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 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.
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
2. Dissolution
3. Naringenin solution administration
4. Blood glucose test
NOTE: Test the blood glucose 1 day prior to the injection and 1 and 2 months after the injection.
5. TRAP staining
6. ELISA
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). 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). 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).
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 µL of ethanol). (C) Naringenin + ethanol (40 mg/mL – 3.52 mg of naringenin in 8.8 µL 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. Please click here to view a larger version of this figure.
Figure 2: Dissolving naringenin in DMSO. (A) Naringenin + DMSO (400 mg/mL – 3.95 mg of naringenin in 9.8 µL of DMSO). (B) Naringenin + DMSO (40 mg / mL- 3.95 mg of naringenin in 98 µL 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. Please click here to view a larger version of this figure.
Figure 3: Dissolving naringenin in DMSO and Tween 80. (A) Naringenin + DMSO (57.2 mg/mL – 6.69 mg of naringenin in 117.7 µL of DMSO). (B) Naringenin + DMSO + Tween (57.2 mg/mL – 6.69 mg of naringenin in 117.7 µL of DMSO and 117.7 µL 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. Please click here to view a larger version of this figure.
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 µm. This figure has been modified from Liu et al.25. Please click here to view a larger version of this figure.
(g) | 0 week | 1 week | 2 weeks | 4 weeks | 5 weeks | 6 weeks | 8 weeks |
Control | 23.7 ± 0.2 | 25.1 ± 1.3 | 26.2 ± 1.0 | 27.7 ± 0.5 | 31.1 ± 0.7 | 31.7 ± 0.8 | 32.7 ± 1.3 |
STZ | 16.8 ± 1.7** | 18.2 ± 2.5** | 18.6 ± 2.5** | 18.2 ± 1.4** | 21.3 ± 1.6** | 22.0 ± 1.4** | 20.8 ± 1.4** |
Naringenin | 16.6 ± 1.1** | 17.6 ± 1.5** | 17.4 ± 1.7** | 15.6 ± 1.4**ΔΔ | 18.4 ± 1.5**ΔΔ | 17.7 ± 1.4**ΔΔ | 15.5 ± 1.0**ΔΔ |
** p < 0.01 vs. Control | |||||||
ΔΔ p < 0.01 vs. STZ |
Table 1: Bodyweight of high-fat diet-fed and STZ-injected (STZ) mice across groups and periods. Data are shown as the mean ± s.d. ** p < 0.01 vs. Control, ΔΔ p < 0.01 vs. STZ.
(mmol/L) | 0 month | 1 month | 2 months |
Control | 4.9 ± 0.9 | 8.4 ± 0.7 | 8.3 ± 0.5 |
STZ | 12.8 ± 4.2** | 22.8 ± 4.3** | 15.5 ± 2.7* |
Naringenin | 13.2 ± 3.5** | 11.0 ± 1.9ΔΔ | 10.1 ± 5.3ΔΔ |
* p < 0.05, ** p < 0.01 vs. Control | |||
ΔΔ p < 0.01 vs. STZ |
Table 2: Fasting blood glucose of STZ mice across groups and periods. Data are shown as the mean ± s.d. * p < 0.05 ** p < 0.01 vs. Control, ΔΔ p < 0.01 vs. STZ.
BV/TV (%) | Tb.N (1/mm) | |
Control | 0.268 ± 0.046 | 5.35 ± 0.31 |
STZ | 0.185 ± 0.081* | 4.74 ± 0.77* |
Naringenin | 0.241 ± 0.032Δ | 5.47 ± 0.19ΔΔ |
* p < 0.05 vs. Control | ||
Δ p < 0.05, ΔΔ p < 0.01 vs. STZ |
Table 3: Bone mass related parameters of STZ mice across groups. Data are shown as the mean ± s.d. * p < 0.05 vs. Control, Δ p < 0.05, ΔΔ p < 0.01 vs. STZ.
1/µm2 | N.oc/T.Ar |
Control | 0.000182 ± 8.84E-05 |
STZ | 0.00024 ± 2.06E-05 |
Naringenin | 0.000156 ± 3.88E-05ΔΔ |
ΔΔ p < 0.01 vs. STZ |
Table 4: Osteoclast activity of STZ mice across groups. Data are shown as the mean ± s.d. ΔΔ p < 0.01 vs. STZ.
ng/mL | CTIX | PINP |
Control | 22 ± 8.98 | 1.64 ± 0.95 |
STZ | 36.98 ± 22.57 | 5 ± 2.33 * |
Naringenin | 5.31 ± 2.09 ΔΔ | 0.85 ± 0.02 ΔΔ |
* p < 0.05 vs. Control | ||
ΔΔ p < 0.01 vs. STZ |
Table 5: Bone resorption rate of STZ mice across groups. Data are shown as the mean ± s.d. * p < 0.05 vs. Control, ΔΔ p < 0.01 vs. STZ.
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 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 °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 µm 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.
The authors have nothing to disclose.
This work was supported by the National Natural Science Foundation of China (81973607 and 81573992).
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 |