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: Tween-80 (final concentration range: 0.5%-1%), DMSO, glycerin (final concentration range: 15%-20%), ethanol (final concentration range: 12% for intramuscular injection)22, and 0.9% PS.</li>
	<li>Estimate the amount of naringenin required based on the dose, number of mice, and injection frequency.
	<ol>
		<li>Order 10 mice (C57BL/6, male, 5 weeks old, SPF) to administer naringenin 5 days a week for 8 weeks. Keep mice in specific pathogen-free (SPF) conditions. 
		NOTE: The dose required for intraperitoneal injection is 20 mg/kg b.w.23.</li>
		<li>Weigh 160 mg of naringenin based on the following calculation: 20 mg/kg x 0.02 kg/mouse x 10 mice x 5 days/week x 8 weeks = 160 mg.</li>
	</ol>
	</li>
	<li>Calculate the concentration of naringenin to be injected in vivo.
	<ol>
		<li>Prepare the recommended volume based on the bodyweight of the mice. The recommended volume of naringenin applied to each mouse is 1% of body weight (0.3 mL). In this experiment, 0.2 mL per mouse was used.</li>
		<li>Calculate the total volume: 0.2 mL per mouse x 10 mice x 5 days/week x 8 weeks = 80 mL.</li>
		<li>Calculate the concentration of the Naringenin in stock: 160 mg/80 mL solvents = 2 mg/mL.</li>
		<li>Calculate the volume for each day: 0.2 mL per mouse x 10 mice = 2 mL.</li>
	</ol>
	</li>
</ol>
2. Dissolution
<ol>
	<li>Ethanol solution
	<ol>
		<li>To prepare naringenin solution at 2 mg/mL, weigh 3.52 mg of naringenin and add it into a 2.0 mL tube. 
		NOTE: To achieve a concentration of 2 mg/mL, the total volume required will be 1760 &#181;L (calculation: 3.52 mg/1760 &#181;L = 2 mg/mL).</li>
		<li>Quickly spin down (2000 x g for 30 s) to make the naringenin powder settle at the bottom of the tube (Figure 1A).</li>
		<li>Add 8.8 &#181;L of 100% ethanol to the tube to prepare a 0.5% (v/v) solution with respect to the total volume required2. Naringenin does not dissolve completely (Figure 1B).</li>
		<li>Continue to add 79.2 &#181;L of 100% ethanol to the tube to prepare a 5% (v/v) solution with respect to the total volume required (calculation: (79.2 &#181;L + 8.8 &#181;L) / 1760 &#181;L x 100% = 5%). Naringenin does not dissolve completely (Figure 1B).</li>
		<li>Add 1672 &#181;L of 0.9% PS to the tube containing 5% ethanol as described in step 2.1.4. This will produce an emulsion (Figure 1D). Centrifuge (2000 x g for 30 s) the solution to check whether naringenin is completely dissolved in the solution. White precipitates of undissolved naringenin appear in the solution (Figure 1E).</li>
	</ol>
	</li>
	<li>DMSO solution
	<ol>
		<li>To prepare naringenin solution at 2 mg/mL, weigh 3.95 mg of naringenin and add into a 2.0 mL tube. 
		&#8203;NOTE: To achieve a concentration of 2 mg/mL, the total volume required will be 1975 &#181;L (calculation: 3.95 mg/1975 &#181;L = 2 mg/mL).</li>
		<li>Quickly spin down (2000 x g for 30 s) to make the naringenin powder settle at the bottom of the tube.</li>
		<li>Add 9.8 &#181;L of DMSO to the tube to prepare a 0.5% (v/v) solution (calculation: 9.8 &#181;L/1975 &#181;L x 100% = 5%). Naringenin dissolves completely (Figure 2A).</li>
		<li>Add 88.2 &#181;L of DMSO to the tube to prepare a 5% (v/v) solution with respect to the total volume required (calculation: (88.2 &#181;L + 9.8 &#181;L) /1975 &#181;L x 100% = 5%). Naringenin dissolves completely (Figure 2B).</li>
		<li>Add 1877 &#181;L of 0.9% PS (v/v percent of 95%) to the solution prepared in step 2.2.4. An emulsion is produced (Figure 1C). Centrifuge (2000 x g for 30 s) the solution to check whether naringenin is completely dissolved in the solution. White precipitates of undissolved naringenin appear in the solution (Figure 1D).</li>
	</ol>
	</li>
	<li>Tween-80 and DMSO solution
	<ol>
		<li>To prepare the naringenin solution at 2 mg/mL, weigh 6.69 mg of naringenin and add into a 5.0 mL tube. 
		&#8203;NOTE: To achieve the final concentration of 2 mg/mL, the total solution volume required will be 3345 &#181;L (calculation: 6.69 mg/3345 &#181;L = 2 mg/mL).</li>
		<li>Quickly spin down (2000 x g for 30 s) to make the naringenin powder settle at the bottom of the tube.</li>
		<li>Add 117.7 &#181;L of DMSO to prepare a 3.5% (v/v) solution with respect to the total volume required (calculation: 117.7 &#181;L /3345 &#181;L x 100% = 3.5%). Naringenin dissolves completely (Figure 3A)</li>
		<li>Add 117.7 &#181;L of Tween 80 to the solution prepared in step 2.3.3 to attain 3.5% (v/v) Tween 80 and 3.5% (v/v) DMSO. Observe the complete dissolution of naringenin (Figure 3B)</li>
		<li>Slowly add the solution prepared in step 2.3.4 to a 5.0 mL tube containing 3109.6 &#181;L of 0.9% PS (v/v percent of 93%) and shake well to obtain an apparent naringenin solution (Figure 3C).</li>
		<li>Let the solution prepared in step 2.3.5 stay at room temperature (RT) for 2 h. The solution is still apparent without any visible precipitates (Figure 3D).</li>
	</ol>
	</li>
	<li>Preparation of naringenin solution for in vivo administration
	<ol>
		<li>According to step 1.2.2, weigh 160 mg of naringenin (160 mg / 2 mg/mL = 80 mL).</li>
