The effect of spray-drying temperature on Centella asiatica extract-β cyclodextrin-maltodextrin nanoparticle characteristics and stability

Centella asiatica extract has low solubility in water. Material modification needs to be conducted to increase the dissolution rate of Centella asiatica extract. The particle size reduction to nano-size was carried out to increase surface contact with aqueous media. High surface contact was expected to increase the solubility and absorption rate. Nanoparticles were prepared with 34% maltodextrin and 6% β-cyclodextrin as a stabilizer and dried by a spray-drying method. High temperature in the spray drying process can affect the physical and chemical characteristics of the nanoparticles, so the inlet temperature in this study was observed as parameter variations, on 140 o C, 150 o C, and 160 o C. The formed nanoparticles then being tested on several parameters, including physical appearance, moisture content, particle size, shape, and morphology. The chemical stability of the active ingredients during the drying process was assessed from the pH value changes and the content of quercetin as an antioxidant post drying process, compared to the initial content. The test results show that the nanoparticles have been formed. The inlet temperature of 160 o C produced the most physically optimum spherical nanoparticles, with a particle size of 191.533 ± 18.791 nm and relatively homogeneous with a polydispersity index (PDI) value of 0.113 ± 0.057. However, temperatures that are too high indicate poor chemical stability. The poor chemical stability can be seen from the quercetin content that decreased significantly after the drying process, until the remaining 53.87 ± 0.55% and 49.52 ± 0.97% for temperatures of 140 o C and 160 o C, respectively. These results indicate that the combination of β-cyclodextrin and maltodextrin can not encapsulate and maintain the stability of the active ingredients during the spray drying process. A significant reduction of inlet temperature is needed to get dry nanoparticles with the most optimum physical mixture and chemical stability.


INTRODUCTION
cyclodextrin is composed of hydrophobic glucose residues, while the outside is hydrophilic due to the presence of hydroxyl groups. In solution, the water molecules in the cyclodextrin cavity are replaced by non-polar molecules or non-polar parts of the guest molecules (drugs) and move back and forth towards the host-guest inclusion complex. The drug molecules in the complex form will quickly be in equilibrium with the free molecules in the solution (Loftsson et al., 2005). Compared to the free molecular form, the guest molecule (drug) complexed with cyclodextrin has new physicochemical properties. One of them is increased solubility in water.
In this study, the researcher made the observations on the inlet temperatures effect of 140 o C, 150 o C, and 160 o C in the spray drying process on the characteristics of the obtained Centella asiatica-β cyclodextrin-maltodextrin nanoparticles, including physical characteristics, particle size, shape, morphology, moisture content, and qualitative densitometric tests. To ensure that the chemical stability of the active ingredients is maintained during the spray drying process, testing also needs to be carried out. The pH of nanoparticles and quercetin content of nanoparticles were determined based on pHmeter determination and the validated assay method with High Performance Liquid Chromatography (HPLC). Quercetin was used based on the target of activity as an anti-hypertensive agent, and thus substance was well known as a kind of flavonoid with purposed activity. The results of the tests will later become the basis for ensuring that the spray drying process with the selected inlet temperature can produce nanoparticles with good physical and chemical characteristics.

Centella asiatica extract preparation
A 500 milligrams of Centella asiatica simplicia was put into an electric stirring macerator. It stirred for 30 minutes in 2.5 L ethanol 70% and macerated for 24 hours. After being filtered, the filtrate obtained was evaporated in the evaporator until a flowing viscous compound was formed (water content of 5-30%) with a yield of maceration >10%. Evaporation is then continued in a porcelain cup over a water bath until a thick extract is obtained for three hours total time at 55 o C.

Centella asiatica nanoparticle formulation
The composition used in the preparation of Centella Asiatica nanoparticles is shown in Table 1. Centella Asiatica extract was weighed as much as 4 grams and dissolved in 50 ml of ethanol. On the other hand, the researcher dissolved β-cyclodextrin and maltodextrin in deionized water. The stabilizer solution is mixed with the extract solution at 5,000 rpm for 5 min until homogeneous. Furthermore, the mixture was dried using a spray dryer with inlet temperature variations of 140 o C, 150 o C, and 160 o C. The researcher set the aspirator rate and airflow of the machine at 90% and 40%, respectively. The flowing rate of the solution mixture was 5.5 mL/min. The nanoparticle of Centella Asiatica extract was formed and surrounded by the hydrophilic matrix as a stabilizer of the spray drying process. However, considering that both phases were in dissolved form (with small dispersion), there might have been a hydrophilic part of the polymer inside the core of nanoparticles. The researcher replicated the formulation process three times and then evaluated the results.

