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. 2023 Jul 26;8(31):28090–28097. doi: 10.1021/acsomega.3c00602

Utilization of Emulsion Inversion to Fabricate Tea (Camellia sinensis L.) Flower Extract Obtained by Supercritical Fluid Extraction-Loaded Nanoemulsions

Nara Yaowiwat †,‡,*, Worrapan Poomanee §, Pimporn Leelapornpisid §, Phanuphong Chaiwut †,‡
PMCID: PMC10413370  PMID: 37576676

Abstract

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This study aimed to obtain tea flower extract (TFE) using supercritical fluid extraction, to determine the compounds present in the TFE and to establish its antioxidant activity. The fabrication of TFE nanoemulsions was also investigated using response surface methodology (RSM). UHPLC-ESI-QTOF-MS/MS and UHPLC-ESI-QqQ-MS/MS analysis showed that the TFE was composed of catechin and its derivatives, flavonols and anthocyanins, suggesting its potential as a free radical scavenger with strong reducing powers. A central composite design was applied to optimize the independent factors of the nanoemulsions. The factors had a significant (p < 0.05) effect on all response variables. The optimum level of factors for the fabrication was a surfactant-to-oil ratio of 2:1, a high hydrophilic–lipophilic balance (HLB) surfactant to low HLB surfactant ratio (HLR) of 1.6:1, and a PEG-40/PEG-60 hydrogenated castor oil ratio of 2:1. The responses obtained from the optimum levels were a 34.01 nm droplet size, a polydispersity index of 0.15, and 75.85% entrapment efficiency. In conclusion, TFE could be an antioxidant active ingredient and has been successfully loaded into nanoemulsions using RSM.

1. Introduction

Tea flowers, a naturally plentiful resource, are completely flowered, which could be reproduced without recultivation.1,2 In the past, tea flowers were considered to be a waste product in the agricultural process, because manufacturers and consumers focused on the tea leaves and buds due to their various health benefits. In recent years, tea flowers have become more interesting, with several researchers reporting that they are as valuable as tea leaves.1,2 Previously, many studies have reported that the antioxidant effects of galloylated catechins were stronger than those of nongalloylated catechins and that the effects of [(−)-epigallocatechin] were also stronger than those of [(−)-epi-catechin] and [(+)-catechin].3 Grzesik et al. also found (−)-epigallocatechin gallate to be the most effective as it had the highest antiradical capacity.4 Tea flowers also contain flavonols in several forms, especially in the form of glycosides, including kaempferol, quercetin, and myricetin, which are a main class of flavonoids.1,2 Many studies have also demonstrated that tea flowers have various kinds of potential health benefits, such as antioxidant, immune-stimulating, and anti-inflammatory activities.1

Many researchers have investigated nanoencapsulation technology to protect the bioactivity and bioactive compounds of tea flowers, because it overcomes the instability of bioactive compounds, reduces unpleasant taste or flavors, and also enhances permeability.5−9 Nanoemulsions are lipid-based nanoparticles consisting of an oil phase mixed with an aqueous phase containing an appropriate ratio of surfactant.8 Nanoemulsions can be fabricated using different techniques, which can be mainly classified as either high energy or low energy emulsifications.8

The aim of this study was to fabricate oil-in-water nanoemulsions containing tea flower extract (TFE) and to evaluate the effects of the emulsifying conditions on the response variables, including the droplet size, size distribution, and entrapment efficacy. The bioactive composition of the TFE and its antioxidant activity were also investigated.

2. Results and Discussion

2.1. Extraction of TFE

The TFE was a viscous semisolid with a greenish-brown color. The extraction yield was 17.06 ± 1.76%. Various studies have reported that supercritical fluid extraction (SFE) requires fewer newer technologies, has higher selectivity, and requires less time, particularly for tea catechin extraction.10

2.2. Total Phenolic Content and Total Flavonoid Content

The total phenolic content (TPC) and total flavonoid content (TFC) of the TFE were 102.77 ± 1.23 mg of gallic acid equivalents (GAE)/g of extract and 27.48 ± 2.18 mg of quercetin equivalents (QE)/g of extract, respectively. The observed phenolic and flavonoid content in the TFE was correlated to the chemical compositions of the TFE, which was reported by Chen et al.2

2.3. Bioactive Composition of TFE

The untargeted assessment of the bioactive compound profile of TFE was performed using UHPLC-ESI-QTOF-MS/MS. The identified compounds are listed in Table 1, along with their retention times, molecular formulas, and molecular weights (m/z). UHPLC-QTOF-MS2 tentatively characterized a total of 24 compounds in the TFE, as shown in Table 1.

