Name: preparation of naringenin solution for in vivo application
Uploaded: 2021-08-10T13:00:00
Description: Watch this Scientific Journal Video about Preparation of Naringenin Solution for In Vivo Application at JoVE.com

这项工作得到了中国国家自然科学基金（81973607和81573992）的支持。

所描述的调查符合国家研究委员会《实验动物护理和使用指南》，并得到上海中医药大学动物护理和使用委员会的批准。进行实验时，出于安全目的，需要实验室外套、一次性丁腈手套和护目镜。
1. 体内 应用所需溶剂的制备和柚皮素的估算
<ol><li>制备以下溶剂：吐温-80（最终浓度范围：0.5%-1%），DMSO，甘油（最终浓度范围：15%-20%），乙醇（最终浓度范围：肌内注射?...

<ol>
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J., Tsiani, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antidiabetic+properties+of+naringenin:+A+citrus+fruit+polyphenol.">Antidiabetic properties of naringenin: A citrus fruit polyphenol.</a> Biomolecules. 9 (3), (2019).</li><li>Tutunchi, H., Naeini, F., Ostadrahimi, A., Hosseinzadeh-Attar, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naringenin,+a+flavanone+with+antiviral+and+anti-inflammatory+effects:+A+promising+treatment+strategy+against+COVID-19.">Naringenin, a flavanone with antiviral and anti-inflammatory effects: A promising treatment strategy against COVID-19.</a> Phytotherapy Research. 34 (12), 3137-3147 (2020).</li><li>Zaidun, N. H., Thent, Z. C., Latiff, A. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Naringenin+on+metabolic+markers,+lipid+profile+and+expression+of+GFAP+in+C6+glioma+cells+implanted+rat's+brain.">Effect of Naringenin on metabolic markers, lipid profile and expression of GFAP in C6 glioma cells implanted rat's brain.</a> Annals of Neurosciences. 18 (4), 151-155 (2011).</li><li>Burke, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naringenin+enhances+the+regression+of+atherosclerosis+induced+by+a+chow+diet+in+Ldlr-/-+mice.">Naringenin enhances the regression of atherosclerosis induced by a chow diet in Ldlr-/- mice.</a> Atherosclerosis. 286, 60-70 (2019).</li><li>Assini, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naringenin+prevents+obesity,+hepatic+steatosis,+and+glucose+intolerance+in+male+mice+independent+of+fibroblast+growth+factor+21.">Naringenin prevents obesity, hepatic steatosis, and glucose intolerance in male mice independent of fibroblast growth factor 21.</a> Endocrinology. 156 (6), 2087-2102 (2015).</li><li>Alifarsangi, A., Esmaeili-Mahani, S., Sheibani, V., Abbasnejad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+citrus+flavanone+naringenin+prevents+the+development+of+morphine+analgesic+tolerance+and+conditioned+place+preference+in+male+rats.">The citrus flavanone naringenin prevents the development of morphine analgesic tolerance and conditioned place preference in male rats.</a> The American Journal of Drug and Alcohol Abuse. 47 (1), 43-51 (2020).</li><li>Sirovina, D., Or&#353;oli&#263;, N., Gregorovi&#263;, G., Kon&#269;i&#263;, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naringenin+ameliorates+pathological+changes+in+liver+and+kidney+of+diabetic+mice:+a+preliminary+study.">Naringenin ameliorates pathological changes in liver and kidney of diabetic mice: a preliminary study.</a> Archives of Occupational Hygiene and Toxicology. 67 (1), 19-24 (2016).</li><li>Nguyen-Ngo, C., Willcox, J. C., Lappas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-diabetic,+anti-inflammatory,+and+anti-oxidant+effects+of+Naringenin+in+an+In+vitro+human+model+and+an+in+vivo+murine+model+of+gestational+diabetes+mellitus.">Anti-diabetic, anti-inflammatory, and anti-oxidant effects of Naringenin in an In vitro human model and an in vivo murine model of gestational diabetes mellitus.</a> Molecular Nutrition &amp; Food Research. 63 (19), 1900224 (2019).</li><li>Ahmed, L. A., Obaid, A. A. Z., Zaki, H. F., Agha, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naringenin+adds+to+the+protective+effect+of+L-arginine+in+monocrotaline-induced+pulmonary+hypertension+in+rats:+favorable+modulation+of+oxidative+stress,+inflammation+and+nitric+oxide.">Naringenin adds to the protective effect of L-arginine in monocrotaline-induced pulmonary hypertension in rats: favorable modulation of oxidative stress, inflammation and nitric oxide.