface.
本发明柠檬酸系列碳点在制备钙结晶抑制剂中的应用，所述柠檬酸系列碳点为柠檬酸系列物质经化学合成得到的碳点，所述柠檬酸系列物质为柠檬酸、羟基柠檬酸及其盐中的至少一种，或者为含有柠檬酸、羟基柠檬酸或其盐的物质中的至少一种。The invention discloses an application of the citric acid series carbon dots in the preparation of a calcium crystallization inhibitor. The citric acid series carbon dots are carbon dots obtained by chemical synthesis of citric acid series substances. The citric acid series substances are at least one of citric acid, hydroxycitric acid and their salts, or at least one of substances containing citric acid, hydroxycitric acid or their salts.
优选的，所述钙结晶可以为草酸钙、磷酸钙、碳酸钙及其水合物中的至少一种，所述水合物包括但不限于单水合物、多水合物中的至少一种。Preferably, the calcium crystals may be at least one of calcium oxalate, calcium phosphate, calcium carbonate and hydrates thereof, and the hydrates include but are not limited to at least one of monohydrate and polyhydrate.
优选的，所述钙结晶即为钙结石包括但不限于尿结石、肾结石、胆结石、牙结石、膀胱结石中的至少一种。Preferably, the calcium crystals are calcium stones including but not limited to at least one of urinary stones, kidney stones, gallstones, dental stones, and bladder stones.
优选的，所述钙结晶抑制剂可以为柠檬酸系列碳点或者为含有柠檬酸系列碳点的物质，所述含有柠檬酸系列碳点的物质可以为某些天然产物比如含有羟基柠檬酸的物质可以为藤黄果粉(含约90％羟基柠檬酸)；当然其也可以为一些人工合成的物质或者为一些人工复配的混合物；进一步的，可通过加入药物制剂所常用的一些辅料形成方便使用的药物或制剂，从而使得所述含有柠檬酸系列碳点的物质成为了一种含有柠檬酸系列碳点的药物或制剂。当然，基于本发明的柠檬酸系列碳点通过药学基本手段可制备得到药学上可接受的各种类型的药物制剂比如口服制剂、注射制剂等等。Preferably, the calcium crystallization inhibitor can be a citric acid series carbon dot or a substance containing a citric acid series carbon dot. The substance containing a citric acid series carbon dot can be some natural products, such as a substance containing hydroxycitric acid, which can be garcinia cambogia powder (containing about 90% hydroxycitric acid); of course, it can also be some artificially synthesized substances or some artificially compounded mixtures; further, it can be formed into a convenient-to-use medicine or preparation by adding some excipients commonly used in pharmaceutical preparations, so that the substance containing a citric acid series carbon dot becomes a medicine or preparation containing a citric acid series carbon dot. Of course, various types of pharmaceutically acceptable pharmaceutical preparations such as oral preparations, injection preparations, etc. can be prepared based on the citric acid series carbon dots of the present invention by basic pharmaceutical means.
本发明所述钙结晶抑制主要是通过本发明的柠檬酸系列碳点促进钙结石成分如草酸钙从一水草酸钙向二水草酸钙甚或向三水草酸钙的转化，也即抑制钙形成热稳定性高的一水草酸钙，促进其形成容易随尿液排出的二水草酸钙，从而可用于预防草酸钙结石的形成。The calcium crystallization inhibition described in the present invention is mainly achieved by promoting the conversion of calcium stone components such as calcium oxalate from calcium oxalate monohydrate to calcium oxalate dihydrate or even to calcium oxalate trihydrate through the citric acid series carbon dots of the present invention, that is, inhibiting calcium from forming calcium oxalate monohydrate with high thermal stability, and promoting it to form calcium oxalate dihydrate that is easily excreted in the urine, thereby being used to prevent the formation of calcium oxalate stones.
