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PG.1 Moringa oleifera Leaves with a New Anticancer Activity



Soluble Extract from Moringa oleifera Leaves with a New Anticancer Activity


  • Il Lae Jung mail
  • Published: April 18, 2014
  • DOI: 10.1371/journal.pone.0095492


Moringa oleifera has been regarded as a food substance since ancient times and has also been used as a treatment for many diseases. Recently, various therapeutic effects of M. oleifera such as antimicrobial, anticancer, anti-inflammatory, antidiabetic, and antioxidant effects have been investigated; however, most of these studies described only simple biological phenomena and their chemical compositions. Due to the increasing attention on natural products, such as those from plants, and the advantages of oral administration of anticancer drugs, soluble extracts from M. oleifera leaves (MOL) have been prepared and their potential as new anticancer drug candidates has been assessed in this study. Here, the soluble cold Distilled Water extract (4°C; concentration, 300 µg/mL) from MOL greatly induced apoptosis, inhibited tumor cell growth, and lowered the level of internal reactive oxygen species (ROS) in human lung cancer cells as well as other several types of cancer cells, suggesting that the treatment of cancer cells with MOL significantly reduced cancer cell proliferation and invasion. Moreover, over 90% of the genes tested were unexpectedly downregulated more than 2-fold, while just below 1% of the genes were upregulated more than 2-fold in MOL extract-treated cells, when compared with nontreated cells. Since severe dose-dependent rRNA degradation was observed, the abnormal downregulation of numerous genes was considered to be attributable to abnormal RNA formation caused by treatment with MOL extracts. Additionally, the MOL extract showed greater cytotoxicity for tumor cells than for normal cells, strongly suggesting that it could potentially be an ideal anticancer therapeutic candidate specific to cancer cells. These results suggest the potential therapeutic implications of the soluble extract from MOL in the treatment of various types of cancers.



Various types of plants have been used for several centuries worldwide not only as dietary supplements but also as traditional treatments for many diseases [1][2][3]. Indeed, the fact that traditional medicines have been widely used worldwide demonstrates the potential of plants as sources of bioactive compounds, including potential antitumor, antioxidant, antiobesity, and antimicrobial molecules. Among these plants, the widely cultivated Moringa oleifera (Moringa or drumstick tree), a rapidly growing perennial tree, was used by the ancient Romans, Greeks, and Egyptians, and has been naturalized from the tropics to the sub-Himalayan regions (e.g., India, Pakistan, Bangladesh, and Afghanistan), Oceania, Latin America, Africa and tropical Asia [4][5][6][7].

For centuries, M. oleifera has been used as a traditional medicinal source. Additionally, besides being edible, all the parts of the Moringa tree (e.g., pods, seeds, and leaves) have long been employed for the treatment of many diseases, and therefore, it was called a “miracle vegetable” [6][8][9]. Since it is a significant source of fats, proteins, beta-carotene, vitamin C, iron, potassium, and other nutrients [7][10], the Moringa tree is highly nutritious. For these reasons, some parts of this plant have drawn much attention and have been studied for its various biological activities, including antiatherosclerotic [11], immune-boosting [12], anticardiovascular diseases [13], antiviral [1][14][15][16], antioxidant [2][17][18][19], antimicrobial [18], anti-inflammatory [20] properties and tumor-suppressive effects in skin papillomagenesis, hepatocarcinoma cancer, colon cancer, and myeloma [1][21][22][23].

However, only a few studies have reported the anticancer activity of M. oleifera leaves (MOL), and most of them have focused on the evaluation of their efficacy with respect to tumor-suppressive activity, but not on the molecular basis of the tumor-suppressive activity. Additionally, most studies have been conducted using solvent extracts of MOL and not their soluble extracts [1][17][18][21][24].

Solvent extraction is the most frequently used technique for the isolation of bioactive compounds from plants. Therefore, the recovery of bioactive compounds from M. oleifera has been typically accomplished using various solvents, such as methanol and ethanol, as well as hot water and buffers [1][17][18][21][24]. Nevertheless, the majority of the studies focused on solvent extracts because the efficacy of solvent extraction is higher than simple water extraction. In fact, the buffer extract of M. oleifera leaves was much less effective than the solvent extracts for hepatocarcinoma cells [1]. Moreover, solvents can dissolve the many useful organic molecules found in plants, such as phenolic compounds.

In the present study, I prepared a cold water-soluble MOL extract and investigated the possibility as anticancer drugs in different types of human cancer cell lines. Finally, the medical value of a water-soluble MOL extract will be discussed.

