INTRODUCTION:

Quercetin is a polyphenol flavonoid compound that is widespread in varieties of fruits, vegetables, leaves and grains¹. Quercetin has many benefits including antihypertensive, anticancer, anti-hypercholesterolemia, antioxidant, antiobesity, antimicrobial, anti-inflammatory, antineoplastic, and immunomodulatory^1-5. Quercetin is difficult to dissolve in water, sensitive to oxygen, pH, temperature, other antioxidants, as well as the presence of metal ions that can affect its stability, and has a short half-life². As an immunomodulator for oral use, quercetin must be protected from the influence of stomach acids that can damage it, so that it can reach the intestines and undergo absorption towards circulation. One of the transmission systems that can be used for such purposes is the microsphere.

The microsphere is a particulate delivery system, in the form of a micron-sized spherical, in which the active agent is entrapped in a solid matrix⁶. In the microspheres form, quercetin is more stable and will be released slowly and gradually so that its effectiveness lasts longer The effectiveness of the microsphere as a delivery system is influenced by its characteristics, including morphology, size, entrapment efficiency, drug loading^7,8. The characteristics of the microsphere are influenced, among others, by the production method of microsphere and the polymer that form matrix.Sodium alginate is a biodegradable, biocompatible, and non-toxic polymer^9,10. Sodium alginate is able to form crosslinking with divalent cation Ca²⁺ to form an eggbox structure, but the resulting microsphere is still porous because only the carboxylic group (COO^-) of the adjacent guluronate block binds only, while the carboxylate group (COO^-) of the manuronate block is still free. The combination of sodium alginate with chitosan results in a strong electrostatic interaction between the chitosan amino group (NH4⁺) and the carboxylic group (COO^-) of the manuronate block forming a chitosan-alginate complex, thereby reducing the porosity of the microsphere and leakage of the encapsulated drug¹¹.

The microencapsulation method used in this study is the ionotropic gelation method of aerosolization technique with a combination matrix of sodium alginate and chitosan. Three formulas were made, namely F1 (sodium alginate 2% - chitosan 0%), F2 (sodium alginate 2% - chitosan 0.5%), F3 (sodium alginate 2% - chitosan 1.0%) using a cross-connecting solution CaCl₂ 0.5M

This study aims to determine the effect of chitosan concentration (0%, 0.5%, and 1.0%) on microsphere characteristics which include: morphology and microsphere size, entrapment efficiency, drug loading, and swelling index of quercetin microspheres.

MATERIALS AND METHODS:

Materials:

Quercetin pharmaceutical grade (Tokyo Chemical Industry CO., LTD), Sodium Alginate pharmaceutical grade (Sigma-aldrich, USA), Chitosan medium viscosity 49.6 cps pharmaceutical grade, Acetic Acid pharmaceutical grade (Sigma-aldrich, Germany), CaCl₂ (EMSURE® ACS Reag. Ph Eur, Merck) pharmaceutical grade, H₂SO₄ pro analysis (Merck), aquademineralisata, Sodium citrate pharmaceutical grade (SAP chemicals), Citric acid pharmaceutical grade, Tween 80 ( SAP Chemicals )

Double-beam Spectrophotometer (UV-1800 Shimadzu), FTIR Spectrophotometer (Perkin Elmer Instrument), Analytical Balance (Chyo Balance Serial 51347), Centrifuge (Rotofix-32), Sonicator (CO-Z 2L Ultrasonic Cleaner), Stirrer Plate (Dragon Lab MSPro),pH meter (Eutech Instrumet pH 700), Spray aerosol with nozzle diameter 1 mm), Scanning Electron Microscope (Hitachi TM3000), Optilab optical microscope, Moisture Analyzer (Metler Toledo), Freeze Dryer.

Qualitative Analysis of Materials:

Qualitative examination of materials including CaCl₂, quercetin, sodium alginate, and chitosan organoleptically and infrared spectra using FTIR byKBr pellet technique.

Quercetin microspheres preparation:

Chitosan (0%, 0.5% and 1.0% w/v) is dissolved in a solution of 1% ad dissolved acetic acid and a viscous liquid is formed. Add a 0.5M solution of calcium chloride to it, and stir at a speed of 500rpm until homogeneous. The dispersion of 2g of sodium alginate into aquademineralisata ad 100mL is stirred until a homogeneous viscous liquid is formed. Quercetin 0.2g is dissolved in 96% ethanol, then mixed into a solution of sodium alginate, and stirred until homogeneous (Table 1).

