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The In Vitro and In Vivo Anticancer Properties of Moringa Oleifera

Academic Editor: Jairo Kennup Bastos

Received 11 Jul 2018 Accepted 28 Oct 2018 Published 14 Nov 2018


Abstract

Moringa oleifera, a fast-growing deciduous tree that is widely cultivated in tropical and subtropical regions of the world, is well known for its abundant uses. The tree is a source of food, shelter, and traditional medicine for many people, especially in developing countries. Many studies have been conducted to evaluate the various claims of traditional medicine practitioners that the moringa tree can improve health and treat various diseases. The tree has a high nutritional profile, especially the nutrient rich leaves. Some reports also support the use of parts of the tree to reduce blood sugar and cholesterol levels. These attractive properties have led researchers to look for other novel uses for the moringa tree, especially as a source of anticancer drugs. Researchers have tested extracts from various parts of the moringa tree both in vitro and in vivo on several types of cancers with varying success. This review explores the state of current research on the anticancer properties of M. oleifera.


1. Introduction

Moringa oleifera, better known as the drumstick tree or simply known as moringa, is native to the foothills of the Himalayan ranges in the Indian subcontinent [1]. The tree, called the ‘miracle tree’ by some, is renowned for its abundant uses. It was first brought to the Mediterranean during the Graeco-Roman period and to Southeast Asia by Indian traders. Later the tree spread around the world with Indian migration, eventually reaching the African continent, the Caribbean islands, South America, and the Southern Pacific islands [2]. Indian migrants tried to grow the tree wherever they settled, and it provided them with food, shelter, and medicine. The moringa tree is very hardy and can grow in most tropical and subtropical climates. It requires minimal care, can withstand droughts, and is able to grow in poor soil. In the century, people are still promoting the growth of M. oleifera in regions with suitable climate. For example, the late revolutionary leader of Cuba, Fidel Castro, actively promoted the cultivation of the moringa tree in Cuba after his retirement from politics [3]. He even sent dignitaries to India to obtain seeds for different varieties to be brought back to Cuba for cultivation. Castro wrote that the tree ‘is the only plant that has every kind of amino acid. With proper planting and management, its green-leaf production can exceed 300 tonnes per hectare in a year. It has dozens of medicinal properties.’ He personally grew the trees in his home garden to provide leaves and pods for his own consumption.


The main use of M. oleifera is as a food source. Its pods are favoured by Indians for use in curries. Special breeds known as ‘PKM-1’ and ‘PKM-2’ were developed by Tamil Nadu University in India specifically to produce pods [4]. These specialty breeds can produce pods six months after planting as compared to regular varieties, which take a year to produce pods. The leaves of the moringa tree are commonly eaten as a vegetable in India, Southeast Asia, and Africa. Mature leaves are not fibrous and are suitable for stir frying. The leaves can also be dried, ground into a fine powder, and used as a supplement by adding small amounts to soup, bread dough, and stews. Moringa leaf powder has been promoted as a health food in many countries due to its high nutritional profile. It has been suggested that leaf powder could be added to food aid provided by the UN to enrich food with vital nutrients and that moringa trees should be planted in areas with low food security as a buffer against malnutrition [5].


The seeds of M. oleifera are also edible. Moringa seeds are high in oil content and can be pressed to extract the oil. Moringa seed oil, which is known commercially as ben oil, has the nutritional profile and characteristics of olive oil [6] and can be used in lieu of olive oil. Ben oil also is used commercially in the production of cosmetics and perfumes due to its stability [7]. Ben oil is very rich in antioxidants and is therefore very stable and does not turn rancid for years. Pressed seed cakes from which the oil has been extracted are still rich in nutrients and can be used as animal fodder or as plant fertilizer. Seed cakes can also be used as a flocculating agent for water treatment. They can be added to turbid water containing suspended solids, and they will clump the solids together and clarify the water. Moringa seed cakes are a viable alternative to conventional flocculating agents such as aluminum sulfate [8]. They have a marked advantage over chemical agents because they are natural, biodegradable products with antimicrobial properties [9]. As seed cakes are a by-product of oil extraction, they have minimal production costs and offer added value to producers who can turn a waste product into a usable product.