Table 1. Bioactive Compounds Identified in TFE by UHPLC-ESI-QTOF-MS/MS.

compound RT (min) molecular formular m/z mass (theoretical) mass (experimental) error (ppm)
caffeine 9.445 C8H10N4O2 195.0878 194.0804 194.0804 –0.24
CATECHINS
(−)-catechin-4beta-ol 5.905 C15H14O7 305.0667 306.074 306.074 –0.22
(+)-catechin-4beta-ol 8.396 C15H14O7 305.0668 306.074 306.074 –0.37
(+)-gallocatechin 8.397 C15H14O7 307.0814 306.074 306.074 –0.58
(−)-catechin 9.324 C15H14O6 289.0718 290.079 290.079 –0.32
(+)-catechin 11.407 C15H14O6 291.0866 290.079 290.079 –0.79
(−)-epigallocatechin gallate 11.601 C22H18O11 457.0781 458.0849 458.0849 –0.82
(+)-epigallocatechin gallate 11.610 C22H18O11 459.0921 458.0849 458.0849 –0.08
(−)-epigallocatechin 3-(3-methyl-gallate) 14.225 C23H20O11 471.0933 472.1006 472.1006 –0.07
(+)-catechin 3′-O-gallate 15.989 C22H18O10 443.0973 442.09 442.09 –0.13
FLAVONOIDS
kaempferol 7-(3G-glucosylgentiobioside) 14.100 C33H40O21 771.1986 772.2062 772.2062 0.31
kaempferol 3-rutinoside-4′-glucoside 15.432 C33H40O20 755.2038 756.2113 756.2113 0.44
quercetin 3-beta-d-glucoside 16.142 C21H20O12 465.1031 464.0955 464.0955 –0.7
kaempferol 3-rhamninoside 16.162 C33H40O19 739.2092 740.2164 740.2164 0.17
kaempferol 7-galactoside 3-rutinoside 16.184 C33H40O20 755.2044 756.2113 756.2113 –0.72
kaempferol 3-laminaribioside-7-rhamnoside 16.185 C33H40O20 757.2191 756.2113 756.2113 –0.48
quercitrin 16.527 C21H20O11 447.0939 448.1006 448.1006 –0.78
narirutin 16.620 C27H32O14 579.1731 580.1792 580.1792 –2.2
ANTHOCYANINS
5-carboxypyrano cyanidin 3-O-beta-glucopyranoside 16.527 C24H20O13 515.0811 516.0904 516.0904 4.24
delphinidin-3-O-glucoside pyruvic acid 16.126 C24H20O14 531.0753 532.0853 532.0853 5.38
petunidin-3-O-arabinoside 16.185 C21H20O11 449.1077 448.1006 448.1006 0.12
petunidin 3-galactoside 16.589 C22H22O12 477.1038 478.1111 478.1111 –0.14
cyanidin 3-(6″-acetylglucoside) 17.156 C23H22O12 489.1041 490.1111 490.1111 –0.85
malvidin 3-glucoside-4-vinylcatechol 17.532 C31H28O14 623.1402 624.1479 624.1479 0.42
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The identities of the flavonoids and anthocyanins were obtained by matching the molecular m/z values from the UHPLC-ESI-QTOF-MS/MS. Eight flavonoids in the form of glycosides formed from the flavonoid were detected in the TFE, along with six anthocyanins (as shown in Table 1). The bioactive compounds found in the TFE were consistent with the data provided by Chen et al.1,2 Therefore, it was hypothesized that the TFE may have beneficial health effects by functioning as an effective antioxidant and could be used as a remedy in the prevention and treatment of various diseases and with regard to its antiaging properties.