A mixture of quercetin-sodium alginate solution is sprayed into the CaCl₂-chitosan solution with a nozzle size of 1mm, a distance of 8cm from the surface of the solution and a pressure of 10 Psi, while stirring at a speed of 1000rpm for 90 minutes. The microsphere is separated from the remaining CaCl₂ by centrifugation at a speed of 2500rpm for 5 minutes and washed using aquademineralisata 2 times. The resulting filtrate is checked with H₂SO₄ when there is no white precipitate, the microsphere is CaCl₂-free. The formed microsphere was dried in a freeze dryer for 72 hours at -40°C^12,13.

Table 1. Quercetin Microsphere Formula

Materials	Function	Formula
Materials	Function	1	2	3
Quercetine	Active ingredient	0.2 %	0.2 %	0.2 %
Ethanol 96%	Quercetine’s solvent	30 ml	30 ml	30 ml
Sodium alginate	polymer	2.0 %	2.0 %	2.0 %
Aquademineralisataad	solvent	100 ml	100 ml	100 ml
Chitosan	Polymer	0.0 %	0.5 %	1.0%
Acetic acid 1 % ad	Chitosan’s solvent	-	30 ml	30 ml
CaCl₂	Crosslinker	0.5M	0.5 M	0.5M
Aquademineralisataad	CaCl₂‘s solvent	100 ml	100 ml	100 ml

Characteristics of Quercetin Microspheres:

a. Organoleptic:

An examination of the shape, color and smell of the formed quercetin microsphere is carried out.

b. Spectrophotometry FTIR Analysis:

Examination of infrared spectra by the KBr pellet method using FTIR spectrophotometry, observed at wave numbers 400 – 4000 cm^-1. The resulting FTIR spectra are compared with the FTIR spectra of each component of the quercetin microsphere.

c. Surface Shape and Morphology:

The evaluation of the shape and surface of the quercetin microspheres was carried out using optical microscopy and Scanning Electron Microscope (SEM).

d. Moisture Content:

Measurement of the moisture content of quercetin microspheres was done using Ohaus MB45 Moisture Analyzer by weighing 1-1.5 grams of sample and adjusting the temperature and time. Next, record of the percentage (%)moisture content displayed on the light emitting diode (LED) of the instrument.

e. Particle Size Determination:

Particle size determination was performed using the microscopic method by measuring 300 microsphere particles and performed three replications for each formula.

f. Determination of Entrapment Efficiency and Drug Loading:

Determination of quercetin concentration in the microsphere was carried out by dissolving 100mg of quercetin microsphere into a pH citrate buffer of 5.0± 0.2 with 2% tween 80 added, then sonicated for 30 minutes until the microsphere dissolved. Furthermore, filtration was carried out and the filtrate was observed absorption at a wavelength of 370nm using a microscope. The entrapment efficiency is calculated using the formula :

EE (%) = (Quercetin concentration in the microsphere / quercetin concentration in the

formula) x 100 %

The drug loading is calculated using the formula:

DL (%) = (Concentration of quercetin in microspheres/total microspheres) x 100 %

g. Determination of Swelling Index:

Determination ofswelling index was carried out based on changes in mass at a certain time interval. After the sample was put into the media, the microspheres were weighed in the amount of ±50mg for 6 samples, then each 5ml of buffer pH 6,0 was added and left at 32± 0.5°C. Changes in weight were observed at 1, 2, 3, 4, 5, 6. 7 and 8 hours.

Swelling index (%) = (expanding weight – dry weight / dry weight) x 100 %

h. Release of Quercetin from Microspheres:¹⁴

Samples of quercetin microspheres equivalent to 10.0 mg of quercetin were put into 100mL of phosphate buffer medium pH 6.8, experimental temperature 37℃± 0.5, stirring speed 100rpm. Sampling of 5.0mL was carried out at 15, 30, 60, 120, 180, 240, 300, 360, 420, 480 minutes. And the same volume was replaced at each sampling. The sample was filtered and the filtrate was checked for absorbance using a UV-Vis Spectrophotometer at a wavelength of 374nm. A release profile curve is made between the % cumulative amount of dissolved quercetin as the y-axis and time as the x-axis¹⁵

i. Determination of Quercetin Release Kinetics:

Release results data are included in zero-order, first order, Higuchi, and KorsmeyerPeppas kinetics calculation then calculated the value of the correlation coefficient.