本发明中，所述柠檬酸系列碳点可采用下述方法制备得到：将柠檬酸系列物质作为反应原料通过高温水热法或微波法反应，然后加水溶解抽滤并透析，再冷冻干燥完成制备；所述反应前驱体中加入或者不加入尿素。碳点合成中，尿素的添加有利于部分碳点结构比如柠檬酸及其盐形成的碳点结构的稳定，既可以提高其合成产率，也改善了其光学性质；而对于某些碳点比如羟基柠檬酸及其盐所形成的碳点则可不添加尿素，是因为该碳点本身就有一个较为稳定的结构，同时拥有较高的合成产率与较为优秀的光学性质，另一方面对于羟基柠檬酸该类碳点也是出于为了使其功能部分更加纯粹的考虑。具体的：In the present invention, the citric acid series carbon dots can be prepared by the following method: using the citric acid series substances as the reaction raw materials to react through a high-temperature hydrothermal method or a microwave method, then adding water to dissolve, filtration and dialysis, and then freeze-drying to complete the preparation; urea is added or not added to the reaction precursor. In the synthesis of carbon dots, the addition of urea is beneficial to the stability of some carbon dot structures, such as the carbon dot structures formed by citric acid and its salts, which can not only improve its synthesis yield, but also improve its optical properties; for some carbon dots, such as the carbon dots formed by hydroxycitric acid and its salts, urea may not be added, because the carbon dots themselves have a relatively stable structure, and at the same time have a higher synthesis yield and better optical properties. On the other hand, for hydroxycitric acid, this type of carbon dots is also out of consideration to make its functional part more pure. Specifically:
本发明中，以含有羟基柠檬酸的物质藤黄果粉制备碳点的方法可以是：In the present invention, the method for preparing carbon dots using Garcinia cambogia powder containing hydroxycitric acid can be:
称取一定量藤黄果粉(含约90％羟基柠檬酸)，转移到小坩埚中，后将其放入马弗炉中，在一定时间下进行热解反应，待反应结束后取出小坩埚中的热解样品，将其转移到小烧杯中；加入适量去离子水到小烧杯中，超声搅拌使其溶解充分，之后静置一段时间；将上述溶液抽滤，收集滤液，装入一定截留分子量的透析袋，在超纯水中透析，透析后的溶液经冷冻干燥后得到深黄色的碳点；优选马弗炉反应温度为160-220℃，反应时间为6-10小时，更优选200℃反应8小时；抽滤时采用孔径为0.22的水性微孔滤膜，透析袋截留分子量为500-2000道尔顿，优选截留分子量为800-1000道尔顿，最优选截留分子量为1000道尔顿，透析时间为72-120小时，冷冻干燥时间为48-72小时。A certain amount of garcinia cambogia powder (containing about 90% hydroxycitric acid) is weighed and transferred to a small crucible, which is then placed in a muffle furnace for a certain period of time for pyrolysis reaction. After the reaction is completed, the pyrolysis sample in the small crucible is taken out and transferred to a small beaker; an appropriate amount of deionized water is added to the small beaker, ultrasonically stirred to fully dissolve it, and then allowed to stand for a period of time; the above solution is filtered, the filtrate is collected, and the filtrate is loaded into a dialysis bag with a certain molecular weight cutoff, dialyzed in ultrapure water, and the dialyzed solution is freeze-dried to obtain dark yellow carbon dots; preferably, the muffle furnace reaction temperature is 160-220°C, the reaction time is 6-10 hours, and more preferably 200°C for 8 hours; when filtration, a water-based microporous filter membrane with a pore size of 0.22 is used, the dialysis bag molecular weight cutoff is 500-2000 Daltons, preferably the molecular weight cutoff is 800-1000 Daltons, and the most preferably the molecular weight cutoff is 1000 Daltons, the dialysis time is 72-120 hours, and the freeze-drying time is 48-72 hours.
本发明中，以柠檬酸钾制备碳点的方法可以是：In the present invention, the method for preparing carbon dots with potassium citrate can be:
称取柠檬酸钾和尿素，加至超纯水中，超声并且搅拌加速溶解完全，将溶液转移至反应釜中，后将其放入马弗炉，在一定时间下进行水热反应，待反应结束后取出反应釜并将溶液转移至小烧杯中，超声搅拌使其混合充分；将上述溶液抽滤，收集滤液，装入一定截留分子量的透析袋，在超纯水中透析，透析后的溶液经冷冻干燥后得到褐色的碳点；优选加入1-2克柠檬酸钾、1-1.5克尿素和20-40毫升超纯水，马弗炉反应温度为160-220℃，反应时间为6-10小时，更优选为160℃反应8小时；抽滤时采用孔径为0.22的水性微孔滤膜，透析袋截留分子量为500-2000道尔顿，优选截留分子量为800-1000道尔顿，最优选截留分子量为1000道尔顿，透析时间为72-120小时，冷冻干燥时间为48-72小时。Weigh potassium citrate and urea, add them to ultrapure water, ultrasonicate and stir to accelerate dissolution, transfer the solution to a reactor, then put it into a muffle furnace, perform hydrothermal reaction for a certain period of time, take out the reactor after the reaction is completed and transfer the solution to a small beaker, ultrasonicate and stir to mix it thoroughly; filter the above solution, collect the filtrate, put it into a dialysis bag with a certain molecular weight cutoff, dialyze it in ultrapure water, and freeze-dry the dialyzed solution to obtain brown carbon dots; preferably, 1-2 g potassium citrate, 1- 1.5 g urea and 20-40 ml ultrapure water, the muffle furnace reaction temperature is 160-220 ° C, the reaction time is 6-10 hours, more preferably 160 ° C reaction for 8 hours; the filtration is performed using an aqueous microporous filter membrane with a pore size of 0.22, the dialysis bag has a molecular weight cutoff of 500-2000 Daltons, preferably a molecular weight cutoff of 800-1000 Daltons, and most preferably a molecular weight cutoff of 1000 Daltons, the dialysis time is 72-120 hours, and the freeze-drying time is 48-72 hours.