		<li>Add 2.8 mL of DMSO to attain 3.5% (v/v) solution (calculation: 2.8 mL / 80 mL x 100% = 3.5%)</li>
		<li>Then, add 2.8 mL of Tween 80 to the solution prepared in step 2.4.2 to attain 3.5% (v/v) Tween 80 and 3.5% (v/v) DMSO.</li>
		<li>Aliquot the solution prepared in step 2.4.3 into four tubes, 1.4 mL per tube [(2.8 mL + 2.8 mL) / 4 = 1.4 mL].</li>
		<li>Aliquot 18.6 mL of 0.9% PS to five 15 mL tubes.</li>
		<li>Store the solution prepared in step 2.4.3 (stock solution) and 2.4.5 at 4 &#176;C (2.8 mL + 2.8 mL + 18.6 mL x 4 = 80 mL).</li>
		<li>Take 140 &#181;L of the stock solution (step 2.4.3) and mix it with 1860 &#181;L of 0.9% PS to prepare 2 mL of naringenin solution for 1 day of administeration.</li>
		<li>Filter the solution through a 0.2 &#181;m filter.</li>
	</ol>
	</li>
</ol>
3. Naringenin solution administration
<ol>
	<li>Handling and restraint
	<ol>
		<li>Spray the cage and hands with 70-75% alcohol before opening the lid. Lift the mouse by the base of the tail and place it on a solid surface to position its tail gently back.</li>
		<li>Grasp the scruff of the neck behind the ears with the left thumb and index finger and position the tail between the little and the ring finger. Keep the mouse in a supine position with its posterior end slightly elevated.</li>
	</ol>
	</li>
	<li>Injection
	<ol>
		<li>Grasp the back skin of the mouse so that the abdominal skin is taut.</li>
		<li>Push the needle (insulin syringe) in at an angle of 10&#176; between the needle and the abdominal surface, in the lower right or left quadrant of the abdomen to avoid hitting the bladder, liver, or other internal organs.</li>
		<li>Run the needle subcutaneously in a cranial direction for 3-5 mm, and then insert it at a 45&#176; angle into the abdominal cavity.</li>
		<li>When the needle passes through the abdominal wall and the resistance disappears, perform an aspiration to confirm that no reflux material is withdrawn. Then, slowly push the solution.</li>
		<li>After the injection, slowly pull the needle out and rotate it slightly to prevent leakage. The recommended volume is 50-100 &#181;L/10 g.</li>
		<li>Dispose of leftover Naringenin in a biohazard container.</li>
	</ol>
	</li>
</ol>
4. Blood glucose test
NOTE: Test the blood glucose 1 day prior to the injection and 1 and 2 months after the injection.
<ol>
	<li>Fast the mice for 15 h before the blood glucose test. Water can continue to be provided.</li>
	<li>Open the test strips and mark the date. Once opened, use the test strips within 3 months. Store the test strips at 2-30 &#176;C. Tightly close the lid after removing the strip to prevent the formation of moisture.</li>
	<li>Place the animal into the induction chamber. Turn on the vaporizer at an induction level of 4% for isoflurane and 4 L/min for oxygen. After the animal is fully anesthetized, maintain the anesthetic with the nose cone and the anesthetic delivery at a level of 1.5% for isoflurane and 0.4 L/min for oxygen.</li>
	<li>Wipe the tail with cotton balls/swabs soaked in 70%-75% alcohol.</li>
	<li>Starting from the lowest point of the tail, make a small puncture on the lateral tail vein with a 25G needle prick and squeeze out a drop of blood.</li>
	<li>Wipe the drop of blood with a tissue paper.</li>
	<li>Squeeze out another drop of blood and collect it on the edge of a test strip.</li>
	<li>Read and record the result displayed on the glucose meter.</li>
	<li>To stop the bleeding, pinch the tail of the mouse with a sterile gauze and wipe the area with 75% alcohol.</li>
	<li>Additional samples may be obtained by a similar technique moving up the tail cranially.</li>
</ol>
5. TRAP staining
<ol>
	<li>Slides preparation
	<ol>
		<li>Perform fasting blood glucose tests on the mice once a week. When the blood glucose levels are &#8805; 11.1 mmol/L (indicative of successful type II diabetes mice model24), euthanize the mice using CO2, followed by cervical disclocation, and collect the Lumbar 4th-6th (L4-L6) (no euthanasia is performed while fasting blood glucose test).</li>
		<li>Fix the L4-L6 samples with 4% paraformaldehyde for 24 h (ensure that the volume of paraformaldehyde is &#62;20x the volume of the tissue), and then wash for 2 h in continuous flow of tap water.</li>
		<li>To decalcify the samples, immerse them in a 10% ethylenediaminetetraacetic acid (EDTA) solution for 2 weeks at RT in static condition until the samples are softened. Ensure that the volume of the decalcifying solution is 20-30 times the volume of the tissue/sample. Change the EDTA solution every other day.</li>
		<li>Dehydrate the sample using a dehydrator.
		<ol>
			<li>Place the specimens in tissue processing embedding cassettes. Number the cassette using a pencil.</li>
			<li>Set the dehydrator program as follows: 75% alcohol for 2 h, 85% alcohol for 1 h, 95% alcohol for 1 h, 95% alcohol for 2 h, anhydrous ethanol (I) for 2 h, anhydrous ethanol (II) for 2 h, anhydrous ethanol (III) for 2 h, xylene (I) for 1 h, xylene (II) for 1 h, xylene (III) for 1 h, paraffin wax (I) for 2 h, and paraffin wax (II) for 2 h, at RT.</li>
		</ol>
		</li>
		<li>Embed the samples in paraffin wax.
		<ol>
