A subscription to JoVE is required to view this content. Sign in or start your free trial.
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.
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.
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 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....
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
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.......
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........
This work was supported by the National Natural Science Foundation of China (81973607 and 81573992).
....Name | Company | Catalog Number | Comments |
1.5 mLÂ microtubes | Corning Science (Wujiang) Co. | 23218392 | Holding liquid |
Automatic Dehydrator | Leica Microsystems (Shanghai) Co. | LEICA ASP 300S | Dehydrate samples |
Blood glucose test strips | Johnson & Johnson (China) Medical Equipment Co. | 4130392 | |
Centrifuge | MIULAB | Minute centrifuge | Centrifugal solution |
Dehydrator | Leica Microsystems (Shanghai) Trading Co. | LEICAÂ ASP300S | Dehydration |
DMSO | Sangon Biotech (Shanghai ) Co.,Ltd. | E918BA0041 | Co-Solvent |
ELISA assay kit | Elabscience Biotechnology Co.,Ltd | Mouse COL1(Collagen Type I) ELISA Kit: E-EL-M0325c Mouse CTX I ELISA Kit: E-EL-M0366c Mouse PICP ELISA Kit: E-EL-M0231c Mouse PINP ELISA Kit: E-EL-M0233c | |
Ethanol absolute | Sinopharm Chemical ReagentCo., Ltd | 10009218 | Co-Solvent |
Ethylene glycol monoethyl ether | Sangon Biotech (Shanghai ) Co.,Ltd. | A501118-0500 | TRAP staining |
Ethylenediaminetetraacetic acid (EDTA) | Sinopharm Chemical ReagentCo., Ltd | 10009617 | Decalcification |
Filter | Merck Millpore LTD. | Millex-GP, 0.22 µm | filter solution |
Glacial acid | Sinopharm Chemical ReagentCo., Ltd | 10000218 | TRAP staining |
Glucose meter | Johnson & Johnson (China) Medical Equipment Co. | One Touch Ultra Vue | Serial number:COJJG8GW |
Grinder | Shanghaijingxin Experimental Technology | Tissuelyser-24 | |
Hematoxylin | Nanjing Jiancheng Bioengineering Institute | D005 | TRAP staining |
Insulin syringe | Shanghai Kantaray Medical Devices Co. | 0.33 mm x 13 mm, RW LB | Intraperitoneal injection |
L-(+) tartaric acid | Sinopharm Chemical ReagentCo., Ltd | 100220008 | TRAP staining |
Microscope | OLYMPUS | sz61 | Observation |
Microtome | Leica Microsystems (Shanghai) Trading Co. | LEICA RM 2135 | Section |
Mini centrifuge | Hangzzhou Miu Instruments Co., Ltd. | Â Mini-6KC | Centrifuge |
Naphthol AS-BI phosphate | SIGMA-ALDRICH | BCBS3419 | TRAP staining |
Naringenin | Jiangsu Yongjian Pharmaceutical Co.,Ltd | 102764 | Solute |
Paraffin Embedding station | Leica Microsystems (Shanghai) Co. | LEICA EG 1150 H, LEICA EG 1150 C | Embed samples |
Pararosaniline base | BBI Life Sciences | E112BA0045 | TRAP staining |
Pipettes | eppendorf | 2–20 µL, 100–1000 µL, 20–200 µL |  transferre Liquid |
Plate reader | BioTek Instruments USA, Inc. | BioTek CYTATION 3 imaging reader | ELISA |
Resin | Shanghai Yyang Instrument Co., Ltd. | Neutral balsam | TRAP staining |
saline (0.9 PS) | Baxter Healthcare (Shanghai) Co.,Ltd | A6E1323 | Solvent |
Sodium acetate anhydrous | Sinopharm Chemical ReagentCo., Ltd | Merck-1.06268.0250 | 250g | TRAP staining |
Sodium nitrite | Sinopharm Chemical ReagentCo., Ltd | 10020018 | TRAP staining |
Tween-80 | Sangon Biotech (Shanghai ) Co.,Ltd. | E819BA0006 | Emulsifier |
Zirconia beads | Shanghaijingxin Experimental Technology | 11079125z 454g | Grinding |
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved