Biological properties of Moringa oleifera: A systematic review of the last decade

1.1 Moringa oleifera

Moringa oleifera is a tree belonging to the Moringaceae family, native to the sub-Himalayan area of India, Pakistan, Bangladesh and Afghanistan whose genus has 13 species distributed from tropical to subtropical regions.⁵ MO is a plant widely studied worldwide for its components since these confer it particularities of interest in areas such as health, aesthetics, and nutrition, among others.

The most studied part is its leaves since these have been attributed different biological properties including antimicrobial, hypotensive, antiulcer, hypocholesterolemic, antispasmodic, antioxidant, anti-inflammatory and antitumor agent potential.⁶ In relation to its chemopreventive role, Sreelatha et al.⁷ evaluated the antitumor effect of Moringa oleifera leaf alcoholic extract (rich in phenolic compounds, such as quercetin and kaempferol) on glandular cervical cancer cells, revealing a potential in the inhibition of cell viability through the induction of apoptosis and oxidative stress through the generation of reactive oxygen species (ROS) and an antiproliferative effect, thus revealing a promising outlook as an antitumor alternative.⁷

MO is one of the most cultivated and important medicinal plants in India and is considered as a staple food in various parts of the world since its valuable nutritional potential⁴ due to the high content of minerals and vitamins such as iron, potassium, copper, zinc, magnesium, manganese, vitamin C, vitamin E, beta-carotene, among others. Different studies have been described associated with the leaf, seed, fruit, bark and flower of Moringa oleifera in several types of cancer, which have revealed the biological antitumor activity both in vitro and in vivo. One of these works was performed by Hussein et al.⁸ in which they determined the components of Moringa oleifera seed essential oil in vitro that act as antioxidants, and showed the cytotoxic potential over tumor cell lines like human breast adenocarcinoma (MCF-7), colon carcinoma (HCT-116), human hepatocarcinoma (HepG2) and HELA cells (8).

1.2 Phytochemical features

This plant has been considered of great curative relevance in relation to the presence of significant phytochemicals⁹ also known as secondary metabolites such as polyphenols, tannins, sterols, terpenoids, flavonoids, saponins, anthraquinones, alkaloids and some reducing sugars mentioned along with anticancer elements, including glucosinolates, isothiocyanates, glycoside compounds and glycerol-1-9-octadecanoate.¹⁰ Phenolic acid derivatives are elements with similarity to flavonoids, which have been related to plant protection against UV rays and also with other defensive mechanisms. One type of phenolic acid, chlorogenic acids, are known for protecting against diseases caused by oxidative stress.¹¹ Some of the main flavonoids found by Sultana and Anwar (2008) present in Moringa oleifera leaves are myricycin, quercetin and kaempferol, at concentrations of 5.8, 0.207 and 7.57 mg/g, respectively.¹² Moreover, Vergara-Jiménez et al.¹³ point out that quercetin is found in MO dried leaves in concentrations of 100 mg/100 g. This phytochemical acts as a strong antioxidant and exhibits hypolipidemic, hypotensive and antidiabetic potential, which has been demonstrated in vivo with rats suffering from metabolic syndrome and rabbits with hyperlipidemia ( Figure 1).¹³

Figure 1. Chemical structures of the most significant secondary metabolites of Moringa oleifera.

Concerning the biological activity of MO against microorganisms, Padla et al.¹⁴ highlight that isothiocyanates isolated from Moringa oleifera seeds showed inhibitory activity at the lowest concentration of 1 mg/ml towards all Gram-positive bacteria tested (Staphylococcus aureus, Staphylococcus epidermidis and Bacillus subtilis) and against the dermatophyte fungi Epidermophyton floccosum and Trichophyton rubrum.¹⁴

1.3 Traditional uses

Several elements of this plant have been used traditionally in India as a preventive and treatment approach for pathologies such as asthma, gout, rheumatism, and infections.¹⁵ Same scenario occurs in Africa where this MO is used to combat arthritis, joint pain, headaches, abdominal pain, otitis, and dental pain and it has also been used as a cardiac and circulatory stimulant, in individuals with asthenia, intestinal parasites, febrile periods, renal and hepatic complications, epilepsy, anemia, ulcers, delirium, and trauma caused by snakes.