			<li>Add paraffin wax to the cassette tray of the paraffin embedding station and heat to 60 &#176;C. Immerse the dehydrated specimens for at least 2 h.</li>
			<li>Place the tissue cassette in the cassette tray and preheat.</li>
			<li>Add paraffin wax to the paraffin reservoir and heat to 60 &#176;C.</li>
			<li>After 2 h, take both tissue cassette and specimens to the work area. Pour the preheated paraffin wax from the paraffin reservoir into the tissue cassette. Place the specimen into the paraffin wax, ensure that the paraffin wax completely covers the tissue, and then immediately move the cassette onto an icing station.</li>
			<li>Use a microtome to cut the paraffin-embedded samples into 5-6 &#181;m sections. Unfold the sections in 40 &#176;C warm water for less than 10 s. Collect the sections on APS (amino silane) coated glass slides. Dry the slides at RT for 1 h, and then move the slides into an oven set at 60 &#176;C for overnight.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>TRAP reagent preparation
	<ol>
		<li>Prepare basic stock incubation solution: Dissolve 9.2 g of sodium acetate anhydrous, 11.4 g of L-(+) tartaric acid, and 2.8 mL of glacial acid in 1000 mL of distilled water. Adjust pH to 4.7-5.0 and store at RT for up to 6 months.</li>
		<li>Prepare Naphthol-ether solution: Dissolve 0.1 g of naphthol AS-BI phosphate in 5 mL of ethylene glycol monoethyl ether. Store at 4 &#176;C for up to 5 weeks.</li>
		<li>Prepare Sodium nitrite solution: Dissolve 1 g of sodium nitrite in 25 mL of distilled water. Store at 4 &#176;C.</li>
		<li>Prepare Pararosaniline dye: Add 1 g of pararosaniline base to 20 mL of 2N HCl (83 mL of HCl in 417 mL of water). Use a stir plate to dissolve the base and filter it before use.</li>
	</ol>
	</li>
	<li>TRAP staining
	<ol>
		<li>Fill two Coplin jars with 50 mL of basic stock incubation solution and place in a 37 &#176;C oven for 2 h.</li>
		<li>Take one Coplin jar and add 0.5 mL of naptol-ether solution.</li>
		<li>Place the slides in the Coplin jar and incubate at 37 &#176;C for 1 h. 
		NOTE: Prepare atleast three slides for each group.</li>
		<li>A few minutes before the incubation time is over, add 1 mL of sodium nitrite solution and 1 mL of pararosaniline dye. Mix gently for 30 s and leave it for 2 min.</li>
		<li>Add the solution prepared in step 5.2.2 to the other preheated Coplin jar containing the basic stock solution. Mix the solution well and insert the slides from the Coplin jar in step 5.3.3.</li>
		<li>Incubate for 15-20 min at RT.</li>
		<li>Rinse the slices in another Coplin jar with 200 mL of PBS for 5 min.</li>
		<li>Counter-stain the slices with 100% hematoxylin for 30 s in a Coplin jar.</li>
		<li>Dehydrate the slices with 85%, 95%, and 100% alcohol (200 mL) for 2 min each and treat in xylene (200 mL) for 2 min for 3x. Use Coplin jars to perform each step.</li>
		<li>Secure the section on the cover glass with a coverslip using resin. Ensure to avoid the trapping of air bubbles.</li>
	</ol>
	</li>
</ol>
6. ELISA
<ol>
	<li>Sample preparation
	<ol>
		<li>Remove the soft tissues from the femur and the tibia of the mouse. Clean the bones with gauze.</li>
		<li>Place the bone samples into a 1 mL microcentrifuge tube and store the sample tubes at -80 &#176;C. 
		NOTE: The samples can also be stored in a liquid nitrogen tank for no more than 6 months.</li>
		<li>Weigh the bone sample. Dilute with 0.9% PS at a ratio of 1:10. For example, dilute 0.1 g of bone with 1 mL of PS.</li>
		<li>Add 3 mm zirconia beads into the tube and grind the samples 3x at 70 Hz for 30 s with 20 s rest in between.</li>
		<li>Centrifuge the samples at 4 &#176;C and 12,000 x g for 5 min. Collect the supernatant.</li>
	</ol>
	</li>
	<li>ELISA assay kit 
	NOTE: Perform ELISA according to the assay protocol specified by the manufacturer.
	<ol>
		<li>Add 100 &#181;L of each dilution of standard, blank, and sample into the appropriate wells. Incubate for 90 min at 37 &#176;C.</li>
		<li>Decant the liquid from each well and add 100 &#181;L of biotinylated detection antibody working solution. Incubate for 1 h at 37 &#176;C.</li>
		<li>Decant the solution, add 350 &#181;L of wash buffer to each well. Soak for 1 min, repeat 3x.</li>
		<li>Add 100 &#181;L of HRP conjugate working solution to each well. Incubate for 30 min at 37 &#176;C.</li>
		<li>Decant the solution. Repeat the wash process 5x as described in step 6.2.3.</li>
		<li>Add 90 &#181;L of the substrate reagent. Incubate for 15 min at 37 &#176;C protected from light.</li>
		<li>Add 50 &#181;L of the stop solution.</li>
	</ol>
	</li>
	<li>Use a plate reader to record the absorbance of each well at 450 nm.</li>
	<li>Standard curve generation
	<ol>
		<li>Plot the respective mean absorbance values against the serially diluted protein concentrations.</li>
		<li>Join the points to create the best fit curve. Use any appropriate computer application (spreadsheet) to generate the standard curve equation.</li>
	</ol>
	</li>
	<li>Substitute the absorbance value of each sample in the standard curve equation to obtain the concentration of the respective sample.</li>
</ol>

