The objective of this research was to create a straightforward and environmentally friendly process to produce small lignin particles. The study aimed to analyze their physical, chemical and structural properties, as well as investigate their ability to encapsulate and release bioflavonoids in simulated gastrointestinal environments. Our methods synthesizes lignin-based particles using cheap and green solvents.
The process is easy, quick, and requires minimal equipment and non-toxic substances. It also has simple methods for characterization encapsulation, capacity determination, and in vitro release potential of poorly water soluble bioactive compounds. The innovative biopolymer microparticles are suitable for oral administration due to the lower risk of gastric irritation.
The submicron particles, due to the small size and significant release potential, could be applied as injectable formulations. To begin, add 125 milligrams of alkali lignin in 25 milliliters of ultrapure water and dissolve it using a magnetic stirrer. Gradually add one milliliter of 96%ethanol into the alkali lignin solution.
Stir the mixture on a magnetic stirrer at 500 RPM for three minutes. Then dissolve 0.5 grams of citric acid in ultrapure water to a final volume of 50 milliliters to prepare 1%citric acid solution. Using a syringe, add seven milliliters of 1%citric acid into the mixture.
Continue stirring the mixture for 10 minutes until a clear brown solution transforms to a cloudy light brown suspension. Now, using an ultrasound homogenizer, homogenize the microparticles for two cycles each lasting four minutes at an intensity of 96%Then lyophilize the microparticles at a temperature of minus 64 degrees Celsius in a freeze dryer and store them in an ex-ek-a-tor for future use. To prepare natural flavonoid-encapsulated lignin micro or submicron particles, weigh 0.04 grams of Quercetin.
Dissolve it in one milliliter of ethanol, and add this ethanolic solution to the alkali lignin aqueous solution. To begin, start an automatic cell counter to assess the particle size and particle size distribution of the prepared sample. Add one microliter of the particle suspension in ultrapure water to the well of the counting slide.
Check the number of particles in one milliliter of the suspension, along with their number and size distribution. Weigh 0.04 grams of unloaded or flavonoid-encapsulated lignin particles. Transfer the weighed particles into an Erlenmeyer flask.
Then add 10 milliliters of 0.1 molar hydrochloric acid. And place the flask on a magnetic stirrer set to 250 RPM. Next, fill a 50 milliliter burette with a 0.1 molar standard solution of sodium hydroxide.
Using a bench pH meter, measure the initial pH of the solution in the Erlenmeyer flask prior to starting the titration. Now start the titration process and measure the pH of the analyzed solution after each 0.5 milliliter addition of the titrant. Record the experimental data in the table, noting the volume of the titrant applied and the corresponding pH value.
Once a nearly constant pH value is observed with increasing volumes of the titrant solution, cease the titration. Illustrate the experimental data in the form of zero, first and second derivative differential titration curves. Prepare 60 milliliters of a 0.1 molar aqueous sodium chloride solution.
Take five stoppered conical flasks. And add nine milliliters of the 0.1 molar sodium chloride solution to each flask. Adjust the pH to initial values of 2, 4, 7, 10, and 12, respectively.
Then add extra sodium chloride solution to adjust the total solution volume to exactly 10 milliliters. Now, add 40 milligrams of dry lignin particles to each flask. And securely cap them.
Attach the flasks upright on an orbital shaker and shake them for 24 hours. Allow the flasks to equilibrate for 30 minutes. Then measure the final pH of the supernatants in each flask.
Plot the final pH values against the corresponding initial pH values. To begin, take 50 milliliters of the simulated enzyme-free gastric medium in a glass batch reactor supplied with a mechanical stirrer. Add 25 milligrams of flavonoid-encapsulated micro or submicron particles into it.
Place the reactor in a thermal water bath set to a constant temperature of approximately 37 degrees Celsius. Submerge the stirrer to a depth of two thirds of the liquid volume to ensure thorough mixing of the solid and liquid phases. Extract one milliliter of the sample from the reactor every 10 minutes until the 90th minute, and immediately pipette one milliliter of fresh simulated fluid solution into the reactor to maintain the total volume and ensure sink conditions.
In vitro release of Morin and Quercetin was studied in enzyme-free, simulated gastric, small intestinal and colon fluids. Small intestinal medium showed double the release of flavanoids compared to the colon and triple compared to the stomach. Quercetin release peaked at 34%in small intestinal fluid at pH 7.4 after 70 to 90 minutes, surpassing the release in gastric fluid and small intestinal fluid at pH 6.8.