Materials and Methods

Sample Preparation

Dried leaves of M. oleifera cultivated in Chinagmai, Thailand, were purchased from GL Networks Co. Ltd. The dried MOL (150 mg) were suspended in 1 mL of cold water (4°C), vigorously vortexed for 30 s, and refrigerated for 5 min to 24 hours. The suspension was vigorously vortexed again for 1 min at room temperature. The water-insoluble parts of the suspension were removed by centrifuging it twice (12,000 rpm, 10 min each), and the supernatants were collected by membrane filtration (0.2-µm filter). The resulting MOL extracts were lyophilized and stored at −20°C for future analysis. For the experiments, the lyophilized MOL extracts were resuspended into DW at a final concentration of 20 mg/mL of protein.

Cell Culture

All the cancer cells and African green monkey kidney cell line COS-7 used in this study were obtained from American Type Culture Collection (ATCC, USA) and Korean Cell Type Collection (KCTC, KOREA), respectively. The cells were grown in RPMI-1640 medium (i.e., A549, H23, and H358) and DMEM (i.e., MCF-7, A431, HT1080, and COS-7) (Hyclone Lab, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone Lab) and 1% penicillin-streptomycin. Cells were inoculated at a density of 1×105 cells in a 6-well plate and were maintained at 37°C in a humidified atmosphere containing 95% air and 5% CO2.

Cell Proliferation Assay (MTT Assay)

The viability of cells was analyzed by a cell proliferation assay method using tetrazolium salt (MTT) [25]. Cells were adjusted to 3×103 cells/well and inoculated in 100 µL of appropriate culture medium/well in 96-well plates. After 1-d incubation, the cells were treated with various concentrations of MOL extract (0–400 µg/mL). After another 1- or 2-d incubation, 10 µL of Cell Counting Kit-8 (cat. No. CK04, Dojindo Laboratories, Japan) or WST assay reagent (Daeil Lab Service Co, Korea) was added per well and incubated for an additional 4 h. The absorbance at 450 nm was measured with a microplate reader (Model 680 microplate reader, Bio-Rad Laboratories, USA).

Flow Cytometric Detection

Cells (1×105) were seeded in a 6-well culture plate for 1 d and treated with the MOL extract. After 2 d, the cells were collected, washed with PBS, and fixed with 70% ethanol at 4°C for 2 h in the dark. Fixed cells were washed twice with PBS and stained with propidium iodide (PI, 50 µg/mL) for 30 min at room temperature. The DNA content was measured with a FACScan system (EPICS XL Flow Cytometry, Beckman Coulter Counter, USA). The percentage of cells in each cell phase was determined using the Phoenix Multicycler Software (Phoenix Flow System).

Colony-formation Assay

Trypsinized cells were collected and seeded in new 6-well culture dishes at a density of 1×103cells/well for 1 d before adding the MOL extract. After 7 d, the cells were stained with 0.1% crystal violet and photographed. The experiments were repeated 3 times, and a representative photograph has been provided.

Measurement of Reactive Oxygen Species (ROS)

Carboxydichlorofluorescein diacetate (DCFH-DA) is a polar compound that is converted into a membrane-impermeable non-fluorescent polar derivative (DCFH) by cellular esterases after its incorporation into the cells. The trapped DCFH is then rapidly oxidized to fluorescent 2′,7′-diclorofluorescein (DCF) by intracellular hydrogen peroxide. Trypsinized cells (approximately 1×105 cells) were washed, resuspended in PBS, and treated with DCFH-DA at a final concentration of 10 µM. The cells were incubated for 30 min in the dark at 37°C, and the ROS level was measured using a FACScan system (EPICS XL Flow Cytometry, Beckman Coulter Counter).


To monitor cell morphology, cells were visualized by light microscopy (Leica Microsystems, Wetzlar, Germany). Images were captured with a Power Shot S45 Canon Digital Camera system.

cDNA Synthesis and PCR Amplification

Cells (1 × 105) were seeded in a 6-well culture plate for 1 d before treatment with the MOL extract. After an additional 2-d incubation, total RNA was isolated from the cells by using the High Pure RNA isolation kit (Roche, Basel, Switzerland). To generate first-strand cDNA from the total RNA (1 µg) by using oligo dT, a cDNA synthesis kit (Maxim RT Premix Kit-Oligo dT Primer, iNtRON Biotechnology, Korea) was employed. The resulting cDNAs were amplified with different primers (Table 1) by using Maxim PCR Premix Kit-iTaq (iNtRON Biotechnology, Korea). The amplified polymerase chain reaction (PCR) products were analyzed by 1.5% agarose gel electrophoresis and then photographed under UV ligh

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