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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. 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+single--and+repeated--dose+toxicity+of+dimethyl+sulfoxide.">The single--and repeated--dose toxicity of dimethyl sulfoxide.</a> Annals of the New York Academy of Sciences. 141 (1), 96-109 (1967).</li><li>Colucci, 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=New+insights+of+dimethyl+sulphoxide+effects+(DMSO)+on+experimental+in+vivo+models+of+nociception+and+inflammation.">New insights of dimethyl sulphoxide effects (DMSO) on experimental in vivo models of nociception and inflammation.</a> Pharmacological Research. 57 (6), 419-425 (2008).</li><li>Kawakami, K., Oda, N., Miyoshi, K., Funaki, T., Ida, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solubilization+behavior+of+a+poorly+soluble+drug+under+combined+use+of+surfactants+and+co-solvents.">Solubilization behavior of a poorly soluble drug under combined use of surfactants and co-solvents.</a> European Journal of Pharmaceutical Sciences. 28 (1-2), 7-14 (2006).</li></ol>

The authors have nothing to disclose.

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. There are many solvent options for dissolving compounds, of which ethanol, DMSO, and PS are the most widely used. Ethanol can dissolve many water-insoluble compounds because of its highly polar properties, allowing hydrogen bonding and thus dissolving both polar and nonpolar substances. Moreover, the concentration of ethanol may determine the properties of the phytochemical&#160;compound. For example, 75 wt.% ethanol/water solvent is considered the best for extracting the highest yield of polyphenols and has the strongest anti-oxidant properties26. Another study found that ethanol concentration could be lowered to 32.5% at 150 &#176;C for polyphenol extracts to express anti-oxidant property27. However, a high concentration of ethanol may cause neurotoxicity and hepatotoxicity28. Ethanol injection (i.p.) in a range of concentrations from 8%-32% v/v is commonly used for behavioral evaluation and may cause conditional taste aversion and hypothermia29. DMSO is a dipolar aprotic solvent of high polarity and is used as a solvent to dissolve numerous organic compounds. A comparative study indicated that DMSO/methanol (50:50 v/v) resulted in the optimum yield of phenolic acids in citrus rinds30. However, dose, concentration, and frequency are not ignorable factors when DMSO is delivered to animals. A 17.7 g/kg dose given intraperitoneally in mice attained LD50 while lowering the dose to 2.5 g/kg for 6 weeks in mice did not cause observable adverse effects31. Although the suggested DMSO concentration is 0.5%-5%, DMSO is not capable of dissolving many compounds. Colucci et al. tested the effects of DMSO and DMSO-containing saline at different concentrations by intracerebroventricular and oral administration in mice. The study demonstrated that a solution of 25% DMSO in saline did not change animal behavioral responses32. Tween 80 is a nonionic surfactant and is widely used as a co-solvent to increase the solubility of poorly soluble drugs and enhance pharmacokinetic features33. A concentration of 1% Tween 80 was chosen considering safety33. Thus, the above solvents and surfactants at different concentrations were used to fully solubilize naringenin for intraperitoneal administration.
Some suggestions are listed here for consideration. First, we suggest starting from a small amount of phytochemical compound for preliminary experiments considering consumption cost. Second, it is necessary to perform comprehensive literature research, especially close studies regarding animal species, diseases, administration routes, and frequency, before preparing the solution. Third, the concentration ranges of solvents and co-solvents such as surfactants are dependent on the available literature, preliminary experiments, and the purpose of the study design. Fourth, using an insulin syringe instead of a regular syringe is recommended to reduce the injection injury from a relatively high frequency of administration. Fifth, to maintain sterile conditions, it is recommended to sterilize the solution using a 0.2 &#181;m filter and use sterile syringes and cotton swabs soaked in alcohol when injecting the solution into live animals.
The advantages of the protocol are its simple operation and low cost. In summary, the protocol demonstrates the preparation of a phytochemical solution for intraperitoneal administration in mice, with naringenin as an example. The protocol will benefit the researchers dealing with drug screening or pharmacology.

