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 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 gavage^9,10,11,12. Besides, other studies have reported supplementing naringenin with chow at 3% (wt/wt) for oral intake at a dose of 3.6 g/kg/d^13,14. Studies have also reported using ethanol (0.5% v/v), PS, and DMSO to dissolve naringenin for intraperitoneal injection at 10-50 mg/kg^15,16,17,18. 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 mice^20,21. 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.

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: 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.
2. Estimate the amount of naringenin required based on the dose, number of mice, and injection frequency.
   1. 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.
   2. 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.
3. Calculate the concentration of naringenin to be injected in vivo.
   1. 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.
   2. Calculate the total volume: 0.2 mL per mouse x 10 mice x 5 days/week x 8 weeks = 80 mL.
   3. Calculate the concentration of the Naringenin in stock: 160 mg/80 mL solvents = 2 mg/mL.
   4. Calculate the volume for each day: 0.2 mL per mouse x 10 mice = 2 mL.

2. Dissolution

1. Ethanol solution
   1. 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 µL (calculation: 3.52 mg/1760 µL = 2 mg/mL).
   2. Quickly spin down (2000 x g for 30 s) to make the naringenin powder settle at the bottom of the tube (Figure 1A).
   3. Add 8.8 µL 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).
   4. Continue to add 79.2 µL of 100% ethanol to the tube to prepare a 5% (v/v) solution with respect to the total volume required (calculation: (79.2 µL + 8.8 µL) / 1760 µL x 100% = 5%). Naringenin does not dissolve completely (Figure 1B).
   5. Add 1672 µL 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).
2. DMSO solution
   1. To prepare naringenin solution at 2 mg/mL, weigh 3.95 mg of naringenin and add into a 2.0 mL tube.
      ​NOTE: To achieve a concentration of 2 mg/mL, the total volume required will be 1975 µL (calculation: 3.95 mg/1975 µL = 2 mg/mL).
   2. Quickly spin down (2000 x g for 30 s) to make the naringenin powder settle at the bottom of the tube.
   3. Add 9.8 µL of DMSO to the tube to prepare a 0.5% (v/v) solution (calculation: 9.8 µL/1975 µL x 100% = 5%). Naringenin dissolves completely (Figure 2A).
   4. Add 88.2 µL of DMSO to the tube to prepare a 5% (v/v) solution with respect to the total volume required (calculation: (88.2 µL + 9.8 µL) /1975 µL x 100% = 5%). Naringenin dissolves completely (Figure 2B).
   5. Add 1877 µL 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).
3. Tween-80 and DMSO solution
   1. To prepare the naringenin solution at 2 mg/mL, weigh 6.69 mg of naringenin and add into a 5.0 mL tube.
      ​NOTE: To achieve the final concentration of 2 mg/mL, the total solution volume required will be 3345 µL (calculation: 6.69 mg/3345 µL = 2 mg/mL).
   2. Quickly spin down (2000 x g for 30 s) to make the naringenin powder settle at the bottom of the tube.
   3. Add 117.7 µL of DMSO to prepare a 3.5% (v/v) solution with respect to the total volume required (calculation: 117.7 µL /3345 µL x 100% = 3.5%). Naringenin dissolves completely (Figure 3A)
   4. Add 117.7 µL 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)
   5. Slowly add the solution prepared in step 2.3.4 to a 5.0 mL tube containing 3109.6 µL of 0.9% PS (v/v percent of 93%) and shake well to obtain an apparent naringenin solution (Figure 3C).
   6. 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).
4. Preparation of naringenin solution for in vivo administration
   1. According to step 1.2.2, weigh 160 mg of naringenin (160 mg / 2 mg/mL = 80 mL).
   2. Add 2.8 mL of DMSO to attain 3.5% (v/v) solution (calculation: 2.8 mL / 80 mL x 100% = 3.5%)
   3. 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.
   4. 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].
   5. Aliquot 18.6 mL of 0.9% PS to five 15 mL tubes.
   6. Store the solution prepared in step 2.4.3 (stock solution) and 2.4.5 at 4 °C (2.8 mL + 2.8 mL + 18.6 mL x 4 = 80 mL).
   7. Take 140 µL of the stock solution (step 2.4.3) and mix it with 1860 µL of 0.9% PS to prepare 2 mL of naringenin solution for 1 day of administeration.
   8. Filter the solution through a 0.2 µm filter.

3. Naringenin solution administration

1. Handling and restraint
   1. 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.
   2. 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.
2. Injection
   1. Grasp the back skin of the mouse so that the abdominal skin is taut.
   2. Push the needle (insulin syringe) in at an angle of 10° 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.
   3. Run the needle subcutaneously in a cranial direction for 3-5 mm, and then insert it at a 45° angle into the abdominal cavity.
   4. 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.
   5. After the injection, slowly pull the needle out and rotate it slightly to prevent leakage. The recommended volume is 50-100 µL/10 g.
   6. Dispose of leftover Naringenin in a biohazard container.

4. Blood glucose test

NOTE: Test the blood glucose 1 day prior to the injection and 1 and 2 months after the injection.

1. Fast the mice for 15 h before the blood glucose test. Water can continue to be provided.
2. Open the test strips and mark the date. Once opened, use the test strips within 3 months. Store the test strips at 2-30 °C. Tightly close the lid after removing the strip to prevent the formation of moisture.
3. 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.
4. Wipe the tail with cotton balls/swabs soaked in 70%-75% alcohol.
5. 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.
6. Wipe the drop of blood with a tissue paper.
7. Squeeze out another drop of blood and collect it on the edge of a test strip.
8. Read and record the result displayed on the glucose meter.
9. To stop the bleeding, pinch the tail of the mouse with a sterile gauze and wipe the area with 75% alcohol.
10. Additional samples may be obtained by a similar technique moving up the tail cranially.

5. TRAP staining

1. Slides preparation
   1. Perform fasting blood glucose tests on the mice once a week. When the blood glucose levels are ≥ 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 4^th-6^th (L4-L6) (no euthanasia is performed while fasting blood glucose test).
   2. Fix the L4-L6 samples with 4% paraformaldehyde for 24 h (ensure that the volume of paraformaldehyde is >20x the volume of the tissue), and then wash for 2 h in continuous flow of tap water.
   3. 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.
   4. Dehydrate the sample using a dehydrator.
      1. Place the specimens in tissue processing embedding cassettes. Number the cassette using a pencil.
      2. 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.
   5. Embed the samples in paraffin wax.
      1. Add paraffin wax to the cassette tray of the paraffin embedding station and heat to 60 °C. Immerse the dehydrated specimens for at least 2 h.
      2. Place the tissue cassette in the cassette tray and preheat.
      3. Add paraffin wax to the paraffin reservoir and heat to 60 °C.
      4. 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.
      5. Use a microtome to cut the paraffin-embedded samples into 5-6 µm sections. Unfold the sections in 40 °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 °C for overnight.
2. TRAP reagent preparation
   1. 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.
   2. Prepare Naphthol-ether solution: Dissolve 0.1 g of naphthol AS-BI phosphate in 5 mL of ethylene glycol monoethyl ether. Store at 4 °C for up to 5 weeks.
   3. Prepare Sodium nitrite solution: Dissolve 1 g of sodium nitrite in 25 mL of distilled water. Store at 4 °C.
   4. 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.
3. TRAP staining
   1. Fill two Coplin jars with 50 mL of basic stock incubation solution and place in a 37 °C oven for 2 h.
   2. Take one Coplin jar and add 0.5 mL of naptol-ether solution.
   3. Place the slides in the Coplin jar and incubate at 37 °C for 1 h.
      NOTE: Prepare atleast three slides for each group.
   4. 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.
   5. 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.
   6. Incubate for 15-20 min at RT.
   7. Rinse the slices in another Coplin jar with 200 mL of PBS for 5 min.
   8. Counter-stain the slices with 100% hematoxylin for 30 s in a Coplin jar.
   9. 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.
   10. Secure the section on the cover glass with a coverslip using resin. Ensure to avoid the trapping of air bubbles.

6. ELISA

1. Sample preparation
   1. Remove the soft tissues from the femur and the tibia of the mouse. Clean the bones with gauze.
   2. Place the bone samples into a 1 mL microcentrifuge tube and store the sample tubes at -80 °C.
      NOTE: The samples can also be stored in a liquid nitrogen tank for no more than 6 months.
   3. 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.
   4. 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.
   5. Centrifuge the samples at 4 °C and 12,000 x g for 5 min. Collect the supernatant.
2. ELISA assay kit
   NOTE: Perform ELISA according to the assay protocol specified by the manufacturer.
   1. Add 100 µL of each dilution of standard, blank, and sample into the appropriate wells. Incubate for 90 min at 37 °C.
   2. Decant the liquid from each well and add 100 µL of biotinylated detection antibody working solution. Incubate for 1 h at 37 °C.
   3. Decant the solution, add 350 µL of wash buffer to each well. Soak for 1 min, repeat 3x.
   4. Add 100 µL of HRP conjugate working solution to each well. Incubate for 30 min at 37 °C.
   5. Decant the solution. Repeat the wash process 5x as described in step 6.2.3.
   6. Add 90 µL of the substrate reagent. Incubate for 15 min at 37 °C protected from light.
   7. Add 50 µL of the stop solution.
3. Use a plate reader to record the absorbance of each well at 450 nm.
4. Standard curve generation
   1. Plot the respective mean absorbance values against the serially diluted protein concentrations.
   2. Join the points to create the best fit curve. Use any appropriate computer application (spreadsheet) to generate the standard curve equation.
5. Substitute the absorbance value of each sample in the standard curve equation to obtain the concentration of the respective sample.

Representative Results

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.

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. 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.

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