Moringa trees have big and deep tap roots that enable them to grow in areas with low rainfall. It is very hardy and adaptable to tropical and subtropical climates. It can tolerate low water environments and requires minimal care to grow well, and it is common to see moringa trees planted as shade trees beside municipal roads in India and Southeast Asia. It is found in gardens in Middle Eastern countries such as Saudi Arabia and the United Arab Emirates, as it can grow in arid regions with minimal water [10]. The trees can also be grown as living fences in gardens or farms. M. oleifera is a good candidate for agroforestry projects in tropical and subtropical climates due to its ease of cultivation and multiple uses [11]. They can be used as a wind break and to prevent soil erosion. They are especially suitable for planting as a wind break at desert edges, to prevent desertification, and in attempts to reclaim areas that have already been turned to desert. Thus, the moringa tree provides ecological services.


The various parts of M. oleifera are used extensively in traditional medicine in many regions of the world. In its native land of India, it is used in ayurvedic medicine and is believed to be able to prevent 300 diseases [12]. It is used for varied functions such as cleansing the blood and liver, strengthening the heart, increasing fat metabolism to promote weight loss, and even removing worms [13]. In other regions of prevalent moringa cultivation, such as Southeast Asia and South Asia, different parts of the moringa plant are thought to have antidiabetic, antibacterial, antitumor, antipyretic, antiepileptic, anti-inflammatory, antiulcer, antispasmodic, diuretic, antihypertensive, cholesterol lowering, antioxidant, hepatoprotective, antibacterial, and antifungal activities [14]. Figure 1 shows some of the medicinal uses of the different parts of the moringa plant. Some of these medicinal uses have been verified by researchers. For example, in a study on the ability of the leaf extract to prevent isoproterenol-induced myocardial damage in Wistar albino rats, Nandave et al. (2009) [15] found that the extract significantly reduced lipid peroxidation in myocardial tissue, thus confirming the cardioprotective effect and antioxidant activity of M. oleifera. Pari and Kumar (2002) [16] described the hepatoprotective effects of moringa leaf extract against liver damage in rats induced by the antitubercular drugs isoniazid, rifampicin, and pyrazinamide. Blood test results and histopathological examination of liver sections showed that rats treated with moringa leaf extract recovered better from hepatic damage induced by antitubercular drugs than did untreated rats. These findings support the use of moringa for some treatments used in traditional medicine. However, scientific interest in the antitumor/anticancer activities of M. oleifera has been growing due to the increasing incidence of cancer, and this will be the main focus of our review.


Figure 1 Medicinal uses of Moringa oleifera (see [1527]).


2. Natural Products as Anticancer Agents

Based on statistics published by the World Health Organization, cancer is the second leading cause of death worldwide, causing an estimated 8.8 million deaths [28]. Cancer incidences are expected to rise by approximately 70% in the next 20 years. About 70% of cancer deaths occur in low- and middle-income countries, likely because of factors such as increasing pollution levels, increased life expectancy, insufficient healthcare facilities, and expensive anticancer drugs. One way to overcome these challenges is to develop anticancer drugs from natural sources such as plants, which might lead to more affordable drugs for low- and middle-income countries. Plants and natural resources such as marine organisms and microorganisms are among the major sources of anticancer drugs. More than 60% of all current anticancer drugs are derived from natural sources [29]. As an example, the anticancer drug camptothecin is derived from the extract of the plant Camptotheca acuminata and its chemical derivatives topotecan and irinotecan [30]. Compared to conventional drugs, plant-derived phytochemicals have fewer adverse effects and lower toxicity, which has led to intensified research on medicinal plants [31]. M. oleifera holds great potential as a source of anticancer drugs due to its low toxicity [32] and the presence of a variety of phytochemicals [33]. There has been extensive interest in exploring the anticancer activities of various parts of the M. oleifera tree, and many published research articles describe promising results of in vitro and in vivo testing of various extracts from the moringa plant.


3. Moringa Leaf Extract: A Potential Anticancer Agent

All parts of the M. oleifera tree have been tested for anticancer activity, including the leaves, seeds, bark, and roots. However, the most extensive research on the anticancer activities of M. oleifera has focused on the leaf extracts. The moringa tree is an evergreen that grows new leaves year-round, with a projected production of six tons per hectare per year [34]. The leaves are rich in polyphenols and polyflavonoids, which are antioxidants and potential anticancer compounds [35].


