Preparation of Naringenin Solution for In Vivo Application

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Second, using an insulin syringe over a regular syringe is recommended To prepare a two milligrams per milliliter naringenin solution in ethanol, weigh 3.52 milligrams of naringenin powder and add it to a two milliliter tube. Quickly spin the tube to settle the powder at the bottom of the tube. Next, add 8.8 microliters of 100%ethanol to the tube to prepare a 0.5%solution.

Then add 79.2 microliters of 100%ethanol to the tube to prepare a 5%solution. Next, add 1.672 milliliters of 0.9%physiological saline to the tube containing the 5%solution. This will produce an emulsion.

Then centrifuge refused the tube to check whether the naringenin is completely dissolved in the solution. White precipitates of undissolved naringenin may appear in the solution. Next, to prepare a two milligram per milliliter naringenin solution in DMSO, weigh 3.95 milligrams of naringenin powder and add it into a two milliliter tube.

After a quick spin, add 9.8 milliliters of DMSO to the tube to prepare 8.5%solution. Then add 88.2 microliters of DMSO to the tube to prepare a 5%solution. Next, add 1.877 milliliters of 0.9%saline to the solution.

This will result in an emulsion. Centrifuge the solution to check whether the naringenin is completely dissolved. White precipitates have undissolved naringenin may appear in the solution.

Next, to prepare a two milligrams per milliliter naringenin solution in Tween 80 and DMSO, weigh 6.69 milligrams of naringenin powder and add it into a five milliliter tube. Quickly spin down the tube, and add 117.7 microliters of DMSO to prepare a 3.5%solution. Then add 117.7 microliters of Tween 80 to the solution to attain a 3.5%Tween 80 and 3.5%DMSO concentration.

Slowly add this solution to another five milliliter tube containing 3109.6 microliters of 0.9%saline and shake well to obtain a clear naringenin solution. After two hours, the solution will remain clear without any visible precipitates. To administer the naringenin solution, lift a five-week-old C57 black six male mouse by the base of its tail and place it on a solid surface positioning its tail back gently.

Next, grasp the scruff of the neck behind the ears with the left thumb and index finger and position the tail between the little and ring finger. Keep the mouse in a supine position with its posterior end slightly elevated. To inject the naringenin solution, grasp the skin on the mouse's back so that the abdominal skin is taut.

Then push the needle into the lower right or left quadrant of the abdomen at a 10-degree angle. to avoid hitting the bladder, liver or other internal organs. Run the needle subcutaneously in a cranial direction for three to five millimeters, then insert it at a 45-degree angle into the abdominal cavity.

Once the needle passes through the abdominal wall, slowly push the solution. After the injection, slowly pulled the needle out, rotating it slightly to prevent leakage. To perform a blood glucose test, open the test strips and mark the date.

Make a small puncture on the lateral tail vein with a 25-gauge needle prick and squeeze out a drop of blood. After wiping the drop of blood with tissue paper, squeeze out another drop of blood and collect it on the edge of the test strip. Read and record the result displayed on the glucose meter.

Body weight of the high fat diet fed and streptozotocin-induced diabetic mice was decreased compared to that of control group mice zero to eight weeks after a streptozotocin treatment. At the fourth week, the weight loss of naringenin-treated mice was significant compared to non-treated mice. Blood glucose level in the diabetic mice dramatically increased within one month after streptozotocin induction.

After two months, it automatically decreased to a level observed two months ago. In contrast, naringenin treatment lowered the blood glucose levels by 51.8%at one month and 34.8%at two months compared to STZ. Compared to controls, the streptozotocin-induced diabetic mice exhibited bone loss as indicated by a decrease in the bone volume by tissue volume and the number of trabecula.

Meanwhile, naringenin treatment significantly rescued the bone loss. Osteoclast activity shown by osteoclast number per trabecular bone area was increased in diabetic mice although no statistical significance was observed between the control and the disease models. TRAP staining of trabecular bone and osteoclasts of control and streptozotocin-induced mice is shown here.

Naringenin treatment significantly decreased osteoclast activities. In the diabetic animals, the C-terminal telopeptide of type one collagen was elevated by 68.09%and the N-terminal propeptide of type one Pro-Collagen was elevated by 204.88%indicating a dramatic increase in the bone resorption rate. However, naringenin treatment significantly decreased both indicators of the bone resorption rate.

so it is challenging to pipette accurate dosage. Cutting the tip off makes pipetting easier and more accurate. Alternatively, the required volume can be converted to a mass, which can be later easily.

The fasting time of all animals before each bladder glucose experiment must remain the thing during the entire experiment.