Preparation of Naringenin Solution for In Vivo Application

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

1. Prepare the following solvents.......

Discussion

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........

Materials

Name	Company	Catalog Number	Comments
1.5 mL  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.	LEICA  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.	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