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 ...

With increasing studies concern the phytochemical components for drug screening approaches to prepare phytochemical solution for evaluation of optimal effects, I was giving attention to the advantages of this protocol as simple operation and low cost. This protocol will benefit the researchers dealing with the drug screening of pharmacology. We suggest starting with a small amount of phytochemical compound for preliminary experiments, considering the consumption cost.Second, using an insulin syringe over a regular syringe is recommended To prepare a two milligrams per milliliter naringenin solution in ethanol, weigh 3.52 milligrams of naringenin powder and add it to a two milliliter tube. Quickly spin the tube to settle the powder at the bottom of the tube. Next, add 8.8 microliters of 100%ethanol to the tube to prepare a 0.5%solution.Then add 79.2 microliters of 100%ethanol to the tube to prepare a 5%solution. Next, add 1.672 milliliters of 0.9%physiological saline to the tube containing the 5%solution. This will produce an emulsion.Then centrifuge refused the tube to check whether the naringenin is completely dissolved in the solution. White precipitates of undissolved naringenin may appear in the solution. Next, to prepare a two milligram per milliliter naringenin solution in DMSO, weigh 3.95 milligrams of naringenin powder and add it into a two milliliter tube.After a quick spin, add 9.8 milliliters of DMSO to the tube to prepare 8.5%solution. Then add 88.2 microliters of DMSO to the tube to prepare a 5%solution. Next, add 1.877 milliliters of 0.9%saline to the solution.This will result in an emulsion. Centrifuge the solution to check whether the naringenin is completely dissolved. White precipitates have undissolved naringenin may appear in the solution.Next, to prepare a two milligrams per milliliter naringenin solution in Tween 80 and DMSO, weigh 6.69 milligrams of naringenin powder and add it into a five milliliter tube. Quickly spin down the tube, and add 117.7 microliters of DMSO to prepare a 3.5%solution. Then add 117.7 microliters of Tween 80 to the solution to attain a 3.5%Tween 80 and 3.5%DMSO concentration.Slowly add this solution to another five milliliter tube containing 3109.6 microliters of 0.9%saline and shake well to obtain a clear naringenin solution. After two hours, the solution will remain clear without any visible precipitates. To administer the naringenin solution, lift a five-week-old C57 black six male mouse by the base of its tail and place it on a solid surface positioning its tail back gently.Next, grasp the scruff of the neck behind the ears with the left thumb and index finger and position the tail between the little and ring finger. Keep the mouse in a supine position with its posterior end slightly elevated. To inject the naringenin solution, grasp the skin on the mouse's back so that the abdominal skin is taut.Then push the needle into the lower right or left quadrant of the abdomen at a 10-degree angle. to avoid hitting the bladder, liver or other internal organs. Run the needle subcutaneously in a cranial direction for three to five millimeters, then insert it at a 45-degree angle into the abdominal cavity.Once the needle passes through the abdominal wall, slowly push the solution. After the injection, slowly pulled the needle out, rotating it slightly to prevent leakage. To perform a blood glucose test, open the test strips and mark the date.Make a small puncture on the lateral tail vein with a 25-gauge needle prick and squeeze out a drop of blood. After wiping the drop of blood with tissue paper, squeeze out another drop of blood and collect it on the edge of the test strip. Read and record the result displayed on the glucose meter.Body weight of the high fat diet fed and streptozotocin-induced diabetic mice was decreased compared to that of control group mice zero to eight weeks after a streptozotocin treatment. At the fourth week, the weight loss of naringenin-treated mice was significant compared to non-treated mice. Blood glucose level in the diabetic mice dramatically increased within one month after streptozotocin induction.After two months, it automatically decreased to a level observed two months ago. In contrast, naringenin treatment lowered the blood glucose levels by 51.8%at one month and 34.8%at two months compared to STZ. Compared to controls, the streptozotocin-induced diabetic mice exhibited bone loss as indicated by a decrease in the bone volume by tissue volume and the number of trabecula.Meanwhile, naringenin treatment significantly rescued the bone loss. Osteoclast activity shown by osteoclast number per trabecular bone area was increased in diabetic mice although no statistical significance was observed between the control and the disease models. TRAP staining of trabecular bone and osteoclasts of control and streptozotocin-induced mice is shown here.Naringenin treatment significantly decreased osteoclast activities. In the diabetic animals, the C-terminal telopeptide of type one collagen was elevated by 68.09%and the N-terminal propeptide of type one Pro-Collagen was elevated by 204.88%indicating a dramatic increase in the bone resorption rate. However, naringenin treatment significantly decreased both indicators of the bone resorption rate.so it is challenging to pipette accurate dosage. Cutting the tip off makes pipetting easier and more accurate. Alternatively, the required volume can be converted to a mass, which can be later easily.The fasting time of all animals before each bladder glucose experiment must remain the thing during the entire experiment.

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Medicine

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

Cataract is the leading cause of blindness in the world 1. The World Health Organization defines cataract as a clouding of the lens of the eye which impedes the transfer of light. Cataract is a multi-factorial disease associated with diabetes, smoking, ultraviolet radiation (UVR), alcohol, ionizing radiation, steroids and hypertension. There is strong experimental 2-4 and epidemiological evidence 5,6 that UVR causes cataract. We developed an animal model for UVR B induced cataract in both anesthetized 7 and non-anesthetized animals 8.
The only cure for cataract is surgery but this treatment is not accessible to all. It has been estimated that a delay of onset of cataract for 10 years could reduce the need for cataract surgery by 50% 9. To delay the incidence of cataract, it is needed to understand the mechanisms of cataract formation and find effective prevention strategies. Among the mechanisms for cataract development, apoptosis plays a crucial role in initiation of cataract in humans and animals 10. Our focus has recently been apoptosis in the lens as the mechanism for cataract development 8,11,12. It is anticipated that a better understanding of the effect of UVR on the apoptosis pathway will provide possibilities for discovery of new pharmaceuticals to prevent cataract.
In this article, we describe how cataract can be experimentally induced by in vivo exposure to UVR-B. Further RT-PCR and immunohistochemistry are presented as tools to study molecular mechanisms of UVR-B induced cataract.

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

Cataract is the leading cause of blindness in the world. Solar ultraviolet radiation (UVR) is the main risk factor for cataract development. An animal model of far UVR-B induced cataract was developed. In this article we describe methods for investigation of cataract formation: exposure to UVR, quantitative RT-PCR and immunohistochemistry.

Cataract is the leading cause of blindness in the world 1. The World Health Organization defines cataract as a clouding of the lens of the eye which ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the brain for visual perception. Damage to RGCs and their axons leads to vision loss and/or blindness. Although the specific cause of glaucoma is unknown, the primary risk factor for the disease is an elevated intraocular pressure. Glaucoma-inducing procedures in animal models are a valuable tool to researchers studying the mechanism of RGC death. Such information can lead to the development of effective neuroprotective treatments that could aid in the prevention of vision loss. The protocol in this paper describes a method of inducing glaucoma - like conditions in an in vivo rat model where 50 &#181;l of 2 M hypertonic saline is injected into the episcleral venous plexus. Blanching of the vessels indicates successful injection. This procedure causes loss of RGCs to simulate glaucoma. One month following injection, animals are sacrificed and eyes are removed. Next, the cornea, lens, and vitreous are removed to make an eyecup. The retina is then peeled from the back of the eye and pinned onto sylgard dishes using cactus needles. At this point, neurons in the retina can be stained for analysis. Results from this lab show that approximately 25% of RGCs are lost within one month of the procedure when compared to internal controls. This procedure allows for quantitative analysis of retinal ganglion cell death in an in vivo rat glaucoma model.

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

Traditional herbal medicine, an alternative medicine in the clinical setting, has received increased attention in recent years. Before delivery to the body, an additional extraction procedure is commonly required to release the active constituents from raw herbs. Water decoction is a classical extraction procedure that is still broadly used in the clinical settings. Here, we propose a detailed protocol for er-xian decoction (EXD) in order to apply herbal decoctions to experimental studies. The calculation of an animal-appropriate dose is described, as well as the four main steps of EXD: soaking, water decoction, filtration, and concentration. In addition, serum-containing EXD is introduced to rats as a means of in vitro validation. Here, rats were orally administered EXD for three days. Blood samples were then collected, inactivated, centrifuged, and filtered. The serum, diluted with the culture medium, can be utilized to treat cells or tissues in vitro. For example, EXD was applied to both in vivo and in vitro studies and demonstrated that EXD enhances osteogenesis. This protocol can be used as a reference for the preparation and application of herbal medicines.

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

Here, we present the preparation of an er-xian decoction (EXD) in four steps&#8212;soaking, decoction, filtration, and concentration&#8212;and demonstrate the administration of a prepared EXD-containing serum to rats. These methods are applicable to the in vivo and in vitro study of herbal decoctions such as traditional Chinese medicines.

Traditional herbal medicine, an alternative medicine in the clinical setting, has received increased attention in recent years. Before delivery to the ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&#8212;Application in Premanifest Huntington's Disease

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy begins many years prior to the motor onset (during the &#34;premanifest&#34; period), but the spatiotemporal pattern of regional atrophy across the brain has not been fully characterized. Here we demonstrate an online cloud-computing platform, &#34;MRICloud&#34;, which provides atlas-based whole-brain segmentation of T1-weighted images at multiple granularity levels, and thereby, enables us to access the regional features of brain anatomy. We then describe a regression model that detects statistically significant inflection points, at which regional brain atrophy starts to be noticeable, i.e. the &#34;change-point&#34;, with respect to a disease progression index. We used the CAG-age product (CAP) score to index the disease progression in HD patients. Change-point analysis of the volumetric measurements from the segmentation pipeline, therefore, provides important information of the order and pattern of structural atrophy across the brain. The paper illustrates the use of these techniques on T1-weighted MRI data of premanifest HD subjects from a large multicenter PREDICT-HD study. This design potentially has wide applications in a range of neurodegenerative diseases to investigate the dynamic changes of brain anatomy.

This paper describes a statistical model for volumetric MRI data analysis, which identifies the &#34;change-point&#34; when brain atrophy begins in premanifest Huntington&#39;s disease. Whole-brain mapping of the change-points is achieved based on brain volumes obtained using an atlas-based segmentation pipeline of T1-weighted images.

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...