Moringa oleifera has been used to treat respiratory diseases such as influenza and asthma, and even SARS-CoV-2, which caused the last pandemic,¹⁶ gastritis, headaches and flatulence¹⁷; thus demonstrating its wide range of uses for a variety of clinical entities. It should be remembered that millions of people in the world do not have access to modern medicine, having to resort to the alternatives provided by ancestral medicine.¹⁸

1.4 Antioxidant effect

The relevance of the antioxidant activities of components that make up the Moringa oleifera plant, such as polyphenols, alkaloids, saponins, carotene, minerals, amino acids, and sterols, has been demonstrated. Such antioxidant features has been identified through several approaches, including colorimetric methods such as DPPHH (2,2-diphenyl-1-picrylhydrazyl), ABTS [2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)], LPO (lipid peroxidation), FRAP (ferric reducing antioxidant power), among others, in order to obtain evidence related to the redox potential of the plant.¹⁹

Polyphenols are phytochemicals found in large quantities especially in Moringa oleifera and are considered natural antioxidants. These are plant compounds that are attributed with the property of indirectly reducing oxidative damage in tissues by strengthening cells or eliminating free radicals.²⁰ It has been demonstrated that consumption of polyphenols protects against chronic pathologies related to oxidative stress, including cardiovascular diseases and cancer.¹³ The presence of polyphenols in high concentrations in this plant leaves opens the door for their use in the aforementioned diseases. It is worth noting that to achieve the greater potential of antioxidants against damage caused by free radicals, the synergistic work of several antioxidants is necessary. A combination of effective antioxidants n the leaves of Moringa oleifera has been found²¹ among then, polyphenols, mostly flavonoids (myricetin, quercetin, and kaempferol), together with phenolic acids (chlorogenic acid, caffeic acid, and gallic acid).²²

Moringa oleifera extracts exhibit strong antioxidant activity and radical scavenging in vivo, protecting animals against oxidative stress-induced diseases. Consumption of leaf and seed extracts has shown significant therapeutic properties against diabetes and hypertension due to the phyto-components, exhibiting mitochondrial redox potential, increasing heme oxygenase-1 and reducing reactive oxygen species (ROS) induction as well as lipid peroxidation, thus conferring protective effects against negative effects associated with obesity.^23,24 It is even suggested that its antioxidant capacity is so promising that the role of Moringa oleífera derivatives should be explored in the treatment of wastewater and as a future source of important nutraceuticals.²¹

1.5 Anticancer activity

The potential of Moringa oleifera as an anticancer agent has been elucidated through the role of ethanolic extracts obtained from the leaves and bark which have effectively inhibited the growth of cancer cells in breast, pancreas, and colorectal tissue. It is believed that the plant’s antitumor capacity is due to the presence of isothiocyanates, glucosinolates, glycosylated compounds, and glycerol 1-9-octadecanoate.²⁵ It has also been proved that Moringa oleifera pods act as a chemo preventive agents in vivo. This protective effect has also been identified by Shaban et al., ²⁶ who observed an in-crease in antioxidant levels and a reduction in the formation of free radicals against several cancer cell lines (lung, liver, colon, and neuroblastoma) treated with the plant.²⁶

Regarding the extract of Moringa oleifera seed, this has shown specific growth inhibitory effects reflected in a 95% inhibitory activity on neuroblastoma cell line.²⁷ In some cases, tumors treated with methanol extracts from fruits and leaves have grown slowly, indicating effective cell degradation. Likewise, it has been found that the extract is more effective in terms of volume, since at a concentration of 500 mg/kg, the doubling time and growth delay indicate tumor-suppressing properties.²⁷

2.1 Inclusion and exclusion criteria

Inclusion criteria included studies that:

2.2 Review process

A search was performed using keywords such as “Moringa oleifera,” “antitumor,” “chronic diseases,” and “infectious diseases.” Following the initial search, titles and abstracts were screened to filter relevant articles. A full reading of the selected articles was then conducted to extract data on biological activity and proposed mechanisms.

2.3 Data analysis

The findings from the selected articles were synthesized, highlighting the most relevant biological properties and methodologies employed in each study. A descriptive approach was used to summarize results and discuss the implications of the findings for human health.