</a> European Journal of Pharmaceutical Sciences. 62, 161-170 (2014).</li><li>Park, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naringenin+ameliorates+kainic+acid-induced+morphological+alterations+in+the+dentate+gyrus+in+a+mouse+model+of+temporal+lobe+epilepsy.">Naringenin ameliorates kainic acid-induced morphological alterations in the dentate gyrus in a mouse model of temporal lobe epilepsy.</a> Neuroreport. 27 (15), 1182-1189 (2016).</li><li>Matsui, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early-stage+Type+2+diabetes+mellitus+impairs+erectile+function+and+neurite+outgrowth+from+the+major+pelvic+ganglion+and+downregulates+the+gene+expression+of+neurotrophic+factors.">Early-stage Type 2 diabetes mellitus impairs erectile function and neurite outgrowth from the major pelvic ganglion and downregulates the gene expression of neurotrophic factors.</a> Urology. 99, 1-7 (2017).</li><li>Sharma, G., Ashhar, M. U., Aeri, V., Katare, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+late-stage+diabetes+mellitus+and+-associated+vascular+complications.">Development and characterization of late-stage diabetes mellitus and -associated vascular complications.</a> Life Sciences. 216, 295-304 (2019).</li><li>Rowe, R. C., Sheskey, P. J., Owen, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Pharmaceutical Excipients. , (2003).</li><li>Fallahi, F., Roghani, M., Moghadami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Citrus+flavonoid+naringenin+improves+aortic+reactivity+in+streptozotocin-diabetic+rats.">Citrus flavonoid naringenin improves aortic reactivity in streptozotocin-diabetic rats.</a> Indian Journal of Pharmacology. 44 (3), 382-386 (2012).</li><li>Liu, F., Yang, H., Zhou, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+characteristics+of+induced+and+spontaneous+db/db+mouse+models+of+type+2+diabetes+mellitus.">Comparison of the characteristics of induced and spontaneous db/db mouse models of type 2 diabetes mellitus.</a> Acta Laboratorium Animalis Scientia Sinica. 22 (6), 54-59 (2014).</li><li>Liu, S., Dong, J., Bian, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual+regulatory+effect+of+naringenin+on+bone+homeostasis+in+two+diabetic+mice+models.">A dual regulatory effect of naringenin on bone homeostasis in two diabetic mice models.</a> Traditional Medicine and Modern Medicine. 03 (02), 101-108 (2020).</li><li>Sun, C., Wu, Z., Wang, Z., Zhang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ethanol/water+solvents+on+phenolic+profiles+and+anti-oxidant+properties+of+beijing+propolis+extracts.">Effect of ethanol/water solvents on phenolic profiles and anti-oxidant properties of beijing propolis extracts.</a> Evidence-Based Complementary and Alternative Medicine. 2015, 595393 (2015).</li><li>Huaman-Castilla, N. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+concentration+of+ethanol+injected+intraperitoneally+on+taste+aversion,+body+temperature,+and+activity.">Effects of concentration of ethanol injected intraperitoneally on taste aversion, body temperature, and activity.</a> Psychopharmacology. 64 (1), 61-65 (1979).</li><li>Magwaza, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+methods+for+extracting+and+quantifying+phenolic+compounds+in+citrus+rinds.">Rapid methods for extracting and quantifying phenolic compounds in citrus rinds.</a> Food Science &amp; Nutrition. 4 (1), 4-10 (2016).</li><li>Smith, E. R., Hadidian, Z., Mason, M. 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植物化学溶液的制备是其在 体内应用的基础。在该协议中，通过使用不同的溶剂（例如乙醇，DMSO，吐温80和0.9%PS）证明了柚皮素溶液的制备。完全溶解状态的溶液需要进一步监测，使其在室温下保持更长时间，然后在 体内使用前过滤。
溶剂测定是该方案中的关键步骤。溶解化合物的溶剂有很多选择，其中乙醇、DMSO 和 PS 使用最广泛。乙醇由于其高极性特性，可...

随着关于使用植物化合物进行药物筛选的研究越来越多，制备植物化学溶液以评估其最佳效果的方法值得关注。制备化合物1时需要考虑许多方面，例如溶出方法、剂量和浓度。
溶剂型溶解广泛用于有机化合物制备1.常用的溶剂有水、油、二甲基亚砜（DMSO）、甲醇、乙醇、甲酸、吐温、甘油等2。虽然当通过胃管饲法施用化合物时，可以使用未溶解物质的悬浮液，但完全溶解的溶质对于静脉内给药至关重要。由于油溶液、混悬液和乳液可引起毛细血管栓塞，因此建议使用水溶液制备化合物，特别是在静脉内、肌肉内和腹膜内注射时3。
有效剂量范围因化合物而异，甚至在用相同化合物治疗的疾病之间也有所不同。有效和安全剂量以及浓度的确定取决于文献和初步实验4.这里以柚皮素化合物的制备为例进行演示。
柚皮素（4，5，7-三羟基黄烷酮）是一种多酚化合物，已被研究用于疾病治疗，其保肝5，抗糖尿病6，抗炎7和抗氧化活性8。对于体内应用，通常使用柚皮素的口服给药。以前的研究报告了在0.5%-1%羧甲基纤维素，0.5%甲基纤维素剂量，0.01%DMSO和生理盐水（PS）中以50-100mg / kg制备柚皮素溶液，通过口服强饲法9，10，11，12给药。此外，其他研究报告了以3.6 g / kg / d的剂量口服摄入的柚皮素（wt / wt）补充柚皮素13，14。研究还报道使用乙醇（0.5% v/v）、PS 和 DMSO 溶解柚皮素，用于腹腔注射，剂量为 10-50 mg/kg15，16，17，18。在颞叶癫痫的研究中，小鼠接受注射悬浮在溶解在PS19中的0.25%羧甲基纤维素中的柚皮素。尽管这些研究报告了使用不同的溶剂来制备柚皮素溶液，但尚未报告进一步的细节，例如溶解状态和动物反应。
该协议介绍了制备柚皮素溶液的程序，用于糖尿病诱导的骨质疏松症的 体内 应用。注射液的制备包括制备溶剂和化合物、剂量估算、溶解过程和过滤。根据文献研究和初步实验，通过监测小鼠每天注射3天后并根据小鼠行为修改剂量来确定剂量。最终选择的浓度（20mg / kg b.w.）在高脂肪饮食和链脲佐菌素（STZ）诱导的糖尿病小鼠中每周5天腹膜内施用8周20，21。通过血糖检测、显微CT、酒石酸盐耐酸性磷酸酶（TRAP）染色和酶联免疫吸附试验（ELISA）评估柚皮素对糖尿病骨质疏松症的影响。
总体而言，观察到浓度范围为40-400mg / mL的柚皮素未完全溶解在乙醇或DMSO或5%（乙醇或DMSO）加95%PS（v / v）中。然而，柚皮素完全溶解在3.52%DMSO，3.52%吐温80和92.96%PS的混合物中。详细的程序将有助于研究人员制备化合物作为 体内 应用的注射溶液。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL&#160; microtubes</td>
			<td>Corning Science (Wujiang) Co.</td>
			<td>23218392</td>
			<td>Holding liquid</td>
		</tr>
		<tr>
			<td>Automatic Dehydrator</td>
			<td>Leica Microsystems (Shanghai) Co.</td>
			<td>LEICA ASP 300S</td>
			<td>Dehydrate samples</td>
		</tr>
		<tr>
			<td>Blood glucose test strips</td>
			<td>Johnson &#38; Johnson (China) Medical Equipment Co.</td>
			<td>4130392</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>MIULAB</td>
			<td>Minute centrifuge</td>
			<td>Centrifugal solution</td>
		</tr>
		<tr>
			<td>Dehydrator</td>
			<td>Leica Microsystems (Shanghai) Trading Co.</td>
			<td>LEICA&#160; ASP300S</td>
			<td>Dehydration</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sangon Biotech (Shanghai ) Co.,Ltd.</td>
			<td>E918BA0041</td>
			<td>Co-Solvent</td>
		</tr>
		<tr>
			<td>ELISA assay kit</td>
			<td>Elabscience Biotechnology Co.,Ltd</td>
			<td>Mouse COL1(Collagen Type I) ELISA Kit: E-EL-M0325c 
			Mouse&#160; CTX I ELISA Kit: E-EL-M0366c 
			Mouse PICP ELISA Kit: E-EL-M0231c 
			Mouse PINP ELISA Kit: E-EL-M0233c</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>10009218</td>
			<td>Co-Solvent</td>
		</tr>
		<tr>
			<td>Ethylene glycol monoethyl ether</td>
			<td>Sangon Biotech (Shanghai ) Co.,Ltd.</td>
			<td>A501118-0500</td>
			<td>TRAP staining</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>10009617</td>
			<td>Decalcification</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Merck Millpore LTD.</td>
			<td>Millex-GP, 0.22 &#181;m</td>
			<td>filter solution</td>
		</tr>
		<tr>
			<td>Glacial acid</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>10000218</td>
			<td>TRAP staining</td>
		</tr>
		<tr>
			<td>Glucose meter</td>
			<td>Johnson &#38; Johnson (China) Medical Equipment Co.</td>
			<td>One Touch Ultra Vue</td>
			<td>Serial number:COJJG8GW</td>
		</tr>
		<tr>
			<td>Grinder</td>
			<td>Shanghaijingxin Experimental Technology</td>
			<td>Tissuelyser-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>D005</td>
			<td>TRAP staining</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>Shanghai Kantaray Medical Devices Co.</td>
			<td>0.33 mm x 13 mm, RW LB</td>
			<td>Intraperitoneal injection</td>
		</tr>
		<tr>
			<td>L-(+) tartaric acid</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>100220008</td>
			<td>TRAP staining</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>OLYMPUS</td>
			<td>sz61</td>
			<td>Observation</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica Microsystems (Shanghai) Trading Co.</td>
			<td>LEICA RM 2135</td>
			<td>Section</td>
		</tr>
		<tr>
			<td>Mini centrifuge</td>
			<td>Hangzzhou Miu Instruments Co., Ltd.</td>
			<td>&#160;Mini-6KC</td>
			<td>Centrifuge</td>
		</tr>
		<tr>
			<td>Naphthol AS-BI phosphate</td>
			<td>SIGMA-ALDRICH</td>
			<td>BCBS3419</td>
			<td>TRAP staining</td>
		</tr>
		<tr>
			<td>Naringenin</td>
			<td>Jiangsu Yongjian Pharmaceutical Co.,Ltd</td>
			<td>102764</td>
			<td>Solute</td>
		</tr>
		<tr>
			<td>Paraffin Embedding station</td>
			<td>Leica Microsystems (Shanghai) Co.</td>
			<td>LEICA&#160; EG 1150 H, LEICA&#160; EG 1150 C</td>
			<td>Embed&#160; samples</td>
		</tr>
		<tr>
			<td>Pararosaniline base</td>
			<td>BBI Life Sciences</td>
			<td>E112BA0045</td>
			<td>TRAP staining</td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>eppendorf</td>
			<td>2&#8211;20 &#181;L, 100&#8211;1000 &#181;L, 20&#8211;200 &#181;L</td>
			<td>&#160; transferre Liquid</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>BioTek Instruments USA, Inc.</td>
			<td>BioTek CYTATION 3 imaging reader</td>
			<td>ELISA</td>
		</tr>
		<tr>
			<td>Resin</td>
			<td>Shanghai Yyang Instrument Co., Ltd.</td>
			<td>Neutral balsam</td>
			<td>TRAP staining</td>
		</tr>
		<tr>
			<td>saline (0.9 PS)</td>
			<td>Baxter Healthcare (Shanghai) Co.,Ltd</td>
			<td>A6E1323</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Sodium acetate anhydrous</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>Merck-1.06268.0250 | 250g</td>
			<td>TRAP staining</td>
		</tr>
		<tr>
			<td>Sodium nitrite</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>10020018</td>
			<td>TRAP staining</td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td>Sangon Biotech (Shanghai ) Co.,Ltd.</td>
			<td>E819BA0006</td>
			<td>Emulsifier</td>
		</tr>
		<tr>
			<td>Zirconia beads</td>
			<td>Shanghaijingxin Experimental Technology</td>
			<td>11079125z 454g</td>
			<td>Grinding</td>
		</tr>
	</tbody>
</table>