The catechins were quantified by UHPLC-ESI-QqQ-MS/MS using calibration curves of the standards, as shown in Table 2. The group of catechin derivatives found in the TFE included catechin, epicatechin, epigallocatechin, gallocatechin, catechin gallate, gallocatechin gallate, and epigallocatechin gallate, and the contents were determined to be 0.05 ± 0.01, 0.14 ± 0.02, 0.12 ± 0.01, 0.16 ± 0.02, 1.75 ± 0.02, 0.02 ± 0.00, and 2.59 ± 0.02 ppm, respectively. Other phenolic acid compounds were also found to be present, including caffeic acid, protocatechuic acid, gallic acid, p-coumaric acid, and ferulic acid, with corresponding contents of 0.06 ± 0.01, 82.52 ± 2.98, 1.58 ± 0.05, 0.04 ± 0.01, and 0.16 ± 0.01 ppm, respectively. The TFE also consisted of 4.33 ± 0.64 ppm of caffeine. The concentration of the group of catechins was slightly higher than the data provided by Chen et al., especially with respect to epigallocatechin gallate,1,2 which is the most abundant catechin derivative reported in green tea infusions and considered to be one of the most active compounds known for its antioxidant properties.11

Table 2. Contents of Bioactive Composition in the TFE by UHPLC-ESI-QqQ-MS/MS.

composition RT (min) linear equation correlation coefficient: R2 concentration (ppm)
caffeine 3.627 y = 48,366,330x + 2,049,601 0.9990512 4.33 ± 0.64
caffeic acid 4.574 y = 12,402,100x + 159497.3 0.9997329 0.06 ± 0.01
protocatechuic acid 2.670 y = 7007.355x + 6762.765 0.8132233 82.52 ± 2.98
gallic acid 1.640 y = 3,183,944x + 36054.58 0.9996324 1.58 ± 0.05
p-coumaric acid 5.535 y = 6,349,098x + 44423.86 0.9995383 0.04 ± 0.01
ferulic acid 5.706 y = 388346.4x + 13558.94 0.9996573 0.16 ± 0.01
catechin 3.967 y = 2,135,389x + 2950.069 0.9994077 0.05 ± 0.01
epicatechin (EC) 4.763 y = 1,426,047x + 24115.47 0.9996847 0.14 ± 0.02
gallocatechin (GC) 2.386 y = 1,182,815x + 25075.34 0.9997533 0.12 ± 0.01
epigallocatechin (EGC) 3.494 y = 2,307,225x + 3103.182 0.9997687 0.16 ± 0.02
catechin gallate (CG) 5.647 y = 3,066,125x + 11638.07 0.9995410 1.75 ± 0.02
gallocatechin gallate (GCG) 5.067 y = 7,645,719x – 68883.98 0.9988892 0.02 ± 0.00
epigallocatechin gallate (EGCG) 5.065 y = 3,942,501x – 39618.69 0.9994777 2.59 ± 0.02
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2.4. Antioxidant Activities of the TFE

The antioxidant activity of the TFE and standards was investigated using the DPPH radical scavenging assay, whereas the reducing capacity was investigated using the ferric reducing antioxidant power (FRAP) assay. The results showed that the TFE possessed an IC50 value of 0.467 ± 0.011 mg/mL. Gallic acid showed the lowest IC50 value of 0.024 ± 0.001 mg/mL among the antioxidants, followed by ascorbic acid (0.038 ± 0.001 mg/mL), quercetin (0.051 ± 0.001 mg/mL), and catechin (0.053 ± 0.001 mg/mL), respectively. The antioxidant activity of the TFE was mostly related to the concentration of catechin and phenolic compounds. Various studies have reported that the antioxidant action of catechin and its derivatives are accepted in various systems4 and also suggest that the scavenging effects of epigallocatechin are stronger than those of epicatechin and catechin. Furthermore, epigallocatechin gallate has the highest antioxidant capacity, which could be correlated to the antioxidant activities of the TFE.

The reducing capacity is another measure of the antioxidant power: the FRAP method measures the direct capacity to reduce ferric ions to ferrous ions. The TFE had a strong reducing power of 1.001 ± 0.001 mM Fe(II)/g, comparative to ascorbic acid (1.550 ± 0.001 mM Fe(II)/g), while catechin presented the highest reducing power (1.998 ± 0.001 mM Fe(II)/g). The presence of the hydroxyl group in the phenolic ring is the main factor of reactivity in the FRAP assay. Previous studies have shown that the reactivity of catechins in the FRAP assay confirms their ability to reduce metal ions due to the hydroxyl groups in the structure.4 Moreover, caffeic acid, which is reported in the TFE, presents two hydroxyl groups, and its ester was also correlated to FRAP reactivity.12

2.5. Optimization of Nanoemulsions

2.5.1. Fitting the Model

The experimental results of the three response variables (the droplet size, polydispersity index (PDI), and entrapment efficiency (EE)) are presented in Tables 4 and 5. The obtained models significantly fitted all response variables and presented a high coefficient of determination (R2), in the range of 0.8947–0.9977. These results confirmed that all values obtained from the experiment were in good agreement with the predicted values. The predicted values of the response variables were calculated using the coefficient of the polynomial equation. The analyses of variance (ANOVA) indicated that the experimental data represent the quadratic polynomial model. The results confirmed that all parameters in the regression models had a probability (p) less than 0.001, thus there was no lack of fit, as shown in Table 3.