Many researchers start by exploring the antioxidant activity and anti-inflammatory activity of the leaf extracts as a preliminary screening for anticancer activity. One of the factors that causes cancer is oxidative stress which is an imbalance in production of free radicals and oxidants and their elimination by antioxidants [36]. Antioxidants can disrupt the formation of free radicals and reduce oxidative stress, which ultimately prevents cancer. The next step often involves testing the effects of the leaf extracts in vitro on cancer cell lines by examining the extract’s impact on growth and proliferation of cancerous cells and on cell morphology. If the leaf extract shows promising anticancer activity for a specific cancer cell line, the researchers usually proceed by identifying the specific pathways disrupted by the extract through molecular analysis. With sufficient evidence, some researchers will continue exploring the anticancer activity of the extract in vivo, usually in mouse or rat models, to observe the actions of the leaf extract in a living mammal, which is a more accurate representation of the human body. The articles reviewed herein will focus on research showing the antioxidant and anticancer activities of moringa leaf extracts in vivo and in vitro and the pathways/mechanisms of action through which these effects may occur.


3.1. Antioxidant Activities

Verma et al. (2009) [37] explored the antioxidant activity of various fractions of moringa leaf extracts. They started by exhaustively extracting moringa leaf powder over three days with 50% methanol. The crude extract was partitioned to obtain four additional fractions. The five fractions produced were the crude extract, diethyl ether extract, phenolic fraction, polyphenolic fraction, and aqueous fraction. The five fractions were tested for their antioxidant activities using several different antioxidant test models because not all compounds will react in the same way in different antioxidant test models [38]. The fractions were tested with eight different antioxidant tests: β-carotene bleaching and linoleic acid assay, ferric-reducing antioxidant power assay, free radical scavenging activity measured with 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), superoxide anion radical scavenging activity, non-site-specific hydroxyl radical scavenging assay, site-specific hydroxyl radical scavenging assay, ferrous ion chelating capacity, and lipid peroxidation. All eight assays gave consistent results and showed that the polyphenolic fraction had the highest antioxidant activity, followed by the crude extract, diethyl ether fraction, nonphenolic fraction, and finally the aqueous fraction with the weakest antioxidant activity.


Once the polyphenolic fraction was determined to have the highest antioxidant activity, another test to determine the polyphenolic fraction’s ability to prevent DNA nicking was conducted. The supercoiled plasmid DNA was incubated with H2O2 and different concentrations of the polyphenolic fraction and then analyzed with agarose gel electrophoresis. The results showed that 10 µg/ml of the polyphenolic fraction provided comparable protection to DNA as 5U of catalase (an enzyme that breaks down H2O2) and 50 µM of quercetin (a strong antioxidant). The results from the antioxidant test and DNA nicking prevention test suggest that the polyphenolic fraction is a strong antioxidant. Based on these results, Verma et al. (2009) [27] proceeded to conduct in vivo testing of antioxidant activity using Sprague-Dawley rats treated with carbon tetrachloride (CCl4), resulting in increased oxidative stress [39]. They found that the polyphenolic fraction could reverse many of the ill-effects of CCl4, including elevated levels of lipid peroxides (markers for oxidative stress) and low levels of glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD), which are strong antioxidants. The antioxidant activity of 100 mg/kg of the polyphenolic fraction was comparable to the antioxidant activity of 50 mg/kg of vitamin E. The researchers also identified the active compounds which contribute to the antioxidant activity which are mainly phenolic acids and flavonoids. In summary, this study showed that moringa leaf extract is a source of strong antioxidants with antioxidant activity both in vitro and in vivo, which indicates that it has potential anticancer activity.


In another study, Sreelatha and Padma (2011) [40] explored the modulatory effect of moringa leaf extracts against H2O2-induced cytotoxicity and oxidative damage in the HeLa-derived KB cell line. They also compared activity between extracts from tender and mature leaves. Tender and mature leaves were extracted with hot water, and the phenolic composition was measured using high performance thin layer chromatography (HPTLC). Quercetin and kaempferol were the main phenolic compounds present, with levels of quercetin at 795 and 436.2 µg/ml in mature and tender leaves, respectively, and levels of kaempferol at 216 and 115 µg/ml in mature and tender leaves, respectively. Mature leaves contained almost twice the amount of phenolic compounds as tender leaves, suggesting that mature leaves might have higher antioxidant activity than tender leaves. The antioxidant activity of the two leaf extracts was measured using the β-carotene bleaching and linoleic acid assay. As expected, the mature leaf extract had higher antioxidant activity (84.6% of the activity of β -carotene) compared to that of the tender leaf extract (63.6%). These extracts were then tested for their effects against H2O2-induced cytotoxicity and oxidative damage in the KB cell line. Single cell electrophoresis to detect DNA damage in KB cells showed significant reduction in DNA damage for cells treated with both mature and tender leaf extracts when compared to untreated cells. These results indicate that moringa leaf extract can prevent H2O2-induced DNA damage in KB cells.