Moisture content
The research did the nanoparticle moisture content test using a moisture content balance 0.5gram sample was placed in the pan and continued with the drying process by a heating halogen lamp at 100 o C. The heating process was stopped when the desired constant weight of the substance reached. The moisture content was obtained in the display due to comparing the weight difference in the pan before and after the drying process.

Particle size and polydispersity index
A particle size analyzer was used to determine the particle size, and polydispersity index 50 mg nanoparticle was dispersed in 20 mL deionized water to avoid multiple scattering effects during analysis. The suspension was then placed into the cuvette, and particle size was observed on the instrument.

Particle shape and morphology
The interaction between the electron beam and the solid specimen is utilized by a scanning electron microscope (SEM) to produce an image. The three-dimensional appearance of the SEM results is useful for describing the particle shape and surface morphology of the nanoparticles (Ayyad, 2011). Centella asiatica solid simplicia and its nanoparticles were placed on double-sided adhesive carbon tape. Furthermore, the sample was coated with gold for 3 minutes and photographed at the appropriate magnification.

pH of nanoparticle
The pH test of nanoparticles was carried out using a pH meter. The testing process begins with the preparation and cleaning of the electrode. After that, the pH meter calibration was carried out with three types of standard buffers and continued with physical mixture and nanoparticle sample testing. Nanoparticles made by 140 o C and 160 o C inlet temperature were chosen as samples with critical manufacturing variation. The dry sample was prepared as 20% w/v in deionized water before observation.

Qualitative determination of nanoparticle
Qualitative determination was started by dissolving 50 mg of nanoparticles in 5 mL of ethanol. 5 µl of the solution and standard were then spotted on the chromatographic system. The area of the saponin spots was measured by densitometry with a UV detector in 254 of wavelength. TLC C18 ODS is used as a fixed phase, while ethyl acetate:toluene: formic acid (60:30:10) is used as a mobile phase. The resulted retention factor (Rf) was compared between the standard and the sample.

Quercetin content of nanoparticle
This research measured the chemical stability of the nanoparticles based on the content of quercetin. Quercetin is one of the flavonoid compounds, was chosen to be a marker. The evaluation was carried out using the validated High Performance Liquid Chromatography (HPLC) method. The mobile phase used in the assay is acetonitrile:2% acetic acid in water 70:30 (v/v) which flew 1.0 mL/min. This study used the column of LiChrospher 100 RP-18 (5µm) (125mm x 4mm). The observed sample was injected with a volume of 20.0 µl, then the quercetin peak was read on a UV detector at 370 nm. All validation parameters, such as linearity, detection limit, quantitative limit, accuracy, precision, and specificity have been carried out and ensured that they meet the requirements before the sample essay. Nanoparticles made at inlet temperatures of 140 o C and 160 o C have weighed as much as 100 mg and dissolved in methanol. The two conditions were chosen as critical parameters (lower and higher temperature) to be evaluated. A physical mixture with the same amount of active substance and excipients was used as a comparison. The researcher compared all observations with the concentration of quercetin as standard, which was calculated as 100% content and repeated three times.

Data Analysis
The research calculated collected results, and the difference between each group was analyzed by descriptive method and one-way ANOVA. Physical appearances and organoleptic of the nanoparticles were expressed in a descriptive method to show the conformity with the expected specifications. Meanwhile, the significance of the data was analyzed using the one-way ANOVA method to see the effect of differences in inlet temperature on the spray drying process on the characteristics of the nanoparticles formed. The powder observed before treatment had a larger size and was coarse, while after the spray drying process, small and fine powder was obtained, as shown in Figure 1. The added stabilizer made the powder color change from dark green during pretreatment to lighter. The distinctive odor of Centella asiatica was smelled in the nanoparticle products. Visually, it can be seen that the improvement of the physical characteristics of the powder occurred after the spray drying process with three different inlet temperatures.  Figure 2 shows the moisture content of nanoparticles formed at three different inlet temperatures, which ranged from 4.43 -5.52%. Based on these results, it can be seen that the difference in inlet temperature used in the spray dryer produced nanocrystals with different moisture content. The higher the inlet temperature used, the lower the moisture content in the resulting nanocrystals is. Moisture in the dosage forms will have an impact on microbial growth and its stability during storage (Zambrano et al., 2019). In addition, the physical characteristics of the product, such as compressibility and crushing strength are also affected by the moisture content value (Liu et al., 2020). The decrease in moisture content occurred due to the high inlet temperature. However, the value was still in line with the expectation. The compressibility and crushing strength of the particles will be good enough to be further formulated into other dosage forms, such as tablets. Microbial growth in the product will also be low enough so that the microbiological stability of the product can be maintained, although further testing still needs to be done. Particle size and polydispersity index of Centella asiatica simplicia powder and nanoparticles were obtained with particle size analyzer and shown in Figure 3. Based on the results, it is seen that nanoparticles with smaller sizes have been formed at the three inlet temperatures. The spray drying process with an inlet temperature of 160 o C resulted in the smallest particle size of 191.533 ± 18.791 nm. These results indicate that an increase in the inlet temperature will dry the droplets faster before the formation of agglomerates in the drying chamber. The three drying processes showed statistically different particle sizes (p<0.05) compared to simplicial powder form. Among the three spray drying process data, there was no significant difference (p>0.05).