1. Zero order kinetic model: graph of relationship between time and cumulative percent of drug released

2. First-order kinetic model: graph of the relationship between time and the log of cumulative percent of drug remaining

3. Higuchi model: graph of the relationship between the square root of time and the cumulative percent of drug released

4. Korsmeyer-Peppas model: graph of the relationship between the log time and the log of cumulative percent of drug released

Data Analysis:

Statistical data is analyzed using the one-way ANOVA method with a degree of confidence of 95% (α = 0.05). The data analyzed include yield, average particle size, MC, swelling index, entrapment efficiency (EP) and drug loading (DL). Then proceed with a post hoc test to find out which formulas differ meaningfully.The FTIR examination results are displayed in the form of overly FTIR spectra from quercetin, sodium alginate, chitosan, and physical mixtures. Swelling index data is presented in the form of a swelling index profile graph for 8 hours of observation.

RESULT:

The yield value describes the efficiency of the methods used in the manufacture of microspheres (Table 2).

Table 2. Yield of Quercetin Microsphere

Formula	Replication	Yield (%)	Mean±SD (%)
F1	1	99.55	97.12 ± 3.41
	2	99.52
	3	92.30
F2	1	107.84	107.38 ± 1.07
	2	105.91
	3	108.40
F3	1	117.97	123.48 ± 5.51

The FTIR is shown in Figure 1.

Figure 1. Comparison of infrared spectra (A) sodium alginate, (B) chitosan, (C) alginate-chitosan physical mixture, (D) F1, (E) F2, (F) F3, (G) quercetin

The moisture content results of the quercetin microsphere in table 3 showed that only F1 met the requirements, which was less than 10%¹⁶.

Table 3. Moisture Content of Quercetin Microspheres

Formula	Replication	Moisture content (%)	Mean ± SD (%)
F1	1	7.37	7.28± 0.40
	2	6.84
	3	7.62
F2	1	9.18	10.28±0.99
	2	10.58
	3	11.09
F3	1	15.49	16.78±1.12
	2	17.27
	3	17.57

The results of morphological observations of quercetin microspheres using scanning electrone microscope (SEM) with a magnification of 2000x in figure 2, only F1 produces microspheres that are less spherical and have undergone agglomeration. This is likely due to Brown's random motion so that particles collide and stick together¹⁷, F2 and F3 microspheres produce spherical shapes and smooth surfaces.

Figure 2. The shape and morphology of quercetin microspheres under SEM observation at 2000x magnification (a) F1, (b) F2, (c) F3

Particle size F1, F2, F3 is qualified for topical use of 1-50μm, small and uniform particles will provide comfort to topical use¹⁷(Table 4).

Table 4. Particle Size of Quercetin Microsphere

Formula	Replication	Average size (µm)	Mean±SD (µm)
F1	1	2.67	2.67 ± 0.09
	2	2.59
	3	2.76
F2	1	2.8	2.72 ± 0.06
	2	2.69
	3	2.69
F3	1	2.93	3.02 ± 0.11
	2	2.99
	3	3.14

The highest entrapment efficiency was obtained in F3 microspheres with a chitosan concentration of 1.0%, namely (94.70±0.78)%, while F2 was (89.0±2.50)% (Table 5).

Table 5. Quercetin microsphere drug entrapment efficiency results

Formula	Replication	Drug entrapment efficiency (%)	Mean ± SD (%)
F1	1	88.18	87.32 ± 0.78
	2	87.10
	3	86.67
F2	1	86.95	89.01 ± 2.50
	2	91.80
	3	88.28
F3	1	95.59	94.70 ± 0.78
	2	94.39
	3	94.12

Increasing the concentration of chitosan can increase the entrapment efficiency and drug loading as shown in table 6, because the porosity of the matrix will decrease and the microspheres formed will be more compact, thereby preventing leakage or release of quercetin from the matrix, resulting in more quercetin entrapped in the matrix¹¹.