本发明中，以柠檬酸制备碳点的方法可以是：In the present invention, the method for preparing carbon dots with citric acid can be:
称取柠檬酸和尿素，加至超纯水中，超声并且搅拌加速溶解完全，将溶液转移至小烧杯中，后将其放入微波炉，在一定时间下进行微波反应，待反应结束后取出小烧杯；加入适量去离子水到小烧杯中，超声搅拌使其溶解充分；将上述溶液抽滤，收集滤液，装入一定截留分子量的透析袋，在超纯水中透析，透析后的溶液经冷冻干燥后得到深褐色的碳点；优选加入4-5克无水柠檬酸、1-1.5克尿素和5-20毫升超纯水，微波炉反应功率为中高火(400-500瓦)，反应时间为4-8分钟；抽滤时采用孔径为0.22的水性微孔滤膜，透析袋截留分子量为500-2000道尔顿，优选截留分子量为800-1000道尔顿，最优选截留分子量为1000道尔顿，透析时间为72-120小时，冷冻干燥时间为48-72小时。Weigh citric acid and urea, add them to ultrapure water, ultrasonically and stir to accelerate dissolution, transfer the solution to a small beaker, put it into a microwave oven, perform microwave reaction for a certain period of time, and take out the small beaker after the reaction is completed; add an appropriate amount of deionized water to the small beaker, ultrasonically stir to make it fully dissolved; filter the above solution, collect the filtrate, put it into a dialysis bag with a certain molecular weight cutoff, dialyze it in ultrapure water, and freeze-dry the dialyzed solution to obtain dark brown carbon dots; preferably add 4-5 grams of free The invention discloses a method for preparing the method of preparing the precipitate of citric acid, 1-1.5 g urea and 5-20 ml ultrapure water, wherein the microwave oven is used for medium-high power (400-500 watts) and the reaction time is 4-8 minutes; the filtration is performed using an aqueous microporous filter membrane with a pore size of 0.22, a dialysis bag with a molecular weight cutoff of 500-2000 Daltons, preferably a molecular weight cutoff of 800-1000 Daltons, and most preferably a molecular weight cutoff of 1000 Daltons, a dialysis time of 72-120 hours, and a freeze-drying time of 48-72 hours.
本发明的上述制备方法中，添加尿素可作为氮掺杂辅助形成碳点以改善碳点性能。In the above preparation method of the present invention, the addition of urea can be used as nitrogen doping to assist in the formation of carbon dots to improve the performance of the carbon dots.