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><tbody><tr><td>1.5 mL&amp;nbsp; 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 &amp;amp; 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&amp;nbsp; 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&lt;br/&gt; Mouse&amp;nbsp; CTX I ELISA Kit: E-EL-M0366c&lt;br/&gt; Mouse PICP ELISA Kit: E-EL-M0231c&lt;br/&gt; 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 &amp;micro;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 &amp;amp; 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>&amp;nbsp;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&amp;nbsp; EG 1150 H, LEICA&amp;nbsp; EG 1150 C</td><td>Embed&amp;nbsp; 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&amp;ndash;20 &amp;micro;L, 100&amp;ndash;1000 &amp;micro;L, 20&amp;ndash;200 &amp;micro;L</td><td>&amp;nbsp; 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 level observed 2 months ago when the animal model was established. Naringenin treatment lowered the blood glucose levels by 51.8% and 34.8% at 1 and 2 months, respectively (Table 2).&#160;STZ-induced diabetic mice exhibited bone loss, as indicated by the decrease in the bone volume/tissue volume (BV/TV) (30.97%) and the number of trabeculae (Tb.N) (11.4%), respectively. The changes in the values of these two parameters suggest that naringenin treatment significantly rescued the bone loss (Table 3).&#160;Osteoclast activity as indicated by N.oc/Tb.Ar (osteoclast number per trabecular bone area) was increased in high-fat diet and STZ-induced diabetic mice, although no statistical significance was observed between the control and the disease models. Naringenin treatment significantly decreased osteoclast activities, as shown in Figure 4 and Table 4. The C-terminal telopeptide of type I collagen (CTIX) and N-terminal propeptide of type I procollagen (PINP) were elevated by 68.09% and 204.88% in diabetic animals, respectively, indicating a dramatic increase in the bone resorption rate. Naringenin significantly decreased both indicators of the bone resorption rate (Table 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62736/62736fig01.jpg" /> 
Figure 1: Dissolving naringenin in ethanol. (A) Naringenin powder in the tube after spin down. (B) Naringenin + ethanol (400 mg/mL - 3.52 mg of naringenin in 8.8 &#181;L of ethanol). (C)&#160;Naringenin + ethanol (40 mg/mL - 3.52 mg of naringenin in 8.8 &#181;L of ethanol) (D) Naringenin in 5% (v/v) ethanol and 95% PS (0.9%). (E) Precipitates in D after spin down. (F) Measurement for obtaining scale bar for Figure 1, Figure 2, and Figure 3. Nar: Naringenin. Scale bar = 1 cm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62736/62736fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62736/62736fig02.jpg" /> 
Figure 2: Dissolving naringenin in DMSO. (A) Naringenin + DMSO (400 mg/mL - 3.95 mg of naringenin in 9.8 &#181;L of DMSO). (B) Naringenin + DMSO (40 mg / mL- 3.95 mg of naringenin in 98 &#181;L of DMSO). (C) Naringenin in 5% (v/v) DMSO and 95% PS (0.9%). (D) Precipitates in C after spin down. Nar: Naringenin. Scale bar = 1 cm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62736/62736fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62736/62736fig03.jpg" /> 
Figure 3: Dissolving naringenin in DMSO and Tween 80. (A) Naringenin + DMSO (57.2 mg/mL - 6.69 mg of naringenin in 117.7 &#181;L of DMSO). (B)&#160;Naringenin + DMSO + Tween (57.2 mg/mL - 6.69 mg of naringenin in 117.7 &#181;L of DMSO and 117.7 &#181;L of Tween 80). (C) Naringenin in the mixture of 3.5% (v/v) DMSO, 3.5% (v/v) Tween 80, and 93% PS (0.9%). (D) No precipitates in C after spin down. Nar: Naringenin. Scale bar = 1 cm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62736/62736fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62736/62736fig04.jpg" /> 
Figure 4: The effect of naringenin on the osteoclast activity of the high-fat diet-fed and STZ-injected (STZ) mice. TRAP staining of trabecular bone and osteoclasts of L4 vertebrae. Triangles indicated osteoclasts. Scale bar = 100 &#181;m. This figure has been modified from Liu et al.25. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62736/62736fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(g)</td>
