Introduction
Plants have long been recognized by the public for their medicinal properties. Traditional medicine is well-known and has been used for generations by Indonesians [1]. Traditional medicine is generally prioritized for maintaining human health, although some applications also aim to treat diseases [2]. One of the medicinal plants frequently used is the curry leaf (Murraya koenigii). This plant, a type of spice in the Rutaceae family, is commonly found in Indonesia, especially in the Aceh and West Sumatra regions [3]. M. koenigii is known as “Temurui” in Aceh and is used as a spice and food flavoring.
Curry leaves contain several chemical components that have been scientifically shown to have beneficial properties, including antidiabetic, larvicidal, antianxiety, antioxidant, and antibacterial effects. Abeysinghe et al. [4] reported that ethanol, methanol, and chloroform extracts of curry leaves exhibit antibacterial and antifungal activity. Crude extracts from curry leaves using solvents, like methanol, petroleum ether, acetone, ethyl acetate, chloroform, and water, have antibacterial and antifungal activity against Candida utilis, Shigella sonnei, Salmonella typhi, Bacillus subtilis, and Escherichia coli [5]. Multiple studies have shown that the diverse bioactive chemicals in curry leaves have therapeutic potential.
Aziman et al. [6] identified flavonoid compounds in the ethanol extract of curry leaves. These substances have the potential to be utilized in the food and pharmaceutical industries due to their recognized therapeutic effects. The various secondary metabolite compounds found in curry leaves have the potential to be further developed as sources of medicinal ingredients [4]. Flavonoids, a broad class of plant polyphenolic chemicals, are found in a wide range of food items [7]. Flavonoids are composed of 15 carbon atoms in the basic carbon skeleton that form a C6-C3-C6 configuration with 2 benzene rings (C6) joined to a propane chain (C3) [8]
Flavonoids act as antibacterials by interfering with the function of bacterial microorganisms. Flavonoids slow bacterial growth by altering the permeability of lysosomes, microsomes, and bacterial cell walls [9]. Quercetin, a flavonol compound and flavonoid derivative, has a 3-hydroxyflavone skeleton. The presence of a hydroxyl group (-OH) in the quercetin structure gives rise to various bioactivities, including antioxidation [10]. Specifically, the radical scavenging mechanism occurs by donating hydrogen atoms from the hydroxyl group, giving compounds with hydroxyl groups antioxidant bioactivity [11].
High-performance liquid chromatography (HPLC) is a technological advance that has facilitated quercetin analysis. HPLC is an instrument that is used in qualitative separation analysis techniques [12]. Given the potential pharmacologic activity of quercetin, there is a need to analyze the quercetin level in curry leaves using HPLC. Therefore, this research aimed to provide scientific data regarding the use of curry leaves as an herbal medicinal ingredient.
Materials and methods
Research design
This study was conducted from August to October 2023. The curry leaf extract was prepared in the Pharmacology Laboratory (Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia). Analysis of quercetin using HPLC was performed in the Pharmacology Laboratory (Faculty of Pharmacy, Universitas Airlangga).
Preparation of curry leaf ethanol extract
Five hundred grams of powdered curry leaves were placed in a maceration vessel. Then, 2000 ml of 96% ethanol were added until the simplicia was submerged. The mixture was kept in a closed vessel for 3 d, protected from direct sunlight, and stirred occasionally. Filtration was performed every morning, afternoon, and evening (3 × 24 h) to obtain the liquid ethanol extract. The residue from the maceration process was re-macerated using 1500 ml of 96% ethanol, stirred, and left for 3 d.
The obtained liquid ethanol extract was mixed with the results of the first re-maceration filtration. The residue from the first re-maceration was re-dissolved in 1200 ml of fresh 96% ethanol, stirred, and left for 3 d. A thick ethanol extract was produced by combining the filtered product and evaporation using a rotating vacuum evaporator.
Determination of the quercetin level in curry leaf ethanol extract using HPLC
The maximum wavelength of quercetin was determined using a UV-Vis spectrophotometer (HACH DR6000, Germany) to run a quercetin solution at a concentration of 3 ppm in the 200–700 nm wavelength range, yielding a maximum wavelength of 260 nm. Qualitative analysis was performed by comparing the retention time of the sample solution chromatogram with the reference standard solution of quercetin under identical HPLC conditions. The quercetin standard was weighed at 10 mg and dissolved in 10 ml of methanol (1000 ppm stock solution). Then, 0.25 ml was pipetted and diluted to the desired concentration (50 ppm) in a 5-ml measuring flask using methanol as the solvent.
The 100-ppm comparison standard solution was prepared by pipetting 0.6, 0.8, 1, 1.2, and 1.4 ml into separate 5-ml measuring flasks and diluted with solvent to the mark line, yielding concentrations of 5, 15, 25, 50, and 100 ppm, respectively. Each solution was passed through a 0.45-μm filter membrane and sonicated for 20 min. Next, 60 μl of each solution was injected into the HPLC apparatus at room temperature using a 50:50 methanol mobile phase at a flow rate of 1 ml/min. The chromatogram was recorded and a calibration curve was created between the peak area and concentration.