Table 4. Significance Probability of Regression Coefficients in the Final Model.
type of effects variables droplet size (Y1, nm)
 PDI (Y2)
 entrapment efficiency (Y3, %)
F-value p-value F-value p-value F-value p-value
main effects Χ1 148.58 <0.0001 1.18 0.3030a 2982.05 <0.0001
Χ2 10.66 0.0085 18.25 0.0016 484.82 <0.0001
Χ3 3.75 0.0817a 0.6976 0.4231a 45.79 <0.0001
quadratic effects Χ12 25.22 0.0005 3.08 0.1099a 640.99 <0.0001
Χ22 6.62 0.0277 25.65 0.0005 50.78 <0.0001
Χ32 0.8976 0.3658a 0 0.9957a 0.7159 0.4173a
interaction effects Χ12 0.0292 0.8678a 21.23 0.0010 113.02 <0.0001
Χ13 0.0099 0.9228a 7.43 0.0214 0.0447 0.8369a
Χ23 0.0443 0.8375a 8.54 0.0152 0.027 0.8727a
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a

Indicates not significant at (p > 0.05).

Table 5. Different Responses of Optimization Experiments.
run droplet size (Y1, nm)
 PDI (Y2)
 entrapment efficiency (Y3, %)
actual value predicted value actual value predicted value actual value predicted value
1 50.59 54.27 0.100 0.132 67.89 67.05
2 102.93 110.55 0.166 0.185 34.37 34.38
3 45.10 40.27 0.112 0.097 74.48 74.22
4 48.26 52.59 0.131 0.119 72.12 70.96
5 99.70 86.95 0.389 0.337 50.23 50.90
6 112.30 122.71 0.160 0.196 31.82 30.55
7 152.80 143.49 0.210 0.197 20.09 20.98
8 62.10 54.27 0.141 0.132 66.12 67.05
9 50.78 57.51 0.220 0.222 69.98 69.46
10 55.29 54.27 0.142 0.132 66.94 67.05
11 57.50 54.27 0.144 0.132 66.64 67.05
12 77.77 69.50 0.140 0.146 62.96 64.47
13 97.79 93.84 0.143 0.145 54.47 54.74
14 47.76 46.70 0.314 0.349 73.15 73.60
15 99.30 103.15 0.334 0.303 51.86 51.16
16 31.55 36.93 0.154 0.162 73.90 73.36
17 39.94 32.32 0.108 0.076 77.10 78.12
18 46.02 54.27 0.111 0.132 67.13 67.05
19 53.42 54.27 0.156 0.132 67.65 67.05
20 49.60 58.41 0.151 0.198 72.33 72.02
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Table 3. Regression Coefficient, R2, Adjusted R2, and Probability Values for the Final Model Equationa.
regression coefficient droplet size (Y1, nm) PDI (Y2) entrapment efficiency (Y3, %)
β0 54.27 0.1325 67.05
Χ1 –31.68 –0.0105 15.57
Χ2 –8.49 –0.0412 6.28
Χ3 5.03 0.0081 –1.93
Χ12 12.71 0.0165 –7.03
Χ22 6.51 0.0476 –1.98
Χ32 2.4 0.0001 0.2349
Χ12 0.58 –0.0581 –3.96
Χ13 –0.3375 –0.0344 –0.0788
Χ23 –0.715 0.0369 0.0612
R2 0.9507 0.8947 0.9977
adjusted R2 0.9063 0.7999 0.9956
regression (p-value) <0.0001b 0.0008b <0.0001b
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a

β0 is a constant; X1, X2, and X3 are the estimated regression coefficients for the main linear effects; X12, X22, and X32 are the estimated regression coefficients for the quadratic effects; X12, X13, and X23 are the estimated regression coefficients for the interaction effects. X1: surfactant-to-oil ratio (SOR), X2: the ratio of high hydrophilic–lipophilic balance (HLB) surfactant to low HLB surfactant (HLR), X3: the effect of PEG-40 and PEG-60 hydrogenated castor oil.

b

Indicates a significant term (p < 0.05).