			<td>0 week</td>
			<td>1 week</td>
			<td>2 weeks</td>
			<td>4 weeks</td>
			<td>5 weeks</td>
			<td>6 weeks</td>
			<td>8 weeks</td>
		</tr>
		<tr>
			<td>Control</td>
			<td>23.7 &#177; 0.2</td>
			<td>25.1 &#177; 1.3</td>
			<td>26.2 &#177; 1.0</td>
			<td>27.7 &#177; 0.5</td>
			<td>31.1 &#177; 0.7</td>
			<td>31.7 &#177; 0.8</td>
			<td>32.7 &#177; 1.3</td>
		</tr>
		<tr>
			<td>STZ</td>
			<td>16.8 &#177; 1.7**</td>
			<td>18.2 &#177; 2.5**</td>
			<td>18.6 &#177; 2.5**</td>
			<td>18.2 &#177; 1.4**</td>
			<td>21.3 &#177; 1.6**</td>
			<td>22.0 &#177; 1.4**</td>
			<td>20.8 &#177; 1.4**</td>
		</tr>
		<tr>
			<td>Naringenin</td>
			<td>16.6 &#177; 1.1**</td>
			<td>17.6 &#177; 1.5**</td>
			<td>17.4 &#177; 1.7**</td>
			<td>15.6 &#177; 1.4**&#916;&#916;</td>
			<td>18.4 &#177; 1.5**&#916;&#916;</td>
			<td>17.7 &#177; 1.4**&#916;&#916;</td>
			<td>15.5 &#177; 1.0**&#916;&#916;</td>
		</tr>
		<tr>
			<td colspan="8">** p &#60; 0.01 vs. Control</td>
		</tr>
		<tr>
			<td colspan="8">&#916;&#916; p &#60; 0.01 vs. STZ</td>
		</tr>
	</tbody>
</table>
Table 1: Bodyweight of high-fat diet-fed and STZ-injected (STZ) mice across groups and periods. Data are shown as the mean &#177; s.d. ** p &#60; 0.01 vs. Control, &#916;&#916; p &#60; 0.01 vs. STZ.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(mmol/L)</td>
			<td>0 month</td>
			<td>1 month</td>
			<td>2 months</td>
		</tr>
		<tr>
			<td>Control</td>
			<td>4.9 &#177; 0.9</td>
			<td>8.4 &#177; 0.7</td>
			<td>8.3 &#177; 0.5</td>
		</tr>
		<tr>
			<td>STZ</td>
			<td>12.8 &#177; 4.2**</td>
			<td>22.8 &#177; 4.3**</td>
			<td>15.5 &#177; 2.7*</td>
		</tr>
		<tr>
			<td>Naringenin</td>
			<td>13.2 &#177; 3.5**</td>
			<td>11.0 &#177; 1.9&#916;&#916;</td>
			<td>10.1 &#177; 5.3&#916;&#916;</td>
		</tr>
		<tr>
			<td colspan="4">* p &#60; 0.05, ** p &#60; 0.01 vs. Control</td>
		</tr>
		<tr>
			<td colspan="4">&#916;&#916; p &#60; 0.01 vs. STZ</td>
		</tr>
	</tbody>
</table>
Table 2: Fasting blood glucose of STZ mice across groups and periods. Data are shown as the mean &#177; s.d. * p &#60; 0.05 ** p &#60; 0.01 vs. Control, &#916;&#916; p &#60; 0.01 vs. STZ.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>BV/TV (%)</td>
			<td>Tb.N (1/mm)</td>
		</tr>
		<tr>
			<td>Control</td>
			<td>0.268 &#177; 0.046</td>
			<td>5.35 &#177; 0.31</td>
		</tr>
		<tr>
			<td>STZ</td>
			<td>0.185 &#177; 0.081*</td>
			<td>4.74 &#177; 0.77*</td>
		</tr>
		<tr>
			<td>Naringenin</td>
			<td>0.241 &#177; 0.032&#916;</td>
			<td>5.47 &#177; 0.19&#916;&#916;</td>
		</tr>
		<tr>
			<td>* p &#60; 0.05 vs. Control</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">&#916; p &#60; 0.05, &#916;&#916; p &#60; 0.01 vs. STZ</td>
		</tr>
	</tbody>
</table>
Table 3: Bone mass related parameters of STZ mice across groups. Data are shown as the mean &#177; s.d. * p &#60; 0.05 vs. Control, &#916; p &#60; 0.05, &#916;&#916; p &#60; 0.01 vs. STZ.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1/&#181;m2</td>
			<td>N.oc/T.Ar</td>
		</tr>
		<tr>
			<td>Control</td>
			<td>0.000182 &#177; 8.84E-05</td>
		</tr>
		<tr>
			<td>STZ</td>
			<td>0.00024 &#177; 2.06E-05</td>
		</tr>
		<tr>
			<td>Naringenin</td>
			<td>0.000156 &#177; 3.88E-05&#916;&#916;</td>
		</tr>
		<tr>
			<td colspan="2">&#916;&#916; p &#60; 0.01 vs. STZ</td>
		</tr>
	</tbody>
</table>
Table 4: Osteoclast activity of STZ mice across groups. Data are shown as the mean &#177; s.d. &#916;&#916; p &#60; 0.01 vs. STZ.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ng/mL</td>
			<td>CTIX</td>
			<td>PINP</td>
		</tr>
		<tr>
			<td>Control</td>
			<td>22 &#177; 8.98</td>
			<td>1.64 &#177; 0.95</td>
		</tr>
		<tr>
			<td>STZ</td>
			<td>36.98 &#177; 22.57</td>
			<td>5 &#177; 2.33 *</td>
		</tr>
		<tr>
			<td>Naringenin</td>
			<td>5.31 &#177; 2.09 &#916;&#916;</td>
			<td>0.85 &#177; 0.02 &#916;&#916;</td>
		</tr>
		<tr>
			<td colspan="3">* p &#60; 0.05 vs. Control</td>
		</tr>
		<tr>
			<td colspan="3">&#916;&#916; p &#60; 0.01 vs. STZ</td>
		</tr>
	</tbody>
</table>
Table 5: Bone resorption rate of STZ mice across groups. Data are shown as the mean &#177; s.d. * p &#60; 0.05 vs. Control, &#916;&#916; p &#60; 0.01 vs. STZ.

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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Universidad San Sebastian

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Medicine

3.2K Views.  Shanghai University of Traditional Chinese Medicine. 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.

Video: Preparation of Naringenin Solution for In Vivo Application

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted mechanism of metabolic activation involves formation of &#945;-hydroxynitrosamines that spontaneously decompose to DNA alkylating agents. Accumulation of DNA damage and the resulting mutations can ultimately lead to cancer. New evidence indicates that &#945;-hydroxynitrosamines can be further oxidized to nitrosamides processively by cytochrome P450s. Because nitrosamides are generally more stable than &#945;-hydroxynitrosamines and can also alkylate DNA, nitrosamides may play a role in carcinogenesis. In this report, we describe a general protocol for evaluating nitrosamide production from in vitro cytochrome P450-catalyzed metabolism of nitrosamines. This protocol utilizes a general approach to the synthesis of the relevant nitrosamides and an in vitro cytochrome P450 metabolism assay using liquid chromatography-nanospray ionization-high resolution tandem mass spectrometry for detection. This method detected N&#8242;-nitrosonorcotinine as a minor metabolite of N&#8242;-nitrosonornicotine in the example study. The method has high sensitivity and selectively due to accurate mass detection. Application of this method to a wide variety of nitrosamine-cytochrome P450 systems will help determine the generality of this transformation. Because cytochrome P450s are polymorphic and vary in activity, a better understanding of nitrosamide formation could aid in individual cancer risk assessment.

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

&#945;-hydroxylation of carcinogenic nitrosamines by cytochrome P450s is the accepted metabolic pathway that produces DNA-damaging intermediates, which cause mutations. However, new data indicates further oxidation to nitrosamides can occur. We describe a general method for detecting nitrosamides produced from in vitro cytochrome P450-catalyzed metabolism of nitrosamines.

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted ...

Cancer Research

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

Epidemiological studies indicate that increased flavonoid intake correlates with decreased mortality due to cardiovascular diseases (CVD) in the United States (US) and Europe. Berries are widely consumed in the US and have a high polyphenolic content. Polyphenols have been shown to interact with many molecular targets and to exert numerous positive biological functions, including antioxidant, anti-inflammatory, and cardioprotective effects. Polyphenols isolated from blackberry (BL), raspberry (RB), and black raspberry (BRB) reduce oxidative stress and cellular senescence in response to angiotensin II (Ang II). This work provides a detailed description of the protocol used to prepare the polyphenol extracts from freeze-dried berries. Polyphenol extractions from freeze-dried berry powder were performed using 80% aqueous ethanol and an ultrasonic-assisted extraction method. The crude extract was further purified and fractionated using chloroform and ethyl acetate, respectively. The effects of both crude and purified extracts were tested on Vascular Smooth Muscle Cells (VSMCs) in culture.

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

This work details a step-by-step method to prepare polyphenol-rich extracts from freeze-dried berry powder. In addition, it provides a thorough description of how to use these polyphenol-rich extracts in cell culture in the presence of the peptide hormone angiotensin II (Ang II) using Vascular Smooth Muscle Cells (VSMCs).

Epidemiological studies indicate that increased flavonoid intake correlates with decreased mortality due to cardiovascular diseases (CVD) in the United ...

Chemistry

Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro enzymatic synthesis of a flavonol. In this method, Atf3h and Atfls1, two key genes in the biosynthetic pathway of the flavonols, are cloned and overexpressed in Escherichia coli. The recombinant enzymes are purified via an affinity column and then a bienzymatic cascade is established in a specific synthetic buffer. Two flavonols are synthesized in this system as examples and determined by TLC and HPLC/LC/MS analyses. The method displays obvious advantages in the derivation of flavonols over other approaches. It is time- and labor-saving and highly cost-effective. The reaction is easy to be accurately controlled and thus scaled up for mass production. The target product can be purified easily due to the simple components in the system. However, this system is usually restricted to the production of a flavonol from a flavanone.

The derivation of a flavonol is crucial for its application in healthcare and the food industry. Here, we provide a detailed protocol for the biosynthesis of a flavonol from a flavanone and discuss the crucial steps and its advantages over other approaches.

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro ...

