The biological response of Carica papaya leaves extract to saponin reduction (O/W) emulsion on human bronchial epithelium cell (BEAS-2B)

2.1.1 Carica papaya leaves

The papaya (Carica papaya L. cv. Eksotika) leaves samples were taken from a papaya orchard located in Bukit Changgang, Dengkil, Selangor. Only a mature green papaya leaf with no insect damage and insecticide-free was used to ensure consistency of the emulsion preparation and healthier cell culture response.

2.1.2 Chemicals

The chemicals used in this study are as follows: Premium virgin coconut oil (VCO) brand of Bio-Asli from Desaku Maju Marketing; Filtered Prime Whey Isolate from Lush Protein Armor ASIA, Singapore; Diaion® WA30 ion exchange resin (Sigma-Aldrich^TM, Germany) and BEAS-2B of the human bronchial epithelial cell line was taken from Advanced Medical and Dentistry Institute (AMDI), Universiti Sains Malaysia (USM). In addition, xanthan gum (Sigma-Aldrich^TM, Malaysia; Catalogue No.: G1253); deionized water (DIW); methylene blue (Sigma-Aldrich^TM, Malaysia; Catalogue No.: 03978); PrestoBlue^TM MTS (ThermoFisher Scientific, Invitrogen^TM; Catalogue No.: A13261); Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich^TM, Malaysia; Catalogue No.: D8437); Trypsin-EDTA solution (Sigma-Aldrich^TM, Malaysia; Catalogue No.: T4049); Phosphate Buffer Saline (PBS) (Sigma-Aldrich^TM, Malaysia; Catalogue No.: P5493); 1 % (v/v) streptomycin (Sigma-Aldrich^TM, Malaysia; Catalogue No.: S6501); 100 μg/mL penicillin (Sigma-Aldrich^TM, Malaysia; Catalogue No.: P3032) and Trypan Blue Solution, 0.4 % (Gibco®, ThermoFisher Scientific; Catalogue No.: 15250061) were used for TPD emulsion preparation, physicochemical and cytotoxicity analysis.

2.2.1 Fresh papaya leaves extract (FPL)

The fresh papaya leaves were extracted according to the Ministry of Health (2015), Nguyen et al. (2016) and Halim et al. (2011) recommendations. Freshly cut papaya leaves were soaked in water for 30 mins, rinsed, air-dried (30 mins) and crushed using mortar. It was then centrifuged using a Gyrozen centrifuge model GZ-406 (BioAstrum Corporation, Maryland, USA) for 5 mins at RCF: 1,006 × g (10 cm of rotor radius). The upper supernatant was collected and stored at −18 °C for further use.

2.5.1 Designing a pseudo-ternary phase diagram

Construction of TPD was performed based on the combined protocols of David & Mary (2019), Wahgiman et al. (2020) and Fadzillah et al. (2020). The first batch of TPD was made with a combination of virgin coconut oil (VCO) and whey protein (WP) in the range of 0: 100 to 100: 0 (VCO: WP (w/w)) is shown in Table 1. These mixtures were then vortexed using Velp Scientifica Vortex Mixture, F202A0173 ZX Classic (Usmate Velate (MB), Italy) for 10 mins for the homogenization process. The theoretical weight is the amount of weight that should be applied to the formulation. Whereas the actual weight is the total weight of the emulsion compound successfully added by approaching the theoretical weight value. Meanwhile, the ‘c’ value was used for the next additional step of deionized water into each test tube. The amount of deionized water to be added (g) into each of these test tubes is calculated using Equation (1) below:

Where,

a = Total weight of deionized water to be added (g).

d = Deionized water added for each round (%, w/w).

c = Actual weight of VCO (Ce) (g) + actual weight of WP (Cf) (g).