2.5.2. Effects of Independent Variables on the Responses

The effects of different levels of independent variables on the responses are presented in Table 5.

2.5.2.1. Droplet Size

As shown in Table 4, the droplet size was mainly affected by the SOR, with linear (p < 0.0001) and quadratic (p < 0.01) effects. Increasing the surfactant concentration led to a decrease in the droplet size. Another factor that significantly affected the linear (p < 0.01) and quadratic (p < 0.01) effects was the HLR, as shown in Table 4. The droplet size decreased as the HLR increased. Therefore, the main factors related to the physicochemical parameters are the oil/surfactant/water ratio and the surfactant blend.13,14

2.5.2.2. Polydispersity Index

As shown in Table 4, the HLR is the factor that significantly affects the linear (p < 0.01) and quadratic (p < 0.01) effects, along with the interactive effects (p < 0.01) of the SOR, as shown in Figure 1a. The response surface plot for the significant interactive effects also verified that the PDI decreased with increasing surfactant concentration and high HLB surfactant concentration, as shown in Figure 1b. Furthermore, significant interactive effects (p < 0.05) between the SOR and the PEG-40/PEG-60 hydrogenated castor oil ratio were also presented, along with significant interactive effects (p < 0.05) between the HLR and the PEG-40/PEG-60 hydrogenated castor oil ratio, as shown in Figure 1c.

Figure 1.

Figure 1

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Response surface plots of the significant (p < 0.05) interaction effects on the studied variations; (a– c) PDI, (d) entrapment efficiency (%).

The PDI, which represents the dispersion of the nanocarrier size distribution, is a highly significant physical characteristics to be considered when developing nanosystems, because the PDI attributes of the lipid-based particle can affect the characteristics, product efficacy, stability, and appearance of the formulation. PDI values of 0.2 and lower are generally accepted in practice for nanoparticle materials.15

2.5.2.3. Entrapment Efficiency

The % EE of the TFE into the nanoemulsion in each run of experiments is presented in Table 5. The EE of the nanoemulsions was mostly affected by the SOR, which had a significant effect on the linear (p < 0.0001), quadratic (p < 0.0001), and interactive effects (p < 0.0001), as shown in Table 4. Increasing the surfactant concentration resulted in an increased % EE, as shown in Figure 1d. Moreover, the HLR also contributed significantly to the linear (p < 0.0001) and quadratic (p < 0.0001) effects. The % EE increased as the high HLB surfactant concentration increased, as shown in Table 4. Another factor that significantly affected the EE was the linear (p < 0.0001) effect of the PEG-40/PEG-60 hydrogenated castor oil ratio. Increasing the concentration of PEG-40 hydrogenated castor oil in the formulation resulted in an increase in the % EE, as shown in Table 4. Polyethylene glycol (PEG) hydrogenated castor oil is a nonionic solubilizer and emulsifying agent obtained by reacting hydrogenated castor oil with ethylene oxide. It is the solubilizer normally selected to solubilize carrier oils, fragrance substances, and hydrophobic active ingredients.16,17 The difference between PEG-40 and PEG-60 hydrogenated castor oil involves the average number of moles of ethylene oxide in the structure and the HLB value. PEG-40 hydrogenated castor oil contains 40 moles of ethylene oxide and has an HLB value between 14 and 16, while PEG-60 hydrogenated castor oil contains 60 moles of ethylene oxide and has an HLB value between 15 and 17.16,17 Therefore, an increasing number of moles of ethylene oxide in the PEG hydrogenated castor oil structure, along with an increasing HLB value, could result in a decrease in the EE of the nanoemulsions.

2.5.3. Optimization of Responses for Formulation of TFE Nanoemulsions

The optimum TFE nanoemulsions (with a minimum droplet size, a PDI not higher than 0.2, and maximum EE) can be fabricated with a SOR of 2:1, indicating a surfactant concentration of 10.0% w/w, an HLR of 1.6:1, and a PEG-40/PEG-60 hydrogenated castor oil ratio of 2:1. Numerical optimization was conducted through Design-Expert software with a maximized desirability of 1. The predicted values for the droplet size, PDI, and % EE obtained from the numeric optimization are illustrated in Table 6.