Sreelatha and Padma (2011) [40] then tested whether the leaf extracts could prevent cytotoxic effects on H2O2 treated cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine cell viability. Mature leaf extracts were slightly better at modulating the effects of H2O2 (i.e., higher cell viability) compared to tender leaf extracts. However, treatment of KB cells with the leaf extracts alone also reduced cell viability of KB cells by 20–40%. This suggests that the leaf extracts have cytotoxic effects on KB cells in the absence of H2O2 but when used in conjunction with H2O2 they reduce H2O2-induced cytotoxicity. The researchers next tested the lipid peroxide levels in H2O2-exposed KB cells and cells treated with leaf extracts. The KB cells were treated and the levels of malondialdehyde (MDA), which is the product of lipid peroxidation and a good indicator of oxidative stress within cells, were measured. Cells treated with H2O2 had increased levels of MDA compared to untreated cells. Cells treated with H2O2 and leaf extract had lower levels of MDA compared to cells treated with H2O2 alone, indicating that the leaf extract reduced lipid peroxidation in H2O2-treated cells. There were no significant differences between the results for mature and tender leaf extracts. However, treatment with leaf extracts alone increased the MDA levels compared to the untreated control as well. In the next experiment, the levels of the antioxidant enzymes SOD and CAT were measured. KB cells treated with H2O2 showed a significant decrease in SOD and CAT levels, as the enzymes were used to remove the H2O2. Cells treated with H2O2 and then leaf extract showed less significant decreases in SOD and CAT levels. Cells treated with leaf extract alone did not show any significant decrease in SOD and CAT levels compared to untreated cells. The active compounds that provide these activities are quercetin and kaempferol. The results of the whole study indicate that moringa leaf extract is a good antioxidant scavenger, as it removes H2O2 and increases viability of KB cells exposed to H2O2. However, moringa leaf extract alone is cytotoxic to KB cells and reduces KB cell viability, but this effect is not caused by compromising SOD or CAT levels.


3.2. In Vitro Anticancer Activities

A number of studies have focused on the anticancer activity of the moringa leaf extract using in vitro screening of cancer cell lines. Parvathy and Umamaheshwari (2007) [41] studied the effects of moringa leaf extracts on the human B-lymphocyte plasmacytoma—U266B1 cell line. U266B1 cells were treated with serial dilutions of the methanol, ethanol, ethyl acetate, and chloroform extracts of the moringa leaf, and cytotoxicity was measured using the neutral red dye uptake assay. The methanol extract had the highest cytotoxic activity against U266B1 cells (IC50: 0.32 µg/ml); this suggests that this extract has high anticancer activity, as a small amount can significantly inhibit the proliferation of U266B1 cells. In another study, Nair and Varalakshmi (2011) [42] tested the anticancer and cytotoxic potential of hot water, methanol, and hexane extracts of the moringa leaf on cervical cancer cells (HeLa cell line) and normal lymphocytes. Results of the MTT assay showed that the aqueous leaf extract caused a dose-dependent decrease in HeLa cell viability (IC50: 70 µg/ml). In contrast, the methanol and hexane leaf extracts caused an increase in HeLa cell viability at higher concentrations. Lymphocytes treated with the different leaf extracts did not exhibit any significant decrease in cell viability. The trypan blue dye exclusion assay was performed for the aqueous leaf extract to verify the results from the MTT assay. The results of this experiment also showed a dose-dependent decrease in cell viability for the aqueous leaf extract. The HeLa cells exhibited increased numbers of detached and dead cells with increasing concentration of the aqueous leaf extract. DNA fragmentation analysis of cells treated with the aqueous leaf extract showed a DNA smear on the agarose gel compared to distinct bands for untreated cells. The DNA smear is indicative of DNA breakage and damage. The last test performed was acridine orange-ethidium bromide staining, which can distinguish viable cells (green fluorescence glow) from nonviable cells (bright orange chromatin). These results showed that the aqueous leaf extract of moringa leaves has significant cytotoxic activity and is capable of fragmenting DNA and killing the HeLa cells.