Biochemistry

Scaled-Up Preparation of an Intermediate of Upatinib, ACT051-3

Upatinib, a Janus kinase inhibitor drug, was developed by a biotech company to treat immune diseases. The compound tert-butyl (5-toluenesulfonyl-5h-pyrrole [2,3-b] pyrazine-2-yl) carbamate (ACT051-3) is an important intermediate of Upatinib. To date, the steady industrial production of this intermediate compound (ACT051-3) has not been reported. In this study, we described the specific synthesis method and process of the compound ACT051-3 in terms of laboratory synthesis, pilot scale-up, and industrial production. During the exploration of the process route for ACT051-3, many appropriate adjustments and improvements were made to the reaction conditions, finally leading to the successful development of the optimal industrial production process for ACT051-3. The reaction time was nearly doubled by changing the state of the potassium carbonate involved in the reaction, which greatly improved the reaction efficiency. Additionally, by introducing N,N-diisopropylethylamine (DIPEA) to the reaction, the amount of the expensive catalyst Pd(OAc)2 was reduced 2.5-fold, significantly lowering production costs, confirming the feasibility of this process route and the industrial production of ACT051-3, and satisfying market demand for this important intermediate.

Here, we present a protocol for the scaled-up synthesis of the intermediate tert-butyl (5-toluenesulfonyl-5h-pyrrole [2,3-b] pyrazine-2-yl) carbamate (ACT051-3) of Upatinib.

Upatinib, a Janus kinase inhibitor drug, was developed by a biotech company to treat immune diseases. The compound tert-butyl ...

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling molecules at low levels and damaging molecules at high levels. Accumulation of ROS in stressed plants can damage metabolites, enzymes, lipids, and DNA, causing a reduction of plant growth and yield. The ability of cerium oxide nanoparticles (nanoceria) to catalytically scavenge ROS in vivo provides a unique tool to understand and bioengineer plant abiotic stress tolerance. Here, we present a protocol to synthesize and characterize poly (acrylic) acid coated nanoceria (PNC), interface the nanoparticles with plants via leaf lamina infiltration, and monitor their distribution and ROS scavenging in vivo using confocal microscopy. Current molecular tools for manipulating ROS accumulation in plants are limited to model species and require laborious transformation methods. This protocol for in vivo ROS scavenging has the potential to be applied to wild type plants with broad leaves and leaf structure like Arabidopsis thaliana.

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...

Bioengineering

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 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 ...

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into nitrite and nitrate by interactions with various oxy-heme proteins and other still not well known pathways. The reverse process, reduction of nitrite and nitrate into NO had been discovered in mammals in the last decade and it is gaining attention as one of the possible pathways to either prevent or ease a whole range of cardiovascular, metabolic and muscular disorders that are thought to be associated with decreased levels of NO. It is therefore important to estimate the amount of NO and its metabolites in different body compartments &#8212; blood, body fluids and the various tissues. Blood, due to its easy accessibility, is the preferred compartment used for estimation of NO metabolites. Due to its short lifetime (few milliseconds) and low sub-nanomolar concentration, direct reliable measurements of blood NO in vivo present great technical difficulties. Thus NO availability is usually estimated based on the amount of its oxidation products, nitrite and nitrate. These two metabolites are always measured separately. There are several well established methods to determine their concentrations in biological fluids and tissues. Here we present a protocol for chemiluminescence method (CL), based on spectrophotometrical detection of NO after nitrite or nitrate reduction by tri-iodide or vanadium(III) chloride solutions, respectively. The sensitivity for nitrite and nitrate detection is in low nanomolar range, which sets CL as the most sensitive method currently available to determine changes in NO metabolic pathways. We explain in detail how to prepare samples from biological fluids and tissues in order to preserve original amounts of nitrite and nitrate present at the time of collection and how to determine their respective amounts in samples. Limitations of the CL technique are also explained.

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

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Biology

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

A Bright NIR-II Fluorescence Probe for Vascular and Tumor Imaging

As an emerging imaging technology, near-infrared II (NIR-II, 1000-1700 nm) fluorescence imaging has significant potential in the biomedical field, owing to its high sensitivity, deep tissue penetration, and superior imaging with spatial and temporal resolution. However, the method to facilitate the implementation of NIR-II fluorescence imaging for some urgently needed fields, such as medical science and pharmacy, has puzzled relevant researchers. This protocol describes in detail the construction and bioimaging applications of a NIR-II fluorescence molecular probe, HLY1, with a D-A-D (donor-acceptor-donor) skeleton. HLY1 showed good optical properties and biocompatibility. Furthermore, NIR-II vascular and tumor imaging in mice was performed using a NIR-II optics imaging device. Real-time high-resolution NIR-II fluorescence images were acquired to guide the detection of tumors and vascular diseases. From probe preparation to data acquisition, the imaging quality is greatly improved, and the authenticity of the NIR-II molecular probes for data recording in intravital imaging is ensured.

The present protocol describes a detailed, real-time NIR-II fluorescence imaging operation of a mouse using a NIR-II optics imaging device.

As an emerging imaging technology, near-infrared II (NIR-II, 1000-1700 nm) fluorescence imaging has significant potential in the biomedical field, owing ...

Preparation of Naringenin Solution for In Vivo Application

Summary

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Chapters in this video

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade

Scaled-Up Preparation of an Intermediate of Upatinib, ACT051-3

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

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

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

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells

A Bright NIR-II Fluorescence Probe for Vascular and Tumor Imaging