Table 6. Predicted and Actual Value of Responses at Optimized Conditions.
response predicted value actual value % prediction error
Y1; droplet size (nm) 32.404 34.01 4.95
Y2; PDI 0.153 0.150 2.00
Y3; entrapment efficiency (%) 78.026 75.85 2.87
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The actual values derived from the experiment and the theoretical predicted values were statistically compared, as shown in Table 6. The percentage of prediction error was less than 5% for all responses, indicating that the model was acceptable.

2.5.4. Morphology of TFE Nanoemulsions

In general, the droplet in the nanoemulsions had a spherical shape and consisted of a hydrophobic oil core surrounded by a thin interfacial layer consisting of a surfactant.8 In this study, the morphology of the TFE nanoemulsions observed by transmission electron microscopy (TEM) also demonstrated a mostly spherical shape surrounded by the adsorbed surfactants, as shown in Figure 2.

Figure 2.

Figure 2

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TEM of the TFE nanoemulsions.

3. Conclusions

Nanoemulsions containing TFE were successfully fabricated using the second degree polynomial model to optimize and explain the effects of independent variables, including the SOR, HLR, and PEG-40/PEG-60 hydrogenated castor oil ratio on the droplet size, PDI, and EE by a central composite design (CCD). TFE nanoemulsions with a minimum droplet size, a PDI not higher than 0.2, and maximum EE were obtained using numerical optimization. Numerical optimization was adopted to find the best formulating conditions, which were a SOR of 2:1, an HLR of 1.6:1, and a PEG-40/PEG-60 hydrogenated castor oil ratio of 2:1. Moreover, an in vitro study indicated that the TFE extracted by SFE possessed a potential scavenging activity against DPPH radicals and also had a great reducing power. Consequently, nanoemulsions containing TFE could be a valuable active substance for the further development of nutraceutical and cosmeceutical products.

4. Materials and Methods

4.1. Chemical Materials

DPPH and TPTZ were purchased from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany). FeSO4•7H2O and C2H9NaO5 were purchased from Loba Chemie Pvt. Ltd. (Mumbai, India). Ethanol, FeCl3·6H2O, methanol, and hexane were purchased from Merck Ltd. (Darmstadt, Germany). Deionized water and sodium hydroxide were purchased from RCI Labscan Limited (Bangkok, Thailand). Polysorbate 80, PEG-40 hydrogenated castor oil, and PEG-60 hydrogenated castor oil were purchased from Chemecosmetics (Bangkok, Thailand).

4.2. Sample Preparation

Dried tea flowers were obtained from 101 Tea Co., Ltd., Mae Fa Luang District, Chiang Rai, Thailand, between September and October 2021. The dried tea flowers were ground using a grinder (Panasonic Co., Ltd., Osaka, Japan) to obtain a fine powder. The sample was kept in an air tight container.

4.3. Supercritical Fluid Extraction

The extraction of the tea flowers was carried out using SFE equipment (SFC-CO2-4000 analytical system, JASCO Inc., Tokyo, Japan). For each experiment, 30 g of tea flower powder was placed in an SFE vessel. The flow rate of CO2 and ethanol (a cosolvent) were both set to 1.0 mL/min, and the extraction was carried out using a pressure of 30 mPa. The extraction was performed in triplicate. The TFE was collected and stored in a container protected from light at 4 °C until required.10,18,19

4.4. Qualification of Polyphenolic Compounds

4.4.1. TPC Determination

The TPC in the TFE was evaluated using the Folin–Ciocalteu reaction, which was slightly modified from the method of Myo et al.20 and Theansungnoen et al.21 In this study, gallic acid was used to prepare the standard curve. The absorbance was measured at 765 nm, and the TPC was expressed as mg GAE per gram of sample. All experiments were performed in triplicate.

4.4.2. TFC Determination

The TFC in the TFE was determined using a method slightly modified from the method of Myo et al.20 and Theansungnoen et al.21 Quercetin was used to prepare the standard curve. The absorbance was measured at 510 nm, and the TFC was expressed as mg QE per gram of sample. All experiments were performed in triplicate.

4.5. Determination of Bioactive Compounds

4.5.1. UHPLC-ESI-QTOF-MS/MS Analysis

The TFE was analyzed using an UHPLC Agilent 1290 Infinity II System coupled to an Agilent 6545 LC-QTOF/MS. The separation was carried out using a Waters XBridge C18 (100 mm × 2.1 mm, 2.5 μm) column. The elution was achieved using a binary gradient system, with 0.1% formic acid in deionized water as eluent A and 0.1% formic acid in acetonitrile (ACN) as eluent B. The gradient steps were 5–17% B at 0–13 min, followed by 17–100% B at 13–20 min, 100% B at 20–25 min, and 100–5% B at 25–27 min, with a flow rate of 0.3 mL/min. Finally, a postrun was set to equilibrate the column for 6 min between analyses.