3.1 Chronic, infectious diseases and cancer

Chronic diseases are characterized by a long course and slow progression, and include cardiovascular diseases, cancer, chronic kidney and liver diseases, rheumatic disease, and diabetes. According to the World Health Organization (WHO), chronic diseases are the leading cause of mortality worldwide, causing approximately 41 million deaths each year, equivalent to 71% of deaths globally.²⁸ These pathologies can be prevented by modifying common behavioral risk factors, such as smoking, alcohol consumption, unhealthy diet, and physical inactivity, which contribute to the development of metabolic changes that usually manifest themselves as hypertension, obesity, hyperglycemia, and hyperlipidemia.²⁹

Specific genetic events and external agents such as physical carcinogens such as ultraviolet (UV) and ionizing radiation, chemical carcinogens such as asbestos, aflatoxins, arsenic, and biological carcinogens (e.g. Helicobacter pylori, viruses, and parasites) increase disease morbidity.³⁰ However, cardiovascular diseases are positioned before cancer, as they are the leading cause of death among chronic diseases (approximately 18 million deaths per year), followed by cancer (9 million deaths per year) and diabetes (1.6 million deaths per year).²⁸ These three pathologies are responsible for more than 80% of premature deaths, that is, fatal events occurring between the ages of 30-69 years.²⁹

Concerning infectious diseases, which are basically foodborne illnesses (FBIs), sexually transmitted infections (STIs) such as HIV/AIDS, acute respiratory infections (ARIs) including the common flu and the current novel coronavirus (COVID-19), and other parasitic and bacterial diseases; a global increase in drug resistance rates has been identified, indicating a loss of effectiveness in usual treatments. Regarding antiviral resistance, there is an increasing concern, mainly for immunocompromised patients, as well as for the emergence of drug-resistant parasites, which constitute one of the greatest threats to malaria control. In the case of drug-resistant fungal infections, the therapeutic situation is already difficult se because patients currently experience treatment problems, such as toxicity, especially in individuals with other underlying infections.³¹ Some infections can be prevented by vaccination; however, the increasing resistance to antimicrobials (antibiotic, antiviral, antifungal, and antiparasitic) due to their inappropriate and excessive use has led to some of these diseases becoming a global public health problem owing to an increase in morbidity and mortality rates, long hospital stays, and high healthcare costs.³¹

Therefore, considering what has already been described, a viable therapeutic alternative is found in natural and complementary medicine based on the use of plant material, its extracts, and derivatives. In recent decades, medicinal plants have become a trend in developing countries, with Moringa oleifera as a promising plant because several studies have demonstrated its therapeutic and pharmacological properties, including neuroprotective, antimicrobial, antiasthmatic, antimalarial, cardioprotective, antidiabetic, antiobesity, hepatoprotective, and anticancer effects.¹

Chronic diseases have had a significant impact worldwide due to their high mortality rates and the fact that their related therapies often have side effects on patients. Based on this, there is a need to explore other therapeutic alternatives, such as natural treatments, including the use of Moringa oleifera, which has shown positive effects regarding the potential treatment of such diseases over time, as revealed in this study. In the following sections, several in vitro and in vivo studies conducted between 2010 and 2021 on the use of Moringa oleifera in the aforementioned clinical events are described.

3.2 Biological activity of Moringa oleifera in chronic diseases

Cardiovascular diseases are chronic diseases and are of great interest because they rank first as the cause of death worldwide. However, in chronic diseases, another clinical entity attracts the attention of the medical community because of the rapid increase in incidence rates and high associated morbidity. According to data from the Diabetes Atlas, 463 million adults currently live with this disease, and the global projection of cases for 2025 is 438 million, but this prediction has already been exceeded by 25 million cases.³² Making the scenario more complex, this pathology is not diagnosed or is misdiagnosed in low-income countries, increasing the risk of serious complications and mortality. Additionally, the lack of availability of diabetes medications is a challenge. The projections for this disease are not only gloomy regarding the well-being of individuals but also in terms of fiscal detriment to global health systems, as expenses related to the disease are estimated to reach over one billion dollars by 2045.³³

