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

This work was supported by the National Natural Science Foundation of China (81973607 and 81573992).

The investigations described conformed to the Guidelines for the Care and Use of Laboratory Animals of the National Research Council and were approved by the Shanghai University of Traditional Chinese Medicine Animal Care and Use Committee. When performing the experiments, lab coats, disposable nitrile gloves, and goggles are required for safety purposes.
1. Preparation of solvents and estimation of naringenin required for in vivo application
<ol>
	<li>Prepare the following solvents...

<ol>
	<li>Stoye, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solvents.">Solvents.</a> Ullmann's Encyclopedia of Industrial Chemistry. , (2000).</li><li>Bouchard, D., 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=Optimization+of+the+solvent-based+dissolution+method+to+sample+volatile+organic+compound+vapors+for+compound-specific+isotope+analysis.">Optimization of the solvent-based dissolution method to sample volatile organic compound vapors for compound-specific isotope analysis.</a> Journal of Chromatography A. 1520, 23-34 (2017).</li><li>Turner, P. V., Brabb, T., Pekow, C., Vasbinder, M. 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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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combating+oxidative+stress+disorders+with+citrus+flavonoid:+Naringenin.">Combating oxidative stress disorders with citrus flavonoid: Naringenin.</a> Life Sciences. 208, 111-122 (2018).</li><li>Tsuhako, R., Yoshida, H., Sugita, C., Kurokawa, 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+suppresses+neutrophil+infiltration+into+adipose+tissue+in+high-fat+diet-induced+obese+mice.">Naringenin suppresses neutrophil infiltration into adipose tissue in high-fat diet-induced obese mice.</a> Journal of Natural Medicines. 74 (1), 229-237 (2020).</li><li>Annadurai, T., 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=Antihyperglycemic+and+anti-oxidant+effects+of+a+flavanone,+naringenin,+in+streptozotocin-nicotinamide-induced+experimental+diabetic+rats.">Antihyperglycemic and anti-oxidant effects of a flavanone, naringenin, in streptozotocin-nicotinamide-induced experimental diabetic rats.</a> Journal of Physiology and Biochemistry. 68 (3), 307-318 (2012).</li><li>Li, 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=Naringenin+improves+insulin+sensitivity+in+gestational+diabetes+mellitus+mice+through+AMPK.">Naringenin improves insulin sensitivity in gestational diabetes mellitus mice through AMPK.</a> Nutrition &amp; Diabetes. 9 (1), 28 (2019).</li><li>Devan, S., Janardhanam, V. 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. L., 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=The+impact+of+temperature+and+ethanol+concentration+on+the+global+recovery+of+specific+polyphenols+in+an+integrated+HPLE/RP+process+on+carm&#233;n&#232;re+pomace+extracts.">The impact of temperature and ethanol concentration on the global recovery of specific polyphenols in an integrated HPLE/RP process on carm&#233;n&#232;re pomace extracts.</a> Molecules. 24 (17), (2019).</li><li>van Thriel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicology+of+solvents+(including+alcohol).">Toxicology of solvents (including alcohol).</a> Reference Module in Biomedical Sciences. , (2014).</li><li>Linakis, J. G., Cunningham, C. 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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The preparation of phytochemical solution is the basis for its application in vivo. In this protocol, the preparation of naringenin solution was demonstrated by using different solvents, such as ethanol, DMSO, Tween 80, and 0.9% PS. The solution in completely dissolved status needs to be further monitored by allowing it to remain at room temperature for some extended hours, and then filtered before being used in vivo.
Solvent determination is a critical step in this protocol....

With increasing studies concerning the use of phytochemical compounds for drug screening, approaches to prepare phytochemical solutions to evaluate their optimal effects are worth giving attention to. Many aspects such as the dissolution methodology, dosage, and concentration&#160;are to be considered when preparing the compound1.
Solvent-based dissolution is widely used for organic compound preparation1. The commonly used solvents include water, oil, dimethyl sulfoxide (DMSO), methanol, ethanol, formic acid, Tween, glycerin, etc2. Although a suspension with undissolved substances is acceptable when the compound is administered by gastric gavage, a fully dissolved solute is critical for intravenous administration. Since oil solution, suspension, and emulsion can cause capillary embolisms, an aqueous solution for compound preparation is suggested, especially when administering intravenous, intramuscular, and intraperitoneal injections3.
The effective dose range varies among compounds and even among diseases treated with the same compound. Determinations of the effective and the safe dose and the concentration are dependant on literature and preliminary experiments4. Here, the preparation of the compound naringenin is demonstrated as an example.
Naringenin (4,5,7-trihydroxy-flavanone), a polyphenolic compound, has been studied in disease treatment for its hepatoprotective5, antidiabetic6, anti-inflammatory7, and anti-oxidant activities8. For in vivo applications, the oral administration of naringenin is commonly used. Previous studies reported preparing naringenin solution in 0.5%-1% carboxymethyl cellulose, 0.5% methylcellulose dose, 0.01% DMSO, and physiological saline (PS) at 50-100 mg/kg, administered by oral gavage9,10,11,12. Besides, other studies have reported supplementing naringenin with chow at 3% (wt/wt) for oral intake at a dose of 3.6 g/kg/d13,14. Studies have also reported using ethanol (0.5% v/v), PS, and DMSO to dissolve naringenin for intraperitoneal injection at 10-50 mg/kg15,16,17,18. In a study of temporal lobe epilepsy, mice received an injection of naringenin suspended in 0.25% carboxymethyl cellulose dissolved in PS19. Though these studies report the use of different solvents to prepare naringenin solutions, further details, such as dissolving status and animal response, have not been reported.
This protocol introduces a procedure for preparing naringenin solution for in vivo application in diabetic-induced osteoporosis. The preparation of the injection solution includes preparing solvents and compounds, dosage estimation, dissolution process, and filtration. The dosage was determined based on literature research and preliminary experiments by monitoring mice after administering injections every day for 3 days and modifying the dosage according to mouse behaviors. The final chosen concentration (20 mg/kg b.w.) was administered intraperitoneally 5 days per week for 8 weeks in a high-fat diet and streptozotocin (STZ)-induced diabetic mice20,21. The effects of naringenin in diabetic osteoporosis were evaluated by blood glucose testing, micro-CT, tartrate-resistant acid phosphatase (TRAP) staining, and enzyme-linked immunosorbent assay (ELISA).
Overall, it was observed that naringenin at a concentration range of 40-400 mg/mL did not completely dissolve in either ethanol or DMSO or 5% (ethanol or DMSO) plus 95% PS (v/v). However, naringenin dissolved completely in a mixture of 3.52% DMSO, 3.52% Tween 80, and 92.96% PS. The detailed procedure will help researchers to prepare the compound as an injection solution for in vivo application.

<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

The preparation of a compound (phytochemical) solution is an overlooked but critical step prior to its application in studies such as drug screening. The complete solubilization of the compound is necessary for its safe use and relatively stable results. Here, a protocol for preparing naringenin solution and its intraperitoneal administration in a high-fat diet and streptozotocin (STZ)-induced diabetic model is demonstrated as an example. A small amount of naringenin (3.52-6.69 mg) was used to test its solubilization in solvents, including ethanol, dimethylsulfoxide (DMSO), and DMSO plus Tween 80 reconstituted in physiological saline (PS), respectively. Complete solubilization of the compound is determined by observing the color of the solution, the presence of precipitates after centrifugation (2000 x g for 30 s), or allowing the solution to stand for 2 h at room temperature (RT). After obtaining a stable compound/phytochemical solution, the final concentration/amount of the compound required for in vivo studies can be prepared in a solvent-only (no PS) stock solution, and then diluted/mixed with PS as desired. The antidiabetic osteoporotic effects of naringenin in mice (intraperitoneal administration at 20 mg/kg b.w., 2 mg/mL) were assessed by measuring blood glucose, bone mass (micro-CT), and bone resorption rate (TRAP staining and ELISA). Researchers looking for detailed organic/phytochemical solution preparations will benefit from this technique.

The bodyweight of the high-fat diet-fed and STZ-induced diabetic mice was found to decrease when compared with that of the control groups from 0-8 weeks after STZ treatment. The weight loss of naringenin-treated mice was significant compared to the nontreated mice (STZ group) at week 4. The control and STZ groups were administered with the same volume of PS (Table 1). The blood glucose level in diabetic mice dramatically increased within 1 month after STZ induction. It then automatically decreased to a l...

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

Here, the protocol presents the preparation of naringenin solution for in vivo intraperitoneal administration. Naringenin is fully dissolved in a mixture of dimethylsulfoxide, Tween 80, and saline. The antidiabetic osteoporotic effects of naringenin were assessed by blood glucose testing, tartrate-resistant acid phosphatase staining, and enzyme-linked immunosorbent assay.

Preparation of Naringenin Solution for In Vivo Application

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

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