The deionized water was added drop by drop into the test tube for each round until it reaches the total weight of water. Then, each test tube was vortexed for 5 mins and centrifuged for 20 mins at RCF: 1,006 × g (10 cm of rotor radius). The layer formation was monitored and only homogeneous emulsions were accounted for in the next step. Each edible O/W emulsion formulation that has successfully formed a homogeneous phase was recorded into CHEMIX School 3.51 (Bergen, Norway) software to generate a pseudo-TPD profile to get an initial depiction of the emulsion behaviour in the formulation mixture.

2.5.2 Ternary phase diagram generation

The above-mentioned steps in Section 2.5.1 were repeated by substituting the deionized water (DIW) component with the FPL and PSLR extracts following to the previous emulsion mixture ratios that have reached a homogeneous phase. The emulsion ratio was obtained from the previous pseudo-TPD data distribution as a working baseline (working template).

2.5.3 Extensive examination of the ternary phase diagram

Further tests were carried out by dropping 10 drops of homogenous TPD formulation with surfactants containing 30 % (w/w) or less at random. A total of 20 g of the selected emulsion was added for each formulation and homogenized using vortex for 15 mins, followed by a homogenizer (T10 Basic Ultra-Turrax homogenizer) for 10 mins at RCF: 1,006 × g (10 cm of rotor radius). After 30 mins of storage, the emulsion stability results were observed, and the best FPL and PLSR extract emulsion formulation were added with 0.2 % (w/w) xanthan gum to increase their stability and shelf-life.

2.6.1 pH analysis

The pH value of the emulsion was determined at 25 °C using a pH meter, PHM 210 analytical radiometer (MeterLab®: LabX, Midland, ON, Canada). The pH meter was calibrated at pH values of 7.01, 4.01, and 10.1 buffer before reading. The reading was taken in triplicate (n = 3).

2.6.2 Creaming index

The creaming index was determined based on the method used by Tangsuphoom & Coupland (2008). Approximately 10 g of the emulsion sample were added into a universal bottle and sealed tightly. It was stored at 25 °C for 24 hrs. The overall height of the emulsion and the height of the bottom layer of droplets formed were calculated based on Equation (2) in triplicate (n = 3):

HL: The height of the droplet layer formed at the bottom.

HE: Total height of emulsion.

2.6.3 Contact angle

The contact angle was measured using a Rame-hart model goniometer (Model 190, USA). Approximately 100 μL of the emulsion was sessile dropped onto a borosilicate glass slide and analysed using a height-width method to measure its angle. The reading was measured in quintuplicate (n = 5) in several different places for consistency.

2.6.4 Particle size measurements

Emulsion particle size was measured based on the method used by Wahgiman et al. (2020) with slight modification using a light scattering laser device (Malvern Zetasizer Ver. 6.00) at 25 °C. and a dynamic light scattering technology. The emulsion extract was first filtered by a 0.45 µm membrane filter. Then, 0.75 mL of the emulsion was inserted into a zeta-sizer device. The intensity distribution was used for mean average (z-average) particle size measurement. The measurement was repeated in triplicate (n = 3).

2.7.1 Cell viability using diphenyltetrazolium bromide (MTT) assay

For the cell toxicity analysis, about 100 µL of the emulsion was added and swirled evenly onto the entire space of BEAS-2B cells. The microplates were then incubated for 24 hrs at 37 °C and 5 % CO₂. Approximately 100 µL of DMEM growth media was set as negative control and a mixture of cells and growth media without extract was set as the positive control. The cell viability (%) was carried out based on the method used by Nguyen et al. (2016). Approximately 10 µL of PrestoBlue^TM solution was added to the microplates that had been treated with emulsion solution and incubated for 24 h at 37 °C and 5 % CO₂. The absorbance for emulsion solubility was measured at 570 nm every 24 hrs using the FluoStar Absorbance Microplate Reader (BMG Labtech, Germany) in triplicate (n = 3) and measured using Equation (3) below. The data was then used to calculate EC₅₀ values.