本发明中柠檬酸系列碳点与常用的柠檬酸在与钙结合方面具有较大区别，具体的：虽然柠檬酸可以直接与钙形成柠檬酸钙络合物，这种物质虽然也不容易解离，但是它可以溶于水，不像草酸钙不溶于水会形成沉淀。但因为柠檬酸从肠道吸收入体内，或者通过直接注射的方法进入体内后都会被肝脏分解成碳酸......能以原型经肾排出的很少。而本发明的柠檬酸系列碳点表面丰富的-COOH可以与钙离子配位，柠檬酸系列碳点周围的钙离子浓度快速增加，在局域部分增大了钙离子与草酸根离子的浓度比值；此外，由于柠檬酸系列碳点表面富集了带正电荷的钙离子，形成一个高能界面，同时被吸附的钙离子自由度降低，能态增加，这种高能界面及高能量环境，均会促进二水草酸钙(COD)的形成。COD晶体与肾细胞膜表面的黏附力远低于一水草酸钙(COM)，相对容易随尿液排出体外。同时柠檬酸系列拥有良好的稳定性不易被分解或破坏，其作用完成之后会以肾清除的形式排出体外，此时还可以通过碳点的荧光性质来研究其代谢情况。The citric acid series carbon dots in the present invention are quite different from the commonly used citric acid in terms of binding with calcium. Specifically: although citric acid can directly form a calcium citrate complex with calcium, this substance is not easy to dissociate, but it can be dissolved in water, unlike calcium oxalate, which is insoluble in water and will form a precipitate. However, because citric acid is absorbed into the body from the intestine, or is decomposed into carbonic acid by the liver after entering the body by direct injection... very little can be excreted through the kidney in its original form. The abundant -COOH on the surface of the citric acid series carbon dots of the present invention can coordinate with calcium ions, and the calcium ion concentration around the citric acid series carbon dots increases rapidly, increasing the concentration ratio of calcium ions to oxalate ions in the local part; in addition, because the surface of the citric acid series carbon dots is enriched with positively charged calcium ions, a high-energy interface is formed, and the degree of freedom of the adsorbed calcium ions is reduced, and the energy state is increased. This high-energy interface and high-energy environment will promote the formation of calcium oxalate dihydrate (COD). The adhesion of COD crystals to the surface of the renal cell membrane is much lower than that of calcium oxalate monohydrate (COM), and it is relatively easy to be excreted from the body with urine. At the same time, the citric acid series has good stability and is not easily decomposed or destroyed. After its function is completed, it will be excreted from the body in the form of renal clearance. At this time, its metabolism can also be studied through the fluorescence properties of carbon dots.
本发明的柠檬酸系列碳点可与游离的钙离子结合生成可溶性配合物，降低尿液中草酸钙的过饱和度，还能与草酸钙晶体的生长位点结合，抑制晶体的生长。柠檬酸系列碳点含-COOH、-OH等酸性基团，且细胞毒性小，稳定性好。因此，柠檬酸系列碳点可用于预防草酸钙结石的形成。The citric acid series carbon dots of the present invention can combine with free calcium ions to form soluble complexes, reduce the supersaturation of calcium oxalate in urine, and can also combine with the growth sites of calcium oxalate crystals to inhibit the growth of crystals. The citric acid series carbon dots contain acidic groups such as -COOH and -OH, and have low cytotoxicity and good stability. Therefore, the citric acid series carbon dots can be used to prevent the formation of calcium oxalate stones.
与现有技术相比，本发明的有益效果是：Compared with the prior art, the present invention has the following beneficial effects:
1)本发明使用的柠檬酸系列碳点水溶性好、稳定性好、生物毒性低、肾清除，可以取代稳定性差、生物毒性较高、难以代谢的常见有机小分子酸来调控草酸钙结石的结晶过程；1) The citric acid series carbon dots used in the present invention have good water solubility, good stability, low biological toxicity, and renal clearance, and can replace common organic small molecule acids with poor stability, high biological toxicity, and difficulty in metabolism to regulate the crystallization process of calcium oxalate stones;
2)本发明制备的柠檬酸系列碳点具有一定的荧光性质，基于碳点的肾清除机制可以通过检测排出液中是否出现荧光来研究其代谢情况，同时该性质也有利于一些荧光检测的进行。2) The citric acid series carbon dots prepared in the present invention have certain fluorescence properties. Based on the renal clearance mechanism of carbon dots, their metabolism can be studied by detecting whether fluorescence appears in the excretion fluid. At the same time, this property is also conducive to the implementation of some fluorescence detection.