preparation of naringenin solution for in vivo application

化合物（植物化学）溶液的制备是将其应用于药物筛选等研究之前被忽视但至关重要的步骤。化合物的完全溶解对于其安全使用和相对稳定的结果是必要的。在这里，以制备柚皮素溶液及其在高脂肪饮食和链脲佐菌素（STZ）诱导的糖尿病模型中腹膜内给药的方案为例进行了演示。使用少量柚皮素（3.52-6.69 mg）测试其在溶剂中的溶解性，包括乙醇，二甲基亚砜（DMSO）和DMSO加在生理盐水（PS）中复溶的吐温80。通过观察溶液的颜色，离心后沉淀物的存在（2000× g 持续30秒）或让溶液在室温（RT）下静置2小时来确定化合物的完全溶解。获得稳定的化合物/植物化学溶液后，可以在仅溶剂（无PS）储备溶液中制备 体内研究所需 化合物的最终浓度/量，然后根据需要与PS稀释/混合。通过测量血糖、骨量（显微CT）和骨吸收率（TRAP染色和ELISA）评估柚皮素对小鼠的抗糖尿病骨质疏松作用（腹膜内给药20mg / kg b.w.，2mg / mL）。寻找详细的有机/植物化学溶液制剂的研究人员将从这项技术中受益。

发现高脂肪饮食喂养和STZ诱导的糖尿病小鼠的体重在STZ治疗后0-8周与对照组相比降低。与未治疗的小鼠（STZ组）相比，柚皮素治疗的小鼠在第4周的体重减轻是显着的。对照组和STZ组用相同体积的PS施用（表1）。糖尿病小鼠的血糖水平在STZ诱导后1个月内显着升高。然后它自动降低到2个月前建立动物模型时观察到的水平。柚皮素治疗在1个月和2个月时分别降低了51.8%和34.8%的血糖水平（<s...

Watch this Scientific Journal Video about Preparation of Naringenin Solution for In Vivo Application at JoVE.com

Preparation of Naringenin Solution for In Vivo Application

在这里，该协议介绍了用于 体内 腹膜内给药的柚皮素溶液的制备。柚皮素完全溶解在二甲基亚砜，吐温80和盐水的混合物中。通过血糖试验、酒石酸盐耐酸性磷酸酶染色和酶联免疫吸附试验评估柚皮素的抗糖尿病骨质疏松作用。

柚皮素溶液的制备

The preparation of a compound (phytochemical) solution is an overlooked but critical step prior to its application in studies such as drug screening. The ...

随着越来越多的研究关注药物筛选方法的植物化学成分，以制备植物化学溶液以评估最佳效果，我注意到该协议作为操作简单和低成本的优点。该协议将使处理药理学药物筛选的研究人员受益。考虑到消耗成本，我们建议从少量植物化学化合物开始进行初步实验。其次，建议使用胰岛素注射器而不是普通注射器 要制备每毫升2毫克的柚皮素乙醇溶液，称取3.52毫克柚皮素粉并将其添加到两毫升的管中。快速旋转管子，将粉末沉降在管底部。接下来，向管中加入8.8微升100%乙醇以制备0.5%溶液。然后向管中加入79.2微升100%乙醇，制备5%溶液。接下来，向含有5%溶液的管中加入1.672毫升0.9%生理盐水。这将产生乳液。然后离心机拒绝试管检查柚皮素是否完全溶解在溶液中。溶液中可能会出现未溶解的柚皮素的白色沉淀。接下来，在DMSO中制备每毫升2毫克的柚皮素溶液，称取3.95毫克柚皮素粉并将其加入两毫升管中。快速旋转后，向管中加入9.8毫升DMSO以制备8.5%的溶液。然后向管中加入88.2微升DMSO以制备5%溶液。接下来，向溶液中加入1.877毫升0.9%盐水。这将产生乳液。离心溶液以检查柚皮素是否完全溶解。溶液中可能出现未溶解的白色沉淀物柚皮素。接下来，在吐温80和DMSO中制备每毫升2毫克的柚皮素溶液，称取6.69毫克柚皮素粉并将其加入5毫升管中。快速旋转试管，加入117.7微升DMSO以制备3.5%的溶液。然后在溶液中加入117.7微升吐温80，以获得3.5%吐温80和3.5%DMSO浓度。慢慢将此溶液加入另一个含有3109.6微升0.9%盐水的5毫升管中，摇匀，得到澄清的柚皮素溶液。两个小时后，溶液将保持透明，没有任何可见的沉淀物。要施用柚皮素溶液，请抬起一只五周大的C57黑六雄性小鼠的尾巴根部，并将其放在坚固的表面上，将其尾巴轻轻地向后放置。接下来，用左手拇指和食指抓住耳朵后面的脖子，并将尾巴放在小指和无名指之间。保持鼠标仰卧位，后端略微抬高。要注射柚皮素溶液，请抓住小鼠背部的皮肤，使腹部皮肤绷紧。然后将针头以 10 度角推入腹部的右下象限或左下象限。避免撞击膀胱、肝脏或其他内脏。将针头沿颅骨方向皮下敷三到五毫米，然后以 45 度角插入腹腔。一旦针穿过腹壁，慢慢推动溶液。注射后，慢慢将针头拉出，稍微旋转以防止泄漏。要进行血糖测试，请打开试纸并标记日期。用 25 号针刺在尾侧静脉上做一个小穿刺，挤出一滴血。用薄纸擦拭血滴后，挤出另一滴血，收集在试纸边缘。读取并记录血糖仪上显示的结果。与链脲佐菌素治疗后0至8周的对照组小鼠相比，喂食和链脲佐菌素诱导的糖尿病小鼠的高脂肪饮食的体重降低。在第四周，与未治疗的小鼠相比，柚皮素治疗的小鼠的体重减轻是显着的。糖尿病小鼠的血糖水平在链脲佐菌素诱导后一个月内显着升高。两个月后，它自动下降到两个月前观察到的水平。相比之下，与STZ相比，柚皮素治疗在一个月时将血糖水平降低了51.8%，在两个月时降低了34.8%。与对照组相比，链脲佐菌素诱导的糖尿病小鼠表现出骨质流失，如组织体积和小梁数量的骨体积减少所表明的那样。同时，柚皮素治疗显著挽救了骨质流失。在糖尿病小鼠中，每个小梁骨区域的破骨细胞数显示的破骨细胞活性增加，尽管对照和疾病模型之间没有观察到统计学意义。本文显示了对照和链脲佐菌素诱导小鼠的小梁骨和破骨细胞的TRAP染色。柚皮素治疗显著降低破骨细胞活性。在糖尿病动物中，1型胶原蛋白的C端端肽升高了68.09%，1型原胶原蛋白的N端端肽升高了204.88%，表明骨吸收率显著增加。然而，柚皮素治疗显著降低了骨吸收率的两个指标。因此，移液准确剂量具有挑战性。切断吸头使移液更容易、更准确。或者，可以将所需的体积转换为质量，以后可以轻松完成。每次膀胱葡萄糖实验前所有动物的空腹时间必须在整个实验过程中保持不变。

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Preparation of Naringenin Solution for In Vivo Application

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