Moisture content
The polydispersity index (PI) value obtained is also quite good. The low value of the PI approaching zero indicates that the obtained nanoparticles not only have a smaller size but were also relatively uniform in one bulk product. Nanoparticle prepared with 160 o C inlet temperature was shown higher PI, even the particle size was the smallest. This result shows that the fast-drying process produced non-uniform agglomerates, although the value is insignificant compared to the others (p>0.05). The success of reducing the particle size into nanoparticles can be a solution to overcome the solubility issue so that the success can increase bioavailability and biological activity (Rachmawati et al., 2016).

Particle Shape and Morphology
The particle shape and morphology of the powder and nanoparticles are shown in Figure 4. Nanoparticles at the three inlet temperatures seemed to form better particle shape and morphology. The more spherical particle with a smooth surface compared to the raw material powder was resulted from the manufacturing process. Spherical shape and smooth surface benefit its physical stability and prevent agglomeration into a larger structure. In addition, this shape also reduces friction between particles in the later process (Mendes et al., 2021).

pH of nanoparticle
The pH of Centella asiatica physical mixture and nanoparticles as one of the chemical parameters was determined and shown in Figure 5. These results showed a pH value ranged from 6.04 -6.53, so it was tolerable to be consumed by oral administration. The values of each pH were not statistically different (p>0.05), both physical mixture without spray drying process and spray dried substance. The difference indicated that the spray drying process had a significant effect neither on the pH of the resulting nanoparticles, nor the pH value changes.

Qualitative determination by densitometry
The sample spots that contained Centella asiatica extract were read on a densitometer with λmax of 301 nm (Sukmasari and Fatimah, 2006). After the researcher carried out the readings, it was obtained as written in Table 2. Both Rf of standard saponins and Centella asiatica extract were 0.24, which indicated that the thick extraction contained saponins. Rf could not be detected at nanoparticles prepared by 150 and 160 o C. This condition could be due to the saponin content that was very small in both products. The high temperature of the drying process caused instability so that the content of the compound was too low to be observed in the weighted sample.

Quercetin content of nanoparticle
Quercetin content assay in physical mixture and nanoparticles began with validation of the analytical method using HPLC. The validation results were shown in Table 3, where the parameters, such as linearity, detection limit, quantitation limit, accuracy, precision, and specificity have met the requirements (United States Pharmaceutical Convention, 2020). Based on these results, the assay method can be used to observe the effect of inlet temperature on the chemical stability of Centella asiatica extraction that is characterized by the quercetin content of the nanoparticles. Figure 6 shows the quercetin content of the physical mixture and nanoparticles that were spraydried at inlet temperatures of 140 o C and 160 o C. From the results, it can be seen that grinding and mixing the component into a physical mixture could decrease the quercetin content. It is shown that the stabilizer (a combination of maltodextrin and β-cyclodextrin) could not maintain the chemical stability of quercetin. An increase in inlet temperature caused a higher degradation of the active ingredient, indicated by a decrease in the quercetin content with a statistically different value (p < 0.05). The reduction of inlet temperature in the spray drying process needs to be carried out while maintaining the optimum physical and chemical characteristics of nanoparticles. This condition considers that the higher inlet temperature (160 o C) is known to produce the best physical characteristics of nanoparticles according to previous tests.  Relative standart deviation (RSD) of retention time: ≤ 1% Relative standart deviation (RSD): 0.037%

CONCLUSION
A nanoparticle of Centella asiatica extract has formed. The three different formulation conditions, namely inlet temperatures of 140, 150, and 160 o C in the spray drying process have improved the physical appearances, moisture content, particle size, shape, and morphology. They were getting better with an increase in the inlet temperature used. However, the chemical characteristics of the nanoparticles show the opposite result. The increase in inlet temperature worsens the active compound content of the nanoparticles, which is indicated by a decrease in the quercetin value. This condition indicates that the use of the stabilizer combination has succeeded in improving the physical characteristics of nanoparticles. However, the inlet temperature value for the spray drying process needs to be further optimized.