3)本发明制备的柠檬酸系列碳点可以促进一水草酸钙向更容易排出体外的二水草酸钙转变，同时可以降低草酸钙晶体的平均晶粒度；柠檬酸系列碳点的加入还提高了草酸钙晶体的亲水性，改善了其在水中的溶解性；该类碳点在钙结石治疗方面展现出了巨大的应用潜力。3) The citric acid series carbon dots prepared by the present invention can promote the transformation of calcium oxalate monohydrate into calcium oxalate dihydrate which is easier to be excreted from the body, and at the same time can reduce the average crystal size of calcium oxalate crystals; the addition of citric acid series carbon dots also increases the hydrophilicity of calcium oxalate crystals and improves their solubility in water; this type of carbon dots shows great application potential in the treatment of calcium stones.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为一水草酸钙晶体和在羟基柠檬酸碳点影响下转化的二水草酸钙晶体的X射线衍射谱图；FIG1 is an X-ray diffraction spectrum of calcium oxalate monohydrate crystals and calcium oxalate dihydrate crystals transformed under the influence of hydroxycitrate carbon dots;
图2为一水草酸钙晶体和在羟基柠檬酸碳点影响下转化的二水草酸钙晶体的扫描电镜图；FIG2 is a scanning electron microscope image of calcium oxalate monohydrate crystals and calcium oxalate dihydrate crystals transformed under the influence of hydroxycitrate carbon dots;
图3为一水草酸钙晶体和在羟基柠檬酸碳点影响下转化的二水草酸钙晶体的红外光谱图；FIG3 is an infrared spectrum of calcium oxalate monohydrate crystals and calcium oxalate dihydrate crystals transformed under the influence of hydroxycitrate carbon dots;
图4为一水草酸钙晶体和在柠檬酸碳点影响下的一水草酸钙晶体的X射线衍射谱图。FIG. 4 is an X-ray diffraction spectrum of calcium oxalate monohydrate crystals and calcium oxalate monohydrate crystals under the influence of citric acid carbon dots.
具体实施方式Detailed ways
下述实施例是对于本发明内容的进一步说明以作为对本发明技术内容的阐释，但本发明的实质内容并不仅限于下述实施例所述，本领域的普通技术人员可以且应当知晓任何基于本发明实质精神的简单变化或替换均应属于本发明所要求的保护范围。The following embodiments are further descriptions of the content of the present invention as an explanation of the technical content of the present invention, but the essential content of the present invention is not limited to the following embodiments. Ordinary technicians in this field can and should know that any simple changes or replacements based on the essential spirit of the present invention should fall within the scope of protection required by the present invention.
实施例1Example 1
羟基柠檬酸碳点影响草酸钙水溶液结晶的应用，具体包括以下步骤：The application of hydroxycitric acid carbon dots to influence the crystallization of calcium oxalate aqueous solution specifically comprises the following steps:
1)称取3克藤黄果粉，研磨充分之后，将其转移到小坩埚当中；1) Weigh 3 grams of Garcinia Cambogia powder, grind it thoroughly, and transfer it to a small crucible;
2)将小坩埚放入到马弗炉当中，在200摄氏度下，反应8小时，使反应物充分反应，待其冷却至室温后取出小烧杯中的焦黄色固体；加入适量去离子水加入小烧杯中，超声搅拌使其溶解充分。2) Place the small crucible into a muffle furnace and react at 200 degrees Celsius for 8 hours to allow the reactants to react fully. After cooling to room temperature, take out the brown solid in the small beaker; add an appropriate amount of deionized water into the small beaker and stir ultrasonically to fully dissolve it.
3)将上述得到的溶液用孔径为0.22微米的有机微孔滤膜真空抽滤，收集滤液，装入截留分子量为1000道尔顿的透析袋，在超纯水中透析72小时，冻干48-72小时得到褐色的碳点。3) The solution obtained above was vacuum filtered through an organic microporous filter membrane with a pore size of 0.22 μm, the filtrate was collected, loaded into a dialysis bag with a molecular weight cutoff of 1000 Daltons, dialyzed in ultrapure water for 72 hours, and freeze-dried for 48-72 hours to obtain brown carbon dots.
4)称取10毫克的羟基柠檬酸碳点将其溶于8毫升去离子水配成羟基柠檬酸碳点溶液，并将其滴加入40毫升A液(22毫摩尔每升的氯化钙溶液)，超声搅拌使其混合均匀；另量取40毫升的B液(22毫摩尔每升的草酸钠溶液)放置在一旁备用；4) Weigh 10 mg of hydroxycitric acid carbon dots and dissolve it in 8 ml of deionized water to prepare a hydroxycitric acid carbon dot solution, and then drop it into 40 ml of solution A (22 mmol/L calcium chloride solution), and stir it by ultrasonic to mix it evenly; measure 40 ml of solution B (22 mmol/L sodium oxalate solution) and set it aside for use;
5)将混合后的A液置于37摄氏度的水浴锅中，将B液缓加入A液中，反应10分钟后获得草酸钙沉淀悬浊液，之后将其静置2小时使其沉淀完全；5) Place the mixed solution A in a water bath at 37 degrees Celsius, slowly add solution B into solution A, react for 10 minutes to obtain a calcium oxalate precipitate suspension, and then let it stand for 2 hours to allow complete precipitation;
6)将上述悬浊液倒入100毫升离心管中，以8000转每分钟离心10分钟；离心完成后倒去离心管中的清液，将沉淀转移至表面皿中，冻干后得到羟基柠檬酸碳点影响后的草酸钙晶体。6) Pour the above suspension into a 100 ml centrifuge tube and centrifuge at 8000 rpm for 10 minutes; after centrifugation, pour out the clear liquid in the centrifuge tube, transfer the precipitate to a watch glass, and freeze-dry to obtain calcium oxalate crystals affected by hydroxycitrate carbon dots.
