In this study, we investigated some bioactive compounds and pharmaceutical qualities of curry leaf (
Plant-derived substances have recently attracted great interest, owing to their versatile applications. Medicinal plants are the richest source of bioactive compounds used in traditional and modern medicine as nutraceuticals and food supplements, pharmaceutical intermediates, and chemical entities for synthetic drugs [
The present investigation was undertaken to determine the bioactive compounds and antioxidant activity in the leaf extracts of
Fresh leaves of curry leaf were collected locally from three different province of Malaysia, namely, Kelantan (Bachok, North), Selangor (BFK, Central), and Johor (Nasuha, South). The samples were identified by Malaysian Agriculture Research and Development Institute (MARDI). Voucher specimens of
Leaf samples (0.25 g) were extracted with 20 mL of methanol on a shaker for 2 h at room temperature. The extract solution was treated with 5 mL of hydrochloric acid (6 M) and refluxed for 2 h at 90°C. The hydrolysed samples were cooled to room temperature and filtered through a 0.45
The content of total phenolics from the curry leaf was evaluated by the Folin-Ciocalteu method [
The total flavonoid content of the curry leaf extract was determined using a modified colourimetric method [
Reversed-phase HPLC was used to assay flavonoid composition. The Agilent HPLC system used consisted of a Model 1100 pump equipped with a multisolvent delivery system, an L-7400 (Hitachi) ultraviolet (UV) detector, and fitted with an Agilent C18 (5
The stock solutions consisted of 300 mM acetate buffer, 10 mM TPTZ (2,4,6-tripyridyl-S-triazine) solution in 40 mM HCl, and 20 mM FeCl3 solution. Acetate buffer (25 mL) and TPTZ (2.5 mL) were mixed, and 2.5 mL FeCl3 was added. Leaf extract (150
The radical scavenging ability was determined using the method described by Mensor et al. [
Human breast carcinoma cell lines (MDA-MB-231) and normal human mammary epithelial cell (MCF-10A) were cultured in 100
The assay was conducted as follows: cancer cells were seeded in 96-well plates at a density of
All analytical values shown represent the means of three replicates. Data were analysed using analysis of variance by Statistical Analysis System (SAS 9.0). Mean separation test between treatments was performed using Duncan multiple range test and a
The results obtained from a preliminary analysis of flavonoid compounds are shown in Table
The concentrations of TF and some flavonoid compounds detected from curry leaf in three locations.
Kelantan | Selangor | Johor | |
---|---|---|---|
TF |
|
|
|
Rutin |
|
|
|
Quercetin |
|
|
|
Epicatechin |
|
|
ND |
Catechin |
|
|
|
Naringin |
|
ND | ND |
Myricetin |
|
|
|
All analyses are the mean of triplicate measurements ± standard deviation. Results expressed in mg/g DW. Means not sharing a common letter were significantly different at
Contrary to epicatechin, catechin was detected in the curry leaf from all locations and the highest catechin concentration was observed in the curry leaf extract from Kelantan (
HPLC chromatogram of curry leaf extracts, Kelantan location. Identification of compounds: catechin (1), epicatechin (2), rutin (3), naringin (4), myricetin (5), and quercetin (6).
Phenolics that exhibit antioxidant activity are known to be mainly flavonoids and phenolic acids. Phenolic acids are the major class of phenolic compounds widely occurring in the plant kingdom, especially in herbs and vegetables. As shown in Table
The concentrations of TP and some phenolic acids detected from curry leaf in three locations.
Kelantan | Selangor | Johor | |
---|---|---|---|
TP |
|
|
|
Gallic acid |
|
|
|
Cinnamic acid | ND |
|
|
Ferulic acid |
|
ND | ND |
Vanillic acid |
|
|
|
All analyses are the mean of triplicate measurements ± standard deviation. Results are expressed in mg/g DW. Means not sharing a common letter were significantly different at
As Table
The FRAP assay is based on the reduction of ferric tripyridyltriazine (Fe(III)-TPTZ) to ferrous tripyridyltriazine (Fe(II)-TPTZ) at low pH. The FRAP assay has been widely used to estimate the antioxidant content/power of dietary polyphenols [
FRAP activity of curry leaf collected from three different locations compared with positive controls’ butylated hydroxytoluene (BHT) and vitamin C. Bars represent standard error of means. **represents significance at
The DPPH radical scavenging by antioxidants is thought to be due to their hydrogen-donating ability. Among the studied locations, the curry leaf extracts from Kelantan exhibited the highest DPPH scavenging activity (66.41%) followed by those from Selangor (60.23%) and Johor (5.76%). In addition, a significant difference in the DPPH activity was observed among the three locations (Figure
DPPH radical scavenging activity of curry leaf collected from three different locations compared with positive controls’ butylated hydroxytoluene (BHT) and vitamin C. Bars represent standard error of means. **represents significance at
Dose-dependent anticancer of curry leaf extracts from 3 different locations (Kelantan, Selangor, and Johor) towards MDA-MB-231 cell line as determined by the MTT assay. Tamoxifen was used as a positive control. Bars represent standard error of means.
Pulido et al. [
Table
Correlation between TF, TP, and antioxidant activity in curry leaf extract.
1 | 2 | 3 | 4 | ||
---|---|---|---|---|---|
1 | TF | 1 | |||
2 | TP | 0.89** | 1 | ||
3 | FRAP | 0.92** | 0.88** | 1 | |
4 | DPPH | 0.85** | 0.91** | 0.95** | 1 |
A preliminary screening showed that curry leaf extracts from the three locations exhibited a significant anticancer activity against MDA-MB-231 cancer cells, with the inhibition rate of 67.2, 59.8, and 53.6% at concentration of 320
This study produced results which corroborate the findings of a great deal of the previous work in this field. Mahenine, a carbazole alkaloid isolated from curry leaf, has been reported to induce apoptosis in human myeloid HL-60 cancer cells by downregulating cell survival factors and disrupting the cell cycle progression [
Previous studies were focused on different varieties and species of
In a herbal supplement, one ingredient may provide the desired therapeutic benefits while others may have toxic effects for humans. Malaysian herbs and spices may also contain certain toxic components which are not well investigated. In this study, the curry leaf extracts were evaluated as nontoxic for normal (MCF-10A) cells as the IC50 values were greater than 320
Effect of curry leaf extracts from three different locations (Kelantan, Selangor, and Johor) on normal cell viability (normal human mammary epithelial cell, MCF-10A.).
The curry leaf with the highest TF and TP contents also showed the highest antioxidant activity as indicated by the FRAP and DPPH assays. Among the three studied locations, Kelantan plants which had high levels of phenolic acids (especially gallic acid) and flavonoids (especially myricetin, epicatechin, and quercetin) also exhibited a significant anticancer activity. Thus, the quantitative and qualitative analyses of major individual flavonoids and phenolics could provide an explanation for the correlation between the TP content and antioxidant capacity in curry leaf extracts. A wide concentration range of flavonoids and phenolic acids and variations in the antioxidant activity in curry leaf extracts could be due to several factors including growing location, altitude, climate, temperature, and diversity of natural vegetation in the area. Furthermore, the composition of phenolic acids and flavonoids and the related antioxidant activity can be useful for standardization of curry leaf extracts for further pharmaceutical applications. One of the more significant findings to emerge from this study is that the curry leaf extracts exhibited a promising anticancer activity on MDA-MB-231 human breast cancer cell line. The extracts contained substantial amounts of effective flavonoid compounds such as myricetin, epicatechin, and quercetin which showed potency in the growth inhibition of breast cancer cells. Subsequently, our MTT assay indicated that curry plants grown in the North of Malaysia (Kelantan) are a potential source of anticarcinogenic therapeutic compounds. More information on other bioactive components in curry leaf would help to further evaluate the anticancer activity of this medicinal plant.
The authors declare that they have no conflict of interests.
The authors would like to thank the Ministry of Higher Education Malaysia and the Research Management Centre, University Putra Malaysia (UPM) for sponsoring this work. They are also grateful to the Malaysian Agriculture Research and Development Institute for collecting of plant samples. The authors would like to acknowledge all staff of Laboratory of Nutrition, Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences for all the help and guidance in order to accomplish this project.