The cell morphology was then observed using a BD-S2 Inverted Biological Microscope (Boshida® optical instrument, Shenzhen, China) to analyse any changes throughout 72 hrs of treatment. Methylene blue was used to stain adherent cells, cultured in 96 well plates to distinguish any dead cells prior to cell morphology analysis.

2.7.2 EC₅₀ value measurement

EC₅₀ is an effective concentration of a chemical compound that is tested on cell culture. The EC₅₀ value ​​of both emulsions and WP was determined based on the cell viability (%) absorbance readings taken at 24, 48, and 72 hrs. This value is determined based on the calculation of the equations obtained from the cell viability (%) versus concentration (log; µg/mL), where the y-value of y = 50 % cell viability.

3.2.1 Fresh papaya leaves extract (FPL) emulsion formulation

In this study, the formulation of a mixed VCO, WP and papaya leaf extract was produced based on the ternary phase diagram (TPD) method. The FPL formulation serves as a control formulation (positive) and this formulation is considered the standard extract preparation based on the Ministry of Health (MOH), Malaysia guidelines. Table 2 shows ten emulsion formulation points which were randomly selected from the TPD homogenized region. Fig. 3 shows the TPD profiles generated from the mixture of FPL emulsion formulations. The grey region shows an emulsion formulation reaching a homogeneous phase after being homogenized and stored for 30 mins. The homogenized emulsion formation was found to be concentrated in the range of 30 % to 90 % (w/w) of the extract concentrations. The VCO and WP concentration was set to be<40 % (w/w) and 30 % (w/w) respectively to produce emulsions with moderate viscosity (Sanjeewani & Sakeena, 2013). The emulsions that produce several phases of emulsion were excluded from this observation. Based on those ten formulations tested, formulation #4 (Fig. 3: H point) showed the best emulsion stability characteristics as compared to the other formulations. At this formulation ratio of 21: 16: 63 % (w/w) (VCO: WP: FPL), it has produced a well-blended and homogenized emulsion that did not produce any immiscible layers after more than 30 mins of storage.

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Fig. 3

Ternary phase diagram for fresh papaya leaf (FPL) of ten emulsion formulation points which was randomly selected from the homogenized region of TPD (grey region). Formulation #4 (H point) was selected prior to physicochemical and cell response analysis.

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3.2.2 Papaya leaves with saponin reduction extract (PLSR) emulsion formulation

At the beginning of the phase formation, the WP was limited to 30 % (w/w) and below. However, the emulsion formulations were randomly assigned to a few points of the increment (WP) to see a full spectrum of formulation behaviour. It was observed that this formulation mixture produces better and broader emulsion stability. Initially, the VCO maximum amount was used (100 %, w/w) with only 60 % (w/w) of WP without the presence of PLSR extract. Next, the highest PSLR extract was used (90 %, w/w) with 9 % (w/w) of WP without the presence of VCO. Finally, the highest WP was utilized (90 %, w/w) with 10 % (w/w) of VCO without the presence of PLSR extract. Ultimately, the construction of the TPD profiles was completed with the generation of a homogenized grey region (stabilized emulsion after 30 mins of storage) that covered almost the entire diagram (Fig. 4). Further analysis was carried out from the homogenized grey region to obtain the best formulation. Table 3 shows ten randomly selected emulsion formulation points and the WP was set below 30 % (w/w) to produce emulsions with moderate viscosity (Sanjeewani & Sakeena 2013). The surfactant (WP) is limited to<30 % (w/w) based on the production cost assessment (economic feasibility study) and health risks factor. High surfactant content (e.g., WP) in any edible food-based formulation can irritate the gastrointestinal tract and serious health issues if taken in a high dosage (Ahmad et al., 2013). Hence, it was observed that formulation #10 (Fig. 4: G point) with a ratio of 19: 16: 65 % (w/w) (VCO: WP: PLSR) shows the best stabilized and well-blended emulsion without any immiscible layer’s formation for more than 30 mins of storage. It was then added with 0.2 % (w/w) xanthan gum to increase its long-term stability and shelf-life.