实施例2Example 2
柠檬酸碳点影响草酸钙水溶液结晶的应用，具体包括以下步骤：The application of citric acid carbon dots to influence the crystallization of calcium oxalate aqueous solution specifically comprises the following steps:
1)称取4.5克的柠檬酸和1.5克尿素，加入10毫升的超纯水中，超声并且搅拌加速溶解完全；1) Weigh 4.5 g of citric acid and 1.5 g of urea, add to 10 ml of ultrapure water, and sonicate and stir to accelerate dissolution;
2)将溶液转移至50毫升小烧杯中，放入微波炉，在中高火(400-500瓦)下，保温反应4分钟，使反应物充分反应，待其冷却至室温后取出小烧杯中的焦褐色固体；加入适量去离子水加入小烧杯中，超声搅拌使其溶解充分。2) Transfer the solution to a 50 ml beaker and place it in a microwave oven at medium-high heat (400-500 watts) for 4 minutes to allow the reactants to react fully. After cooling to room temperature, remove the burnt brown solid in the beaker; add an appropriate amount of deionized water to the beaker and stir ultrasonically to fully dissolve it.
3)将上述得到的溶液用孔径为0.22微米的有机微孔滤膜真空抽滤，收集滤液，装入截留分子量为1000道尔顿的透析袋，在超纯水中透析72小时，冻干48-72小时得到深褐色的碳点。3) The solution obtained above was vacuum filtered through an organic microporous filter membrane with a pore size of 0.22 μm, the filtrate was collected and placed in a dialysis bag with a molecular weight cutoff of 1000 Daltons, dialyzed in ultrapure water for 72 hours, and freeze-dried for 48-72 hours to obtain dark brown carbon dots.
4)称取10毫克的柠檬酸碳点将其溶于8毫升去离子水配成柠檬酸碳点溶液，并将其滴加入40毫升A液(22毫摩尔每升的氯化钙溶液)，超声搅拌使其混合均匀；另量取40毫升的B液(22毫摩尔每升的草酸钠溶液)放置在一旁备用；4) Weigh 10 mg of citric acid carbon dots and dissolve it in 8 ml of deionized water to prepare a citric acid carbon dots solution, and then dropwise add the solution into 40 ml of solution A (22 mmol/L calcium chloride solution), and stir the mixture by ultrasonication to make it uniform; measure 40 ml of solution B (22 mmol/L sodium oxalate solution) and set aside for use;
5)将混合后的A液置于37摄氏度的水浴锅中，将B液缓加入A液中，反应10分钟后获得草酸钙沉淀悬浊液，之后将其静置2小时使其沉淀完全；5) Place the mixed solution A in a water bath at 37 degrees Celsius, slowly add solution B into solution A, react for 10 minutes to obtain a calcium oxalate precipitate suspension, and then let it stand for 2 hours to allow complete precipitation;
6)将上述悬浊液倒入100毫升离心管中，以8000转每分钟离心10分钟；离心完成后倒去离心管中的清液，将沉淀转移至表面皿中，冻干后得到柠檬酸碳点影响后的草酸钙晶体。6) Pour the above suspension into a 100 ml centrifuge tube and centrifuge at 8000 rpm for 10 minutes; after centrifugation, pour out the clear liquid in the centrifuge tube, transfer the precipitate to a watch glass, and freeze-dry to obtain calcium oxalate crystals after the effect of citric acid carbon dots.
实施例3Example 3
柠檬酸钾碳点影响草酸钙水溶液结晶的应用，具体包括以下步骤：The application of potassium citrate carbon dots to influence the crystallization of calcium oxalate aqueous solution specifically comprises the following steps:
1)称取3克的柠檬酸钾和1克尿素，加入30毫升的超纯水中，超声并且搅拌加速溶解完全；1) Weigh 3 grams of potassium citrate and 1 gram of urea, add them to 30 milliliters of ultrapure water, and stir and sonicate to accelerate the dissolution;
2)将溶液转移至反应釜中，放入马弗炉，在160摄氏度下，保温反应8小时，使反应物充分反应，待其反应完全后；取出内衬，并将溶液转移至烧杯中。2) The solution was transferred to a reactor, placed in a muffle furnace, and kept warm at 160 degrees Celsius for 8 hours to allow the reactants to react fully. After the reaction was complete, the liner was removed and the solution was transferred to a beaker.
3)将上述得到的溶液用孔径为0.22微米的有机微孔滤膜真空抽滤，收集滤液，装入截留分子量为1000道尔顿的透析袋，在超纯水中透析72小时，冻干48-72小时得到深褐色的碳点。3) The solution obtained above was vacuum filtered through an organic microporous filter membrane with a pore size of 0.22 μm, the filtrate was collected and placed in a dialysis bag with a molecular weight cutoff of 1000 Daltons, dialyzed in ultrapure water for 72 hours, and freeze-dried for 48-72 hours to obtain dark brown carbon dots.
4)称取10毫克的柠檬酸钾碳点将其溶于8毫升去离子水配成柠檬酸钾碳点溶液，并将其滴加入40毫升A液(22毫摩尔每升的氯化钙溶液)，超声搅拌使其混合均匀；另量取40毫升的B液(22毫摩尔每升的草酸钠溶液)放置在一旁备用；4) Weigh 10 mg of potassium citrate carbon dots and dissolve it in 8 ml of deionized water to prepare a potassium citrate carbon dot solution, and then dropwise add the solution to 40 ml of solution A (22 mmol/L calcium chloride solution), and mix evenly by ultrasonic stirring; measure 40 ml of solution B (22 mmol/L sodium oxalate solution) and set aside for use;
5)将混合后的A液置于37摄氏度的水浴锅中，将B液缓加入A液中，反应10分钟后获得草酸钙沉淀悬浊液，之后将其静置2小时使其沉淀完全；5) Place the mixed solution A in a water bath at 37 degrees Celsius, slowly add solution B into solution A, react for 10 minutes to obtain a calcium oxalate precipitate suspension, and then let it stand for 2 hours to allow complete precipitation;
6)将上述悬浊液倒入100毫升离心管中，以8000转每分钟离心10分钟；离心完成后倒去离心管中的清液，将沉淀转移至表面皿中，冻干后得到柠檬酸钾碳点影响后的草酸钙晶体。6) Pour the above suspension into a 100 ml centrifuge tube and centrifuge at 8000 rpm for 10 minutes; after centrifugation, pour out the clear liquid in the centrifuge tube, transfer the precipitate to a watch glass, and freeze-dry to obtain calcium oxalate crystals affected by potassium citrate carbon dots.
图1为一水草酸钙晶体和在羟基柠檬酸碳点影响下转化的二水草酸钙晶体的X射线衍射仪能谱图，可以看到羟基柠檬酸碳点的加入显著改变了晶体XRD信号。根据对照发现，羟基柠檬酸碳点的加入明显促进了一水草酸钙晶体向二水草酸钙的转变,客观上有利于尿结石晶体向体外的排出。Figure 1 shows the X-ray diffractometer energy spectrum of calcium oxalate monohydrate crystals and calcium oxalate dihydrate crystals transformed under the influence of hydroxycitrate carbon dots. It can be seen that the addition of hydroxycitrate carbon dots significantly changed the crystal XRD signal. According to the comparison, the addition of hydroxycitrate carbon dots significantly promoted the transformation of calcium oxalate monohydrate crystals into calcium oxalate dihydrate, which objectively facilitated the excretion of urinary stone crystals out of the body.
图2为一水草酸钙晶体和在羟基柠檬酸碳点影响下转化的二水草酸钙晶体的扫描电镜图，从中可以得知羟基柠檬酸碳点的加入促进了草酸钙结晶由一水草酸钙向二水草酸钙转化，左图a为草酸钙晶体原样的扫描电镜，其主体呈现片状晶体，归属于一水草酸钙；右图b是受碳点影响后的草酸钙晶体，可以看到其主要是立方体状晶体，归属于二水草酸钙；因为二水草酸钙更容易排出体外，所以此种转化有利于抑制草酸钙晶体在体内的聚集。Figure 2 is a scanning electron microscope image of calcium oxalate monohydrate crystals and calcium oxalate dihydrate crystals transformed under the influence of hydroxycitrate carbon dots. It can be seen from the scanning electron microscope image that the addition of hydroxycitrate carbon dots promotes the transformation of calcium oxalate crystals from calcium oxalate monohydrate to calcium oxalate dihydrate. The left figure a is a scanning electron microscope image of the original calcium oxalate crystals, the main body of which is flaky crystals, belonging to calcium oxalate monohydrate; the right figure b is the calcium oxalate crystals after being affected by carbon dots. It can be seen that it is mainly cubic crystals, belonging to calcium oxalate dihydrate; because calcium oxalate dihydrate is easier to be excreted from the body, this kind of transformation is beneficial to inhibit the aggregation of calcium oxalate crystals in the body.