For the LC–MS system, a dual Agilent Jet System electrospray ionization (ESI) was used as an interface, with specific parameters including sheath gas temperature, 250 °C; sheath gas flow rate, 12 L/min; gas flow rate, 11 L/min; gas temperature, 300 °C; and nebulizer pressure, 45 psig. The LC–MS full-scan mode was operated using positive and negative ionizations. The scan range was 50–1050 m/z, and the scan rate was 1 spectra/s. Auto-MS2 was managed using fixed collision energies of 10, 20, and 40 eV. The MS/MS scan range was set from 50 to 1100 m/z, with a scan rate of 3 spectra/s. The isolation width MS/MS was set at ±4 m/z. The reference solutions were incorporated to provide internal reference masses for mass correction in positive and negative modes of operation.22,23

4.5.2. UHPLC-ESI-QqQ-MS/MS Analysis

Bioactive compounds from the TFE were determined using a Shimadzu Nexera X2 UHPLC system (Kyoto, Japan) equipped with a CBM-20A controller, DGU-20A5R degasser, LC-30 AD binary gradient pumps, a SIL-30 AC autosampler, and a CTO-20 AC column oven. Each bioactive compound was analyzed using a C18 reversed-phase Avantor ACE Excel C18-PFP (100 mm × 2.1 mm, 1.7 μm) column. The injection volume was 1 μL. Elution was performed using a binary gradient system with eluent (A) 0.2% formic acid diluted in deionized water and eluent (B) ACN. The gradient steps were 10% B at 0–0.30 min, then 10–15% B at 0.30–2.40 min, 15–20% B at 2.40–3.25 min, 20% B at 3.25–3.60 min, 20–95% B at 3.60–6.20 min, 95% B at 6.20–7.00 min, 95–10% B at 7.00–7.50 min, and 10% B at 7.50–11.0 min, with a flow rate of 0.3 mL/min.

For MS detection, both positive and negative ionization modes were operated using a Shimadzu LCMS-8060 (Kyoto, Japan) equipped with an ESI source. A triple quadrupole system was used for detection, under multiple reaction monitoring (MRM) mode. Nitrogen was used as a drying and nebulizing gas, at a flow rate of 10.0 and 3.0 L/min, respectively. The heating gas flow rate was 10.0 L/min. The ESI temperature was set at 300 °C with the temperature of the DL and the heat block set at 250 and 400 °C, respectively. The bioactive compounds were characterized by comparing the precursor ions (m/z), product ions (m/z), and retention times (RT, min). LabSolutions software (Kyoto, Japan) was used to verify and process the bioactive compounds. The concentration of each compound was expressed as ppm compared to the standards.20,24 The selected standard compounds are detailed in Table 7.

Table 7. List of Quantified Standard Compounds.
compounds [M – H] – (m/z) product ion (m/z) polarity
caffeine 195.00 138.15 positive
caffeic acid 178.80 135.20 negative
protocatechuic acid 153.25 109.05 negative
gallic acid 169.10 125.20 negative
p-coumaric acid 162.85 119.00 negative
ferulic acid 193.00 134.10 negative
catechin 289.25 245.20 negative
epicatechin (EC) 289.00 245.30 negative
gallocatechin (GC) 304.95 125.20 negative
epigallocatechin (EGC) 304.80 125.10 negative
catechin gallate (CG) 441.10 169.20 negative
gallocatechin gallate (GCG) 456.95 169.20 negative
epigallocatechin gallate (EGCG) 457.00 169.20 negative
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4.6. Antioxidant Activity Assays

4.6.1. DPPH Radical Scavenging Assay

Diluted TFE concentrations were prepared in ethanol in order to determine the radical scavenging activity using the DPPH assay, which was slightly modified from Nantarat et al.25 Gallic acid, ascorbic acid, and quercetin were used as the standards. Briefly, the TFE solution was incubated with 167 μM DPPH• in ethanol in the dark at room temperature for 30 min. The absorbance was measured at 517 nm using a spectrophotometer microplate reader (SPECTROstar Nano, Ortenberg, Germany). All experiments were performed in triplicate. The inhibiting effect of the TFE was calculated using the following equation:

4.6.1. 1

where Ac is the absorbance of the blank and As is the absorbance of the test sample. The IC50 was then calculated using a calibration curve of the TFE by plotting the sample concentration and the % inhibition.