Table 6. Quercetin microsphere drug loading results

Formula	Replication	Drug loading (%)	Mean ± SD (%)
F1	1	8.11	8.21 ± 0.31
	2	7.97
	3	8.55
F2	1	5.97	6.14 ± 0.26
	2	6.44
	3	6.02
F3	1	5.13	4.73 ± 0.35
	2	4.55
	3	4.50

The swelling index value at F1 at 7 and 8 hours could not be determined, because the matrix system inside the microspheres had been eroded and dissolved in citrate buffer pH 4 media, but at 6 hours it had a higher swelling index value compared to F2 and F3 (Table 7).

Table 7. Swelling index results of quercetin microspheres at pH 6

Formula	Time (hour)	Swelling index Mean ± SD (%)
F1	1	628.76 ± 41.51
	2	-
	3	-
	4	-
	5	-
	6	-
	7	-
	8	-
F2	1	614.44± 37.43
	2	642.26± 23.99
	3	657.08± 31.74
	4	708.28± 47.28
	5	737.07± 73.74
	6	768.50± 40.46
	7	828.10± 22.64
	8	913.20± 29.75
F3	1	310.48± 32.49
	2	365.40± 5.50
	3	463.13± 51.78
	4	573.37± 55.29
	5	634.95± 73.25
	6	653.95± 61.26
	7	668.97± 46.98
	8	704.40± 57.18

Table 7 and Figure 3 show that the swelling index values of quercetin microspheres F2 and F3 at pH 6 decreased with an increase in chitosan concentration.

Figure 3. Swelling index comparison chart for each formula at pH 6

The results of the release test at pH 6.0±0.2 showed a significant difference in the percentage of cumulative quercetin released in the third formula (Figure 4).

Figure 4. Quercetin release profile at pH 6.0

DISCUSSION:

The results of statistical analysis of yield show that there are significant differences in the three formulas, and meaningful differences occur between F1 and F3, F2 and F3 but there is no meaningful difference between F1 and F2. The higher the polymer concentration, the yield value also increases because the number of microspheres obtained is more due to the increasingly intensive cross-linking reaction^18,19,20. The yield value of F2 and F3 is greater than 100%, possibly due to the presence of solvents that have not completely disappeared; this can be seen in the high content of moisture in both formulas, which is more than 10%.

The absorption of the COO^- asymmetric group of sodium alginate that originally existed at the wave number 1594.91 cm^-1 became lost in all three formulas (Figure 1). The vibration of the COO^- asymmetric group is most sensitive in the presence of Ca²⁺ ions as a crosslinker²¹. The COO^- symmetric group also underwent a shift, and the most distant shift was given by F3 which shifted from 1405.94 cm^-1 to 1422.22 cm^-1. The loss of uptake and shift of the COO^- group-indicates a cross-linking reaction between the divalent cation of CaCl₂and the COO^- group- of the sodium alginate guluronate block from adjacent chains forming an egg box structure and the presence of electrostatic interactions forming a complex between the protonation amino group (NH3⁺) and the carboxylic group (COO^-) of the manuronate block of sodium alginate²². The formation of the complex is evidenced by the shift in the wave number of absorption of the OH^- and NH₂ groups that originally existed at the wave number 3200-3400 cm^-1 shifted to a weaker wave number of 3100 cm^-1. In the fingerprint area, a specific absorption belonging to the aromatic C-H bond of quercetin was obtained at a wave number of 600-900 cm^-1which showed that the microsphere contained quercetin.

The high moisture content will affect the stability of the microsphere because it can accelerate the growth of microorganisms, and active agent that are unstable to moisture will degradation²³.

The high value of moisture content is likely due to insufficient drying process. The freezing and drying process in the freeze drying technique the temperature used must be below the glass transition temperature (Tg)⁷. Ethanol as a solvent, in moderate cooling has a Tg value ≈ 95°K (-176.15°C)¹⁶, while when drying the microsphere by freeze dry the temperature used only reaches -40°C.

Chitosan concentration affects the morphology of the quercetin microsphere. Increasing chitosan concentration will increase the bond area between the chitosan amino group (NH3⁺) and the carboxylic group (COO^-) of the sodium alginate manuronic block, so that it can produce finer microspheres due to reduced microsphere porosity¹¹.