图3为一水草酸钙晶体和在羟基柠檬酸碳点影响下转化的二水草酸钙晶体的红外光谱图，一水草酸钙和二水草酸钙的νas(COO-)分别为1 617和1 647波数，νs(COO-)分别为1318和1 328波数。羟基柠檬酸碳点的加入使得草酸钙晶体的νas(COO-)和νs(COO-)各蓝移了24波数和7波数，这表明羟基柠檬酸碳点的加入促进了一水草酸钙向二水草酸钙晶体转变，与图1的XRD数据和图2的扫描电镜数据相吻合。Figure 3 shows the infrared spectra of calcium oxalate monohydrate crystals and calcium oxalate dihydrate crystals transformed under the influence of hydroxycitrate carbon dots. The νas (COO-) of calcium oxalate monohydrate and calcium oxalate dihydrate are 1 617 and 1 647 wavenumbers, respectively, and the νs (COO-) are 1 318 and 1 328 wavenumbers, respectively. The addition of hydroxycitrate carbon dots causes the νas (COO-) and νs (COO-) of calcium oxalate crystals to blue shift by 24 wavenumbers and 7 wavenumbers, respectively, indicating that the addition of hydroxycitrate carbon dots promotes the transformation of calcium oxalate monohydrate into calcium oxalate dihydrate crystals, which is consistent with the XRD data in Figure 1 and the scanning electron microscope data in Figure 2.
图4为一水草酸钙晶体和在柠檬酸碳点影响下的一水草酸钙晶体的X射线衍射图，可以看到柠檬酸碳点的加入显著降低了晶体衍射峰的信号强度。根据谢乐公式D＝Kλ/Bcosθ,可以计算出平均晶粒度(D)，由图4中XRD数据计算获得的一水草酸钙晶体的平均晶粒度由原先的365纳米降低到了受碳点影响后的292纳米，抑制率为20％。Figure 4 shows the X-ray diffraction diagram of calcium oxalate monohydrate crystals and calcium oxalate monohydrate crystals under the influence of citric acid carbon dots. It can be seen that the addition of citric acid carbon dots significantly reduces the signal intensity of the crystal diffraction peak. According to the Scherrer formula D = Kλ/Bcosθ, the average grain size (D) can be calculated. The average grain size of calcium oxalate monohydrate crystals calculated from the XRD data in Figure 4 is reduced from the original 365 nanometers to 292 nanometers after being affected by carbon dots, and the inhibition rate is 20%.
应当说明的是，以上实施例仅是对于本发明技术内容的一般性验证例举，并不能简单地认为本发明就仅仅限定于上述实施例所述。本发明的实质保护范围应以权利要求书所述之为准。本领域技术人员应当知晓，凡基于本发明的实质精神所作出的任何修改、等同替换和改进等，均应在本发明的实质保护范围之内。It should be noted that the above embodiments are only general examples of verification of the technical content of the present invention, and it cannot be simply considered that the present invention is limited to the above embodiments. The substantive protection scope of the present invention shall be subject to the claims. Those skilled in the art should know that any modification, equivalent substitution and improvement based on the substantive spirit of the present invention shall be within the substantive protection scope of the present invention.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310140031.6ACN118059125B (en) | 2023-02-13 | 2023-02-13 | Citric acid series carbon dots and application thereof in preparation of calcium crystallization inhibitor |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310140031.6ACN118059125B (en) | 2023-02-13 | 2023-02-13 | Citric acid series carbon dots and application thereof in preparation of calcium crystallization inhibitor |
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| CN118059125B CN118059125B (en) | 2025-07-29 |
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| CN202310140031.6AActiveCN118059125B (en) | 2023-02-13 | 2023-02-13 | Citric acid series carbon dots and application thereof in preparation of calcium crystallization inhibitor |
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| CN114940487A (en)* | 2022-06-14 | 2022-08-26 | 上海交通大学医学院附属第九人民医院 | Ginseng carbon dots and preparation method and application thereof |
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| CN118059125B (en) | 2025-07-29 |
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