4.6.2. FRAP Assay

The FRAP values for the TFE were evaluated compared to standard ferrous sulfate solution, using a method that was slightly modified from Nantarat et al.25 The TFE samples were prepared in ethanol and mixed with the FRAP reagent. The mixtures were then incubated for 5 min. The absorbance was measured at 593 nm. The FRAP values of each sample were calculated using the regression equation derived from the standard curve. The calibration curve was linear, with a regression coefficient (R2) of 0.9999 (data not shown). All experiments were performed in triplicate.

4.7. Preparation of the TFE-Loaded Nanoemulsions

The TFE nanoemulsions were prepared using emulsion inversion point (EIP) methods. Camellia sinensis seed oil was selected as the oil phase, and propylene glycol dissolved in deionized water was selected as the continuous phase. In this study, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, and polysorbate 80 were selected as the surfactant system. Briefly, using a magnetic stirrer, the oil and surfactant were slowly mixed with an aqueous phase containing propylene glycol, Spectrastat BHL, and deionized water, at room temperature. Stirring was continued until the nanoemulsion was completely formed.26

4.8. Characterization of Nanoemulsions

4.8.1. Droplet Size Analysis

A zetasizer (Malvern Instruments Ltd., Malvern, UK) was used to analyze the nanoemulsion droplet size following a previous protocol.26 Each formulation was diluted with water at a ratio of 1:100, at 25.0 ± 0.1 °C. All measurements were analyzed in triplicate. The size (nm) and size distribution (PDI) were reported.26

4.8.2. Entrapment Efficiency

The EE of each TFE nanoemulsion run was investigated using a centrifugal filtration device with a 100 kDa molecular weight cutoff filter (Microcon Millipore, Billerica, MA). Briefly, each formulation was added to the sample reservoir and then centrifuged at 1500 × g at 4 °C for 30 min to separate the entrapped and untrapped components. The EE of the TFE nanoemulsion was evaluated using the Folin–Ciocalteu reaction to measure its TPC.27 All experiments were analyzed in triplicate.

4.8.3. Morphology of Nanoemulsions by TEM

The morphology of the TFE nanoemulsions was investigated according to the method reported by Nantarat et al.26 Each sample was prepared in a 300 mesh copper grid, and 2% phosphotungstic acid was used to adjust the contrast of the image. The derived sample was analyzed using a JEOL JEM-1200 EXII electron microscope (Japan) operated at 80 kV at 40,000× magnification.

4.9. Experimental Design

The effects of independent variables including X1 (SOR), X2 (HLR), and X3 (the PEG-40/PEG-60 hydrogenated castor oil ratio) on Y1 (droplet size), Y2 (PDI), and Y3 (EE) were studied using a response surface methodology (RSM) design. The coded independent variables are given in Table 8. CCD was applied along with the quadratic model.28

Table 8. Levels of Independent Variables.

independent variables coded level
axial (−α) low center high axial (+α)
surfactant-to-oil ratio (SOR) (X1) 0.6591 1 1.5 2 2.3409
the ratio of high HLB surfactant to low HLB surfactant; HLR (X2) 0.6591 1 1.5 2 2.3409
the effect of PEG-40 and PEG-60 hydrogenated castor oil; RH-40:RH-60 (X3) –1.68 –1 0 1 1.68
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The second degree polynomial equation (as follows) was employed to express Y1 (droplet size), Y2 (PDI), and Y3 (EE) as a function of the independent variables.

4.9.

where Yi represents the responses, β0 indicates a constant, and βi, βii, and βij are linear, quadratic, and interactive coefficients, respectively. The coefficients were provided using Design-Expert software (version 7.1; Stat-Ease Inc., Minneapolis, MN, USA).

4.10. Statistical Analysis

The experimental procedure and analysis were carried out in triplicate. The results were analyzed using one-way ANOVA with 95% confidence level (p < 0.05). The data are shown as the mean ± standard deviation.

Acknowledgments

This work was financially supported by a grant from Mae Fah Luang University with grant no. 651A02001 and partially supported by the funding from Reinventing University Program 2021. The authors thank the scientific and technological instrument center, Mae Fah Luang University and Faculty of Pharmacy, Chiang Mai University for the instrument support.

The authors declare no competing financial interest.

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