Physico-Chemicals Characterization of Quercetin from the Carica papaya Leaves by Different Extraction Techniques

Carica papaya leaf extracts from four different extraction 
methods (cold-water, hot-water, sonication and supercritical fluid), 
were characterized using physico-chemical analysis. Sonication extraction 
showed the highest percentage yield of crude extracts (6.76%) and the lowest 
from the supercritical fluid extraction (1.83%). The Infrared (IR) spectrum of 
cold-water extract demonstrated the most similarity of functional group to quercetin. Likewise, the 
thermal analysis indicated that cold-water extract gave a quite similar 
Endothermic peak to quercetin 
with the obtained value which was 137°C and onset 
value which was 118°C. The 
liquid chromatography (LC) profile indicated that only the cold-water extract 
exhibited 0.203 ppm of quercetin.

Quercetin is a biflavonoid that is usually found in many plants including Carica papaya. [4] reported that the existence of quercetin in Carica papaya leaves promotes anti-dengue activities. The leaf extract is generally prescribed for patients with dengue fever, but scientific pieces of evidence for the anti-dengue activities are still limited. Flavonoid such as quercetin was reported to possess a high inhibitory effect towards dengue [5]. Quercetin has the ability to inhibit the platelet aggregation, which promotes anti-dengue activities [6]. Several analytical techniques have been developed for identification and quantification of flavonoids such as gas chromatographic (GC), mass spectrometry, thin layer chromatography, UV spectrometry, high-performance liquid chromatography (HPLC) and infrared spectroscopy [7].
FTIR is techniques based on the vibrations of the atoms of a molecule which are no two compounds produce the exact same infrared spectrum because each different material is a unique combination of atoms [8]. Furthermore, measurements made by FTIR are accurate and reproducible due to their sensitivity, speed, internally calibrated and mechanical simplicity. DSC is a technique for determining thermodynamic properties of biomacromolecules with the ability to provide detailed information about both the physical and energetic properties of a substance [9].
HPLC is the most widely used of all of the analytical separation techniques due to its sensitivity, its ready adaptability to accurate quantitative determinations, its ease of automation, its suitability for separating non-volatile species or thermally fragile ones as well as its widespread applicability to substances that are important to the industry, many fields of science and the public [10]. Therefore, the combination of several spectroscopy methods is proposed to enrich the identification and quantification of the compound. In this study, we combined three spectroscopic techniques (FTIR, DSC and HPLC) to identify and quantify quercetin from the extract of Carica papaya leaves. Thus, identifying the amount of quercetin in Carica papaya leaves extract could further explain the pharmacological properties of the plant.

Extraction
To determine the optimal extraction method of Carica papaya leaves, four dif-Open Journal of Physical Chemistry ferent methods were tested, which were cold-water extraction, hot-water extraction, sonication and supercritical fluid extraction (SFE).

Cold Water Extraction
1 kg of ground leaves samples was added with 5 L ultra-pure water and the extract was filtered and transferred into a plastic bag. The extract was stored in −20˚C freezer. The frozen sample was then transferred into a freeze-dry vessel followed by lyophilization process to powder using a freeze dryer machine (CHR-iST ® , ALPHA 1-2 LD). The powder was then collected, weighted and stored in a labeled bottle and kept at −20˚C for further usage.

Hot Water Extraction
1 kg of ground leaves samples was added with 5 L ultra-pure water and heated to 40˚C for 3 hours and let it cooling down to room temperature and filtered using cloth. The extract was packed into a plastic bag and stored in a −20˚C freezer.
The frozen sample was then transferred into a freeze-dry vessel followed by lyophilization process to powder using a freeze dryer machine (CHRiST ® , ALPHA 1-2 LD). The powder extracts was then collected, weighted, stored in a labeled bottle and kept at −20˚C for further usage.

Sonication
1 kg of the leaves was grounded with 1.25 L ultra-pure water. The ground sample was transferred into 4 units of 1000 mL beaker and sonicated for 1 hour. The extract was filtered and transferred into a plastic bag. The filtered extract was stored in −20˚C freezer and further lyophilized to powder. The powder was then collected, weighted and stored in a labeled bottle and kept at −20˚C for further usage.

Supercritical Fluid Extraction (SFE)
109 g of leaves samples was sent to an analytical service company, Renetech Sdn Bhd (734686-H) (formerly known as Renetech Scientific Sdn Bhd) for the supercritical fluid extraction (DELTA, Taiwan) service. In this study, fixed extraction parameters were optimized at temperature of 50˚C, pressure of 3626 psi for 5 hours.

Differential Scanning Calorimetry (DSC) Analysis
Diamond DSC (Perkin Elmer, USA) was used for thermal analysis. The type of pan used was standard aluminium pans (158003) and covers (160955). The scan-

Sample Preparation
All extracts were dissolved in methanol HPLC grade to obtain 1000 ppm concentration in methanol and filtered using 0.45 µm PTFE filter prior to HPLC analysis.

Standard Curve
Stock solution of quercetin standard (SIGMA-ALDRICH, USA) with ≥95% was prepared at 10 ppm and the standard curve was constructed (Table 1) [10].

1) Specificity
Specificity of the HPLC method was observed by the separation of the analytes. A volume of 10 µL of quercetin was injected and the chromatogram was recorded.

2) Linearity
The linearity was conducted based on a standard curve by plotting peak area versus concentration of quercetin where the square of the correlation coefficient R 2 > 0.99 is indicative of the linearity.

3) Repeatability
The repeatability of the proposed method was conducted by injecting three replicates of 0.4 ppm concentration. The peak area was calculated based on the standard curve within the Beer's range. Percentage of relative standard deviation (RSD) was calculated based on the peak area.

4) Limit of detection (LOD) and limit of quantification (LOQ)
The LOD and LOQ were determined by using the formula based on the standard deviation of response and slope.
where σ is the standard deviation of response and S is the slope of the calibration curve.

Quantification of Quercetin in Sample
The quantification of quercetin in the cold-water extract, hot-water extract, sonication water extract and supercritical fluid extraction was based on the standard curve.

Extraction of Carica papaya Leaves
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

Fourier Transform Infrared (FT-IR) Analysis
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].

Differential Scanning Calorimetry (DSC) Analysis
DSC was used to determine the thermal characteristics of each sample to suggest Open Journal of Physical Chemistry the possibility of incompatibilities and interactions [14]. 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 (TGA) Analysis
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].

High Performance Liquid Chromatography (HPLC) Analysis of Quercetin
The validation method of HPLC analysis comprises specificity, linearity, repeatability, limit of detection and limit of quantification. Figure 4 (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].

Conclusion
To this end, the study found that the cold water extraction method showed the Open Journal of Physical Chemistry   highest spectral similarities to quercetin from DSC thermogram and FTIR spectral. The quantification of quercetin in the cold-water extract indicated a 0.203 ppm yield, but was not determined in the other three extracts. Quercetin is sensitive to heat, thus no heat was applied using this method where active chemical constituent inside the sample can be preserved. Also, this method uses minimal electricity cost and time consumption. However, both hot water extract and sonification extract did not possess this flavanoid due to high temperature. Higher temperature causes the change in the protein structure and protein unfolding with the loss of activity which promotes to denaturation of the protein [22]. Even though supercritical fluid extraction (SFE) provides a range of benefits such as low critical temperature (31˚C), selectivity, inertness, non-toxicity, and capability to extract thermally labile compounds, but SFE also offers some of the limitations which include very expensive and complex equipment operating at elevated pressures, no polar substances are extracted and high power consumption. Hence, the cold-water extraction method was found to be the optimal extraction method and the identification and quantification of the extract are enriched from all three analytical techniques by showing the highest spectral analysis to quercetin. The extraction method and the combination of the analytical techniques can be used to standardize the extract, which is can be used in developing nutraceutical product specifically to the anti-dengue properties.