Plant samples of Caricapapaya were supplied by d’Cultivate Resources Sdn. Bhd. and obtained from Desasiwa RST, Universiti Sains Malaysia (USM), Penang. The plant was identified by Dr. Rahmad Zakaria, botanist from School of Biological Sciences, Universiti Sains Malaysia, Penang with the voucher specimen is IPNAT203A. The leaves of plant samples were washed and packed in 1 kilogram per bag for each different extraction method.

To determine the optimal extraction method of Caricapapaya leaves, four different methods were tested, which were cold-water extraction, hot-water extraction, sonication and supercritical fluid extraction (SFE).

2 mg of each extract (hot, cold, sonication and supercritical fluid) and standard compound, quercetin (SIGMA-ALDRICH, USA) with ≥95% were analyzed using Attenuated Total Reflectance (ATR) crystal, Perkin Elmer FT-IR C103470 (Spectrum Two DTGS) with a scan (16 scans) range from 4000 to 450 cm⁻¹ (mid-infrared) at resolution of 4 cm⁻¹.

Diamond DSC (Perkin Elmer, USA) was used for thermal analysis. The type of pan used was standard aluminium pans (158003) and covers (160955). The scanning program used for all components was Isothermal for 1 minute at −20˚C and scanning from −20˚C to 300˚C at the scanning rate of 10˚C/min. Every sample was scanned three times and both mean and SD were calculated for the melting characteristic namely the melting point (˚C) and the energy used (Delta H) in the melting process (J/g).

20 mg of four sample carica papaya extract and one standard compound quercetin were send to USAINS Biomics Laboratory Testing Services Sdn. Bhd (937636-D) (formerly known as Usains Biomics Sdn Bhd). Thermogravimetric analysis using Perkin Elmer TG/DTA analyzer (STA 6000, Massachusetts, U.S.A) operating in the range of 25˚C to 300˚C with at temperature rise of 10.00˚C/min and flow of nitrogen gas is 10 mL/min.

Four different extraction methods involved in this study are hot water, cold water, sonication and supercritical fluid. Table 2 shows the sonication method produced the highest percentage yield, which is 6.76% followed by cold water (5.78%), hot water (3.22%) and supercritical fluid extraction (1.83%) respectively. Even though the sonication method produced the highest yield, the cold-water method is more preferable for further analysis because no heat was applied that

may change the morphological structures as well as an active chemical constituents inside the sample, which may result in compound degradation [11].

The spectral data of the comparison between the four different extraction methods and the standard compound, quercetin can be found in Figure 1 and the summary of each spectrum is listed in Table 3.

The FTIR spectroscopy was used to investigate the possible chemical compounds in the sample because it is the most suitable technique of the non-destructive spectroscopic methods [12]. Quercetin is a plant polyphenol from the flavonoid group. The functional group of quercetin is phenol with the hydrogen bonded O-H stretch from the range 3600 - 3100 cm⁻¹ where it can be found in all extracts (Table 3) but not in supercritical fluid extraction method. Among the functional groups observed in all extracts, -OH group was found to be present uniformly in the water extracts. Based on the spectrum, the cold-water extract showed the most similar functional group to quercetin. Meanwhile, the hot-water extract showed the missing one functional groups of C-O stretch and C-C=C asymmetric stretch as well as sonication extract. This can be deduced from the hot water extraction may denature the protein structure and cause the permanent structure denaturation and reducing the flavonoid property of quercetin [13]. Meanwhile, for the supercritical fluid extract, there is missing peak of O-H stretch due to no solvent was applied in the extraction process. In addition, there is an additional C-H₃ bend which was suggested from the CO₂ supply

during extraction process. Hence, the cold-water extraction method shows an enriched identification of quercetin as an active ingredient in the Caricapapaya leaves.

DSC was used to determine the thermal characteristics of each sample to suggest the possibility of incompatibilities and interactions [14]. The results are predictive by interpreting the thermogram of each component and the mixture of the components [15]. The diagram (Figure 2) depicts the thermogram of each extraction method.

The DSC curves for SFE extract show that thermal processes occurred at onset temperature 62.61˚C. The presence of endothermic event at 66.49˚C (D = 3.2761 J∙g⁻¹) is observed, which is probably related to the loss of volatile constituents of the sample, such as water [16]. DSC curves for sonication extract shows that thermal processes occurred at onset temperature 112.90˚C with the presence of endothermic event at 135.02˚C (D = -26.3375 J∙g⁻¹) was observed. DSC curves for cold-water and hot-water extract shows that thermal processes occurred at near onset temperature which is 118.77˚C and 117.04˚C respectively but with different endothermic event at 137.71˚C (D = -33.11 J∙g⁻¹) for cold water extract and endothermic event at 118.16˚C (D = 1.4177 J∙g⁻¹) for hot-water extract.

When compared to quercetin, the cold-water extraction method was the optimal method. The Endothermic peak between the method and quercetin standard is quite similar, which they obtained value is 137˚C and onset value is 118˚C. Despite, the thermogram of each extraction method shows reproducibility, and the cold-extraction method demonstrates the optimal reproducibility.

Thermogravimetric analysis (TGA) is an evaluation technique that measures different substance masses as their temperature is changed or at a constant temperature over a given time. It is used to analyze decomposition and evaporation rates, oxidation, material purity and many other properties. The thermal behavior of the different Carica papaya extract is shown in Figure 3 that represents thermal behavior of the standard compound (quercetin) as a marker.

Overall, the TGA curve shows that the sample decomposes in three major steps within the temperature range 25˚C - 300˚C. The first mass loss, takes place between 30˚C - 98˚C, results in 0.7% may be attributed to the loss of adsorbed and structural water of excipient or due to desorption of moisture as hydrogen-bond water to the polysaccharide structure [17].

The second weight loss event take place between 99˚C - 113˚C, resulted in weight loss of about 1.8%, may be attributed to the excipient decomposition (maximum oxidation or decomposition temperature) [18].

The third weight loss event takes place between 115˚C - 300˚C, results in a weight loss of about 97.5%. A major weight loss (97.5%) takes place attributed to the complete decomposition of plant extract. Therefore, the calcination temperature for the preparation of the extract was chosen to be 250˚C that cover the temperature region from above the major decomposition region at 200˚C to 300˚C, where a stable mass was obtained [19].

The validation method of HPLC analysis comprises specificity, linearity, repeatability, limit of detection and limit of quantification. Figure 4(b) indicates a single peak of standard quercetin detected at 12.568 min at wavelength 372 nm. Based on Figure 4(a), the correlation coefficient, R² was found to be 0.9996 with the slope at 36.247 and the intercept at 1.551. LOD and LOQ were found to be 0.0277 ppm and 0.0841 ppm respectively. The percentage of RSD (Table 4) is 0.371% for repeatability of quercetin. The acceptance criterion for precision is less than 2% of RSD value.

All extracts were analyzed using HPLC method to detect the presence of quercetin. The quantification of each extract was determined respectively (Table 5). Based on the HPLC profile (Figures 4(c)-(f)), only the cold water extract exhibited 0.203 ppm quercetin compares to hot-water extract, sonication extract and supercritical fluid extract which indicate none of the compound itself. The difference of extraction conditions will affect the yield of quercetin. [20] reported that isolation of quercetin using the cold condition is more effective, contradicts to the thermal processing condition, which could also cause the loss of natural bioactive properties [21].