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Fig. 4

Ternary phase diagram for fresh papaya leaf with saponin reduction (PLSR) of ten emulsion formulation points which was randomly selected from the homogenized region of TPD (grey region). Formulation #10 (G point) was selected prior to physicochemical and cell response analysis.

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Furthermore, based on both FPL and FPSR emulsion formulation profiles, the stability of a protein mixture emulsion depends heavily on the interaction between the protein layers absorbed by the oil molecules. The main thermodynamic absorption force is when the removal of excess hydrophobic molecules occurs by replacing them with water molecules in the aqueous phase (Andrews et al., 2005; Asenjo & Andrew, 2011; Loi et al., 2019). During the homogenization process, the molecular droplets are disturbed due to strong shear forces and form a new surface around the molecules. Currently, WP is required in large amounts to cover the surface of the molecules (McClements, 2004). Realistically, as the usage of WP is<10 % (w/w), the emulsion system shows the formation of heterogeneous emulsion formulations. At this point, the presence of WP is quite insufficient to cover the entire surface of the droplet, but in fact, the droplet is likely to clump and produce larger-sized droplets. Nevertheless, as the WP increased to 15 % (w/w) and above, the emulsion began to show a thinner separation layer, until it reaches a desired homogeneous phase. The presence of WP at this point has successfully covered almost all droplets on the new surface and stabilized the oil droplets in the emulsion system (Qian et al., 2011).

In the presence of high emulsifier content (%, w/w), WP can form several layers covering the emulsion droplets. The formation of several layers can alter the interface rheology of emulsion droplets due to the formation of a stable interface layer that is resistant to the occurrence of re-agglomeration (McClements, 2004). Besides, there is an improvement in droplet molecular composition and the reduction of collision interactions between droplets occurring due to the mobilization of each emulsion droplet in the new emulsion system, causing the emulsion concentration to increase in the continuous phase (Tesch & Schubert, 2002).

3.3.1 The pH profiles

The pH value in Table 4 indicates that both FPL and PLSR extract emulsion was slightly acidic and very near to neutral pH (p greater than 0.05). In fact, the removal of saponin (PLSR) did not attribute to any significant difference as compared to FPL extract emulsion (p greater than 0.05). As these emulsions serve as therapeutic supplements for dengue fever patients, neutral pH is seen to be suitable for promoting cell tissue growth (e.g., fibroblast: blood vessels) and at the same time helping other emulsion components (e.g., VCO and WP) to respond well to the human body microenvironment systems. Besides, excessively acidic or alkaline emulsification conditions can cause emulsion instability and might affect the gastrointestinal tract. However, the VCO pH value shows high acidic when they reach a value of as low as 5.40 due to the VCO's fatty acids content (predominantly containing 48.40 % to 52.80 % of lauric acid) which acts as antiviral, anti-inflammatory and wound healing precursors (Carandang, 2008; Nevin & Rajamohan, 2010; Subermaniam et al., 2014). However, the acidity of the VCO does not affect the pH reading of the homogenized emulsion as it has been stabilized by the presence of the WP.

Furthermore, the phase separation (immiscible layers) of the WP mixture emulsion system can occur at pH 5.0 and below due to the formation of flocculation between proteins (Laplante et al., 2005). Adjusting the emulsion pH to 5.5 and above might have resulted in the formation of stable and smooth lipid droplets. The optimal stability of both emulsions was finally obtained ranging from pH 6.0 to 7.0, where WP synergistic effect might have been the main contributor to the ultimate stabilized emulsion formation. However, protein oxidation (WP) can still happen at pH 4.5 in this VCO-based emulsion and produce separate protein deposits in the emulsion system (Chen & Diosady, 2003). This is because the protein contained in VCO-based emulsion can easily turn into sediment and clots at pH 4.0 (Tangsuphoom & Coupland, 2008). Surprisingly, under this nearly neutral pH emulsion condition, the decomposition of casein (WP) can be prevented from occurring in the protein mixtures emulsion and simultaneously prolong its shelf-life for food-based safety purposes (Bos & Van Vliet, 2001).