Table 4 also shows that an increase in chitosan concentration results in an increase in particle size. This can be attributed to an increase in the micro-viscosity of polymer dispersion due to increased chitosan concentrations, which eventually leads to the formation of larger beads²⁴. An increase in chitosan concentration will also increase the bond area between the chitosan amino group (NH3⁺) and the carboxylate group (COO^-) of the sodium alginate manuronate block, so that the number of entrapped quercetin is higher and the particle size becomes bigger¹⁹.

The results of the One Way Anova statistical analysis with a value of Sig = 0.003 (<0.05) show that there is a significant difference. The results of the post hoc tukey HSD test showed that there was a significant difference between F1 and F3, and F2 and F3, where F1 which was not combined with chitosan produced the lowest entrapment efficiency (87.32±0.78) %. This is due to the cross-linking reaction that occurs only between Ca²⁺ ions and the carboxylic group of the adjacent guluronic block, while the manuronate group is still free.

Increasing the concentration of chitosan can increase the efficiency of entrapment, because a higher concentration of polymer in the protonated state results in intensive cross-linking, the greater the polymer concentration, will increase drug encapsulation²⁴. There is no significant difference between F1 and F2, this could be due to the fact that the number of protonated amino groups in F2 with 0.5% chitosan concentration is not fully sufficient to bind to the COO^- group of sodium alginate.

F1 microsphere with a sodium alginate matrix that is not combined with chitosan, produces porous microspheres, making it easier for water to enter the matrix, experiencing faster swelling, then dissolving²⁵. The results of the post hoc tukey HSD test showed that there was a significant difference between F1 and F3, and F2 and F3, but between F1 and F2 there was no significant difference, this was because the number of protonated amino groups in the F1 and F2 microspheres was not significantly different.

F2 and F3 microspheres containing chitosan swelling index process still last until the 8th hour, this is because the pKa of the amino group in chitosan is about 6.5, they tend to remain protonated at acidic and neutral pH and produce microsphere stabilization and lower swelling index values^26-28. The presence of protonation amino groups in F2 and F3 results in repulsion with H⁺ ions from the acidic pH so as to prevent hydrolysis of the polymer^29,30. The number of protonation amino groups in F3 is greater than F2, resulting in a lower swelling index value at F3 than F2

In F1 that is not combined with chitosan, the swelling index is already very high, even at the 2^nd to 8^th hour the microsphere has dissolved, this is likely because the –COOH group of sodium alginate has been ionized, so that the bonds in the matrix weaken and undergo erosion.

In F2 and F3 the swelling process is still ongoing until the 8^th hour, an increase in chitosan concentration results in a protonation amino group also increasing. At pH 6 ± 0.2 the amino group is still protonation and the intrusion of water into the microsphere can be inhibited so that the swelling index value is lower and still lasts until the 8^th hour.

The release of quercetin in F1 occurred for 1 hour, F2 for 6 hours, and F3 for 8 hours, this was is related of differences in chitosan concentration in the microsphere matrix. The greater concentration of chitosan in the matrix, the slower the release of the drug as happened in F3. The greater the chitosan concentration, the matrix density also increases and the matrix becomes more compact, so that drug release from the matrix is more difficult^31-33. Meanwhile, in the F1 formula which does not contain chitosan (0%) release occurs the fastest, because the microspheres formed have high porosity and the microsphere matrix is less compact, so the drug release will be faster than other formulas.

CONCLUSION:

Increased chitosan concentrations (0 %, 0.5 %, and 1.0 %) resulted in increasingly compact, spherical quercetin microspheres with smooth surfaces, increasing particle size, yield value, moisture content, entrapment efficiency, but lowering drug loading. The swelling process lasts longer and the release time is extended.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank Universitas Airlangga for the funding support by PUF grant scheme 2021 and Faculty of Pharmacy Universitas Airlangga for research facilities.

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Received on 04.01.2024 Revised on 08.06.2024

Accepted on 12.09.2024 Published on 20.01.025

Available online from January 27, 2025

Research J. Pharmacy and Technology. 2025;18(1):44-50.

DOI: 10.52711/0974-360X.2025.00007

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