Charoensin (2014) [43] compared the anticancer activity of the leaf extract among different cancer cell lines (hepatocarcinoma (HepG2), colorectal adenocarcinoma (Caco-2), and breast adenocarcinoma (MCF-7)). First, the methanol and dichloromethane leaf extracts of M. oleifera were tested for their antioxidant activity using the DPPH and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) assays. The results of both assays indicated that the methanol extract had better antioxidant activity. The anticancer activity of the leaf extracts was then tested on the three cell lines. The MTT assay, conducted to assess the effects on cell proliferation, showed that the dichloromethane leaf extract (IC50: 112–133 µg/ml) was more effective than the methanol leaf extract (250 µg/ml) at killing cancer cells. This means that a lower dose of dichloromethane is needed to inhibit the proliferation of cancer cells by 50% compared to the methanol leaf extract. In vitro chemoprevention was also tested using the quinone reductase (QR) induction assay on the hepatoma (Hepa-1c1c7) cell line. The dichloromethane leaf extract induced significant QR activity, whereas the methanol leaf extract exhibited no significant inductive activity. These results indicate that the methanol extract of moringa leaves has better antioxidant activity, but the dichloromethane leaf extract has better anticancer activity on HepG2, Caco-2, and MCF-2 cells as well as better chemoprevention activity.


Khalafalla et al. (2010) [44] tested the anticancer effects of M. oleifera leaf extracts on primary leukemia cells harvested from 15 patients with acute myeloid leukemia (AML) and 10 patients with acute lymphoblastic leukemia (ALL). They also tested the effects of cold water, hot water, and 80% ethanol leaf extracts on the HepG2 cell line. The leaf extracts were first tested for their antioxidant activity using the DPPH assay. The hot water and 80% ethanol extracts had the highest antioxidant activity. They showed 63% and 77% inhibition of radical formation, respectively, compared to 49% inhibition by the cold water extract. The MTT assay was then performed to determine if the extracts could inhibit the proliferation of cancerous cells. The leaf extracts showed promising results, causing 72–82% of AML cells and 77–86% of ALL cells to die after 24 hours of incubation with 20 µg/ml of the extract. After the same treatment, 69–81% of HepG2 cells died. The ethanol extract had the highest anticancer activity in both AML and ALL cells, followed by the hot water extract and then the cold water extract. For HepG2 cells, the hot water extract resulted in the strongest inhibition and the cold water extract had the weakest anticancer activity. Overall, the results showed that moringa leaf extracts had good anticancer activity in vitro against AML, ALL, and HepG2 cells.


3.3. In Vivo Anticancer Activity

Once in vitro studies show promising results, the next step is to determine whether the results can be replicated in vivo. Because humans are complex multicellular organism, in vitro results can only be used as indications of the anticancer activity of the moringa leaf extract. In vivo studies are required to move the research to the next level, as they can demonstrate the anticancer activity of the moringa leaf extract in a complex organism. Jung et al. (2015) [45] tested the effects of a water soluble moringa leaf extract in vitro and in vivo using a mouse model. The study started with tests of effects of moringa leaf extract anticancer activity on human hepatocellular carcinoma (HepG2) cells. The moringa leaf extract was extracted with cold water, and the HepG2 cells treated with leaf extract were tested by flow cytometry to determine the effects of the extract on DNA content and cell cycle stage of the cells. A dose-dependent increase of cells in the sub-G1 phase occurred as leaf extract concentration increased. The MTT assay also showed that cell proliferation was inhibited as leaf extract concentration increased. In the colony forming assay, cells treated with 50 µg/ml of leaf extract (highest tested dose) showed a 70% reduction in colonies compared to the negative control. The Annexin V-fluorescein isothiocyanate (FITC)/PI flow cytometric assay showed that the apoptotic cell ratio was five times higher in the treatment group compared to the untreated control. Western blot analysis detected an increase in cleaved and deactivated PARP, which indicated that cells were dying due to accumulation of DNA damage. The levels of B-cell lymphoma-extra large (Bcl-xL) increased significantly. Because Bcl-xL is an antiapoptotic protein, its downregulation showed that more cells were undergoing apoptosis. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was performed to detect DNA strand breaks in apoptotic cells. The assay causes cells with DNA breaks and undergoing apoptosis to give off a bright green glow. As leaf extract concentration increased, the number of cells glowing and the intensity of the glow increased, indicating that more cells were undergoing apoptosis.