A total of 0.25 g of curry leaf ethanol extract was carefully weighed and placed in a 100-ml round bottom flask. The sample was then ultrasonicated for 2 h at room temperature, followed by the addition of 5 ml of 6 M HCl and refluxed for 2 h at 90 °C. After cooling to room temperature, the sample was filtered through a 0.45-μm nylon membrane into an HPLC vial. The quercetin content in the sample was then calculated.
Results and discussion
This study aimed to determine the quercetin level in curry leaf ethanol extract using HPLC. An extraction process was carried out to extract quercetin from the curry leaf samples. Curry leaf simplicia was extracted using the maceration method with 96% ethanol as the solvent. The use of 96% ethanol is based on the similar solubility properties with quercetin [13], which is a polar polyphenolic compound that requires a polar solvent for dissolution.
The maceration procedure is particularly advantageous because in addition to being inexpensive and simple to apply heat-sensitive compounds are preserved [14]. This method is modified to account for the chemical and physical characteristics of flavonoids, the class of compounds found in curry leaves [15]. Quercetin, a member of the flavonol group, is a flavonoid that is easily oxidized at high temperatures and lacks heat resistance [16].
A rotary vacuum evaporator was used to evaporate the maceration results until a thick extract was produced. As shown in Table 1 , the thick extract obtained weighed 52.613 g, yielding a soaking result of 9.50%. The percent immersion method calculates the amounts of secondary metabolites carried by the solvent, not the type of chemical carried [17].
Percentage Yield of Curry Leaf Ethanol Extract
| Sample | Simplicia Weight (g) | Extraction Yield (g) | Amount of Ethanol Solvent (ml) | Extract Yield (%) |
|---|---|---|---|---|
| Curry leaves | 500 | 52.613 | 4700 | 9.50% |
The first step in the HPLC measurement of quercetin in curry leaf ethanol extract is to ascertain the maximum wavelength, which is the wavelength at which the substance absorbs light the most [18]. The maximum wavelength measured was 260 nm, which was previously determined using a UV-Vis spectrophotometer operating in the 200–700 nm range.
Quantitative analysis begins with preparing a standard quercetin solution at a concentration of 50 ppm, which is then diluted to create a series of concentrations (5, 15, 25, 50, and 100 ppm). Each standard solution was filtered with a 0.45-μm filter membrane and sonicated to remove air bubbles. Then, 60 μl of each solution was injected into the HPLC apparatus at room temperature with a flow rate of 1 ml/min using a 50:50 methanol mobile phase at a wavelength of 260 nm. The chromatogram was recorded and a calibration curve was created between the peak area and concentration. The results of standard quercetin measurements are shown in Table 2 .
Standard Measurements of Quercetin
| Standard Solution (μl) | Added μl | PPM | Area |
|---|---|---|---|
| 5 | 1000 | 5.0898 | 163.48793 |
| 15 | 1000 | 15.2694 | 873.67419 |
| 25 | 1000 | 25.4490 | 1352.55347 |
| 50 | 1000 | 50.8980 | 3310.36597 |
| 100 | 1000 | 101.7960 | 7602.09912 |
The analysis of curry leaf extract samples revealed that samples 1 and 2 had a quercetin level of 0.080 mg/g 0.079 mg/g, respectively. The average quercetin level in the ethanol extract was 0.03% ( Table 3 ).
Results of Quercetin Measurements from Curry Leaf Ethanol Extract Samples
| Sample | Retention Time | Sample Area | Sample Weight (mg) | Sample ppm | Quercetin Level (mg/g) | Quercetin Content (%) | Average (%) |
|---|---|---|---|---|---|---|---|
| Curry leaf extract | 6.584 | 218.98546 | 258.8 | 8.033 | 0.080 | 0.03 | 0.03 |
| 6.634 | 207.46503 | 250.6 | 7.887 | 0.079 | 0.03 |
The retention time (RT) was obtained in repeated HPLC measurements of the reference compound and quercetin was detected in the chromatogram from the ethanol extract of curry leaves. The chromatogram showed that quercetin in sample 1 had an RT of 6.584 min ( Figure 1 ), while quercetin in sample 2 had an RT of 6.634 min ( Figure 2 ). These results indicated that the curry leave ethanol extract contains flavonoid compounds, especially quercetin.
Flavonoid compounds, such as quercetin, are powerful antioxidants [16]. The ability of antioxidants to scavenge free radicals is associated with an ability to donate protons [19]. The quantity and orientation of the aromatic or hydroxyl groups in the phenolic component affect the number of hydrogen protons that can be donated [20]. The more aromatic hydroxyl groups present, the more effective the aromatic hydroxyl groups are at inhibiting chain reactions in the oxidation process by donating hydrogen atoms or acting as free radical acceptors [21, 22].
As an antioxidant, quercetin can be used in the treatment of cancer and heart disease [23, 24]. Antioxidant activity is found in the leaves, stems, and roots of plants [25, 26]. Curry leaves were selected due to the high chlorophyll content, which is believed to provide potent antioxidant properties [27, 28]. Additionally, quercetin has diverse pharmacologic activities, such as coronary artery dilation, blood fat level reduction, and anti-platelet, anti-cancer, antioxidant, anti-anemia, anti-inflammatory, and anti-anaphylaxis effects [29, 30].
The results of the analysis with respect to the quercetin level in curry leaf ethanol extract are essential for the development of future medicines and public knowledge. The results of this study provided a basis for further research to expand the use of medicinal plants, especially curry plants, in the future.