3.3.2 Creaming index (CI)

The creaming index was commonly used to predict the emulsion formulation stability. Both nano emulsion mixtures of FPL and PLSR extract formulation produced a creaming index of 0 indicating a very stable emulsion has been successfully developed (p greater than 0.05). In fact, both formulations did not show any apparent separation phases (immiscible layers) which indicates that flocculation and creaming conditions were not produced in this emulsion. The mixture remained homogenous which indicates its high stability at 25 °C for 24 hrs of storage. Thus, the stability of both emulsion formulations was high because the mechanical structure of the emulsion system can withstand the occurrence of gravity deposition and creaming precipitation. However, other emulsion formulations (Table 2 and Table 3) began to show separate oil layer formation in<24 hrs of storage. Cream precipitation can occur when the density of oil droplets is heavier or lighter than the density of the aqueous phase, decomposition of the emulsion composition, gravitational or centrifugal forces and lipid oxidation. Moreover, flocculation can also occur when oil droplet molecules overshared some of the protein molecules in the emulsification process due to the number of protein molecules available is quite insufficient to cover the entire oil–water interface layer (Chanamai & McClements, 2000; McClements, 2004; Thevene, 2006; Tadros, 2010).

3.3.3 Contact angle profiles

The contact angle describes the wetting value of where the liquid–vapour interface meets on an untreated/treated solid flat surface. There was an almost 2-fold decrease in PLSR contact angle values than the FPL extract emulsion (p < 0.05) (Table 4). In fact, both emulsions contact angles were<55.70° (water contact angle on an uncoated micro slide) which indicates that the emulsion particles were mostly hydrophilic-based liquid on the surface of the micro slide glass (Gao et al., 2006). Moreover, a low contact angle indicates that the surface is high in wetting, meaning that the water droplet spreads out more on the surface (Aydar, 2020; Ramlan et al., 2022). Thus, PLSR demonstrated two times higher wetting capability than FPL extract emulsion to indicate that the removal of saponin bioactive compound (aescin) from the extract has changed its hydrophobicity/hydrophilicity profoundly (p < 0.05). Furthermore, FPL was prepared from fresh papaya leaves without the addition of any water or solvent. Unlike PLSR, the presence of high-water content in the extract produces extra intermolecular interactions and repulsive forces between the particles producing lower surface tension between the particles (Tadros et al., 2004; Snoeijer & Andreotti, 2008). Surfaces with high energy interactions will produce better absorption for later mixing and formulating (Muster & Prestidge, 2002; Ramlan et al., 2018).

3.3.4 Particle size measurement

Particle size measurement is used to measure the particle size distribution for each component in an emulsion system. It was observed that the particle size distribution of PLSR was four times smaller than the FPL emulsion formulation (p < 0.05) (Table 4). In fact, both formulations were also categorized as nanoemulsions since the size range is between 100 and 500 nm (Kabri et al., 2011). Nanoemulsion has good suspension stability at −10 to 55 °C, can withstand the physical instability of the emulsion caused by gravity, flocculation and/or coalescence, is easily prepared at low surfactant content of as low as 20 % or less and has a better wettability and absorbability (Tadros et al., 2004; Anton et al., 2008; Khor et al., 2014). The production of nanoemulsions has always been a major selection in any medicinal product development. Those special features are considered an added value to the developed emulsion by enhancing the drug absorbability into the body’s cellular functions. It helps by increasing the efficiency of the drug delivery system to patients which can be taken orally or by injection (Sanjeewani & Sakeena, 2013). For that reason, good characteristics of papaya leaf extract nanoemulsion will eventually help its absorption into targeted cells and biological systems so that any immune responses that help the body to fight those viruses (e.g., dengue virus) can be activated efficiently.