Based on these promising in vitro results, Jung et al. (2015) [45] conducted in vivo testing on immunodeficient nude mice using the hollow fiber assay. HepG2 and A549 cells were grown inside hollow fibers with 1 mm inner diameter. The fibers were then implanted into the subcutaneous cavity of the mice, and the mice were allowed to recover for three days before being fed leaf extract for four days. The positive control mice were given an intravenous injection of doxorubicin (an anticancer drug). After treatment, the fibers were recovered, and the cells were analyzed using the trypan blue exclusion assay, in which dead cells are stained blue. HepG2 cells were more susceptible to the moringa leaf extract than A549 cells. At the maximum tested dose of 200 mg/kg, the survival of HepG2 and A549 cells decreased by 60% and 50%, respectively. The moringa leaf extract decreased the survival of HepG2 cells to levels lower than those achieved by the doxorubicin control. These results illustrate the potential of using the moringa leaf extract as an anticancer drug.


Krishnamurthy et al. (2015) [46] conducted in vitro and in vivo studies to identify and characterize the potent anticancer fraction of the moringa leaf extract. The leaf extract was extracted with successive Soxhlet extractions using n-hexane, chloroform, ethyl acetate, and 50% methanol. The ethyl acetate leaf extract was further separated into 15 fractions with a silica gel column. The ethyl acetate and fraction 1 (F1) were subjected to standardization with HPTLC and qualitative phytochemical analysis. The HPTLC produced the distinct profile of the two extracts, and the phytochemical analysis identified steroid, flavonoid, and phenolic compounds in the ethyl acetate extract and steroids and phenolic compounds in F1. All of the different extracts and fractions were tested using the Hep2 human epidermoid cancer cell line to determine their anticancer activity. The cells were incubated with the extracts and fractions for 72 hours, and morphological changes were observed and noted every 24 hours. After 72 hours of incubation, the cells were fixed and stained with sulforhodamine B, which binds to cellular protein, to determine cell viability. Among all extracts and fractions tested, F1 had the best antiproliferative activity against Hep2 cells (IC50: 12.5 µg/ml). The in vivo test was conducted in Swiss albino mice using F1. The acute oral toxicity of F1 on the mice was tested by giving them a 2000 mg/kg dose and observing them for 14 days before they were culled for gross necropsy analysis. Results indicated that F1 had very low toxicity, as no mice died and no abnormalities were observed in their internal organs. The IC50 of F1 was determined to be > 2000 mg/kg, which is category 5 in terms of acute toxicity which is relatively low in acute toxicity. The in vivo anticancer activity of F1 was tested using Dalton’s lymphoma ascites (DLA) model. Mice were inoculated with the DLA via intraperitoneal injection and given treatment orally for 10 days. Mice given FI treatment had significantly longer survival compared to untreated mice and even slightly longer survival than mice treated with the anticancer drug 5-fluorouracil. This study showed that the F1 of the ethyl acetate moringa leaf extract had very high anticancer activity both in vitro and in vivo.


3.4. Mechanisms of Action

A number of researchers have also tried to identify the pathways and molecular changes involved in moringa leaf extract-induced cancer cell death. In a follow-up to Sreelatha and Padma’s (2011) [40] study showing that the moringa leaf extract had cytotoxic effects on KB cells (HeLa contaminant cells), Sreelatha et al. (2011) [47] decided to do an in-depth study on the cytotoxic effects of moringa leaf extract on KB cells and tested the antiproliferative effects as well as induction of apoptosis by M. oleifera leaf extracts on cancerous KB cells. The MTT cell proliferation assay showed that 100 µg/ml of the hot water leaf extract reduced cell viability by ~38% and increasing the concentration to 200 µg/ml reduced cell viability by 60%. Thus, the leaf extract significantly inhibited KB cell proliferation in a dose-dependent manner. Some of the morphological changes observed in treated cells were cytoplasmic membrane shrinkage, loss of contact with neighboring cells, membrane blebbing, and apoptotic body formation, which are signs of cells undergoing apoptosis. Propidium iodide (PI) is a fluorescent intercalating agent that can bind to DNA and allows visualization of cell nuclei. PI can also be used as an index of the extent of apoptosis in cells because it is too big to pass through the intact cell membrane of living cells but is increasingly able to permeate cells undergoing apoptosis. When treated cells were stained with PI to detect any changes to the nuclei of live and dead cells, those treated with 200 µg/ml of moringa leaf extract showed changes, including nuclear shrinkage, DNA condensation, and fragmentation. Thus, the extract appears to induce apoptosis in KB cells.


Sreelatha et al. (2011) [47] did further tests to quantify the apoptosis in the treated cells by determini