3.2.1 Therapeutic properties for chronic diseases

In India, the native country of the plant, Aju et al.³⁴ evaluated the protective role of the methanolic extract of Moringa oleifera leaves (MOME) in the hearts of diabetic rats and demonstrated that treatment with MOME at a dose of 300 mg/kg/day significantly reduced hyperglycemia and oxidative stress in the hearts of the animals.³⁴ The potential antidiabetic and antioxidant properties of MOME were attributed to the presence of various biologically active compounds, such as heptadecanoic acid, hexadecanoic acid, DL-alpha-tocopherol, 11-14-17-eicosatrienoic acid, and 9-12-15-octadecatrienal, which have been previously associated with the antiarthritic and anticoronary activities of the plant.³⁴

In line with the antidiabetic study, Khan et al.³⁵ used the aqueous extract of Moringa oleifera leaf (AEMOL) at a dose of 100 mg/kg in streptozotocin-induced diabetic rats to determine the hypoglycemic potential to assess the enzymatic activity, seeking to identify the antioxidant capacity due to the presence of compounds such as phenols.³⁵ However, the evaluation of inhibitory activities and potential radical scavengers of two promising plants (M. oleifera and T. occidentalis) on the enzymes highlighted in diabetes mellitus revealed enzyme inhibition,³⁶ with Moringa oleifera being the major contributor to this event in a dose-dependent manner. Another relevant finding of this study was related to the identification of a greater number of phytoconstituents in Moringa oleifera than in Telfairia occidentalis.³⁶

Concerning the antidiabetic role of Moringa oleifera leaf powder, the chemical composition of this substance has been evaluated with regard to the inhibition of α-amylase enzyme, testing the inclusion of 20 g of Moringa oleifera leaf powder in natural foods and studying in diabetic and healthy patients. From these analyses, it was found that Moringa oleifera leaf powder contains a high amount of protein, fiber, and trace elements, in addition to known polyphenols.³⁷

Approaches focused on demonstrating an association between the potential of leaf extracts and therapy for vascular diseases have evaluated the antihypertensive mechanisms of the aqueous extract of Moringa oleifera leaves (AEMOL). Aekthammarat et al.³⁸ concluded that AEMOL induces the synthesis of Nitric Oxide (NO) in endothelial cells, thus reducing peripheral vascular tone and blood pressure (BP), a dependent event on soluble guanylate cyclase (sGC). This induces vasorelaxation through the activation of the eNOS-NO-sGC pathway, allowing us to understand the pharmacological properties of AEMOL against arterial hypertension.³⁸

The seeds of Moringa oleifera seeds have also shown potential for exhibiting antidiabetic effects. Al-Malki and Rabey³⁹ tested low doses of Moringa oleifera seed powder (50 and 100 mg/kg) in rats with induced diabetes. Using various methodological approaches, such as biochemical, immunological, physiological, and histological analyses they observed that low doses decreased lipid peroxidation to a greater extent than the 100 mg/kg concentrations. They also found that antioxidant enzyme levels increased in the treated groups. Specifically, regarding glycosylated hemoglobin, rats treated with M. oleifera showed significantly reduced values, although the most drastic change was associated with the dose of 100 mg/kg.³⁹

The methanolic extract of Moringa oleifera seeds (MEMOS) has been evaluated for its ability to alter serum lipid profiles. An example of this is the assessment carried out by Ajayi et al., ⁴⁰ where weight gain was observed in untreated animals compared to those treated with this extract. It was also noted that there was a decrease in total cholesterol (<0.05) in experimental animals administered MEMOS at 100 mg/kg, but this effect was not significant at 200 mg/kg. Regarding high-density lipoprotein (HDL), it was found that in groups treated with both concentrations of MEMOS, this parameter increased, but this increase was more significant at 200 mg/kg.⁴⁰

Table 1 shows some in vivo and in vitro concerning the potential of extracts obtained from different parts of the plant to counteract some chronic diseases.

3.3 Biological activity of Moringa oleifera on cancer

The International Agency for Research on Cancer (IARC) states that in 2020 there were 19.3 million new cases of cancer and nearly 10 million deaths.⁵⁹ According to the Global Cancer Observatory (GCO), lung cancer is the most commonly diagnosed cancer in men (14.3%), followed by prostate cancer (14.1%) and colorectal cancer (10.6%). In women, the most common type of neoplasia and the one with the highest mortality rate was breast cancer (24.5%), followed by colorectal cancer (10.6%) and lung cancer (8.4%).⁵⁹ It is predicted that by 2030, there will be a 32% