The scope of our research focuses on the development of natural reactive compounds. So using an in vitro method, we aim to evaluate the activity of elastase, an enzyme involved in the degradation of elastin and the loss of elasticity, in order to restore the homeostasis of the tissue. Quantifying natural plant extracts, the activity, presents several challenges, mainly related to their complex chemical composition and their often strong coloration that can interfere with common measurements.
Developing reliable adaptive protocols is vital for advancing this research field. Our study offers several benefits to the fields, including simplicity, sensitivity, fast result of tension, and adaptability to different research needs. To begin, weigh the appropriate amount of Tris base using an analytical balance and transfer the weighed Tris base to a beaker.
Using a graduated cylinder, add deionized water to the beaker. Stir the solution with a magnetic stirrer until the Tris base is completely dissolved. Then adjust the solution's pH to eight using four normal hydrochloric acid.
Once the desired pH is reached, transfer the solution to a volumetric flask. Using deionized water, fill the flask to the marked volume. Transfer the prepared buffer to a labeled storage bottle and store it at room temperature.
For sample preparation, accurately weigh the required amount of sample using an analytical balance. Dissolve the sample in the prepared reaction buffer to achieve the desired concentration. Use a vortex mixer to fully dissolve the sample.
And store the prepared sample on ice. Next, prepare a working solution of elastase enzyme in reaction buffer to a final concentration of 10 micrograms per milliliter. Store the elastase solution on ice until ready for use.
Now prepare a 0.8 millimolar SANA solution in reaction buffer. Protect the solution from light and store it at four degrees Celsius until use. For the phenylmethanesulfonyl fluoride, or PMSF stock, prepare a 100 millimolar PMSF solution in isopropanol.
Store the prepared solution on ice. Prepare all the reactions in triplicate using microcentrifuge tubes. Incubate all the tubes at room temperature for 20 minutes.
Then add 100 microliters of the SANA substrate solution to each tube, except for the color controls. Gently invert the tubes several times to mix the samples and transfer 300 microliters from each tube to a 96-well plate, ensuring triplicate measurements for each sample. Finally, place the 96-well plate in a microplate reader set to 410 nanometers, and measure the absorbance periodically, every minute for 20 minutes, or until the signal stabilizes at room temperature.
Extract one showed weak but significant elastase inhibitory activity at concentrations of one, 0.75, and 0.5 milligrams per milliliter, with no significant effect at 0.25 milligrams per milliliter. Extract two demonstrated high elastase inhibitory activity at all tested concentrations, with inhibition levels similar to the positive control at one, 0.75, and 0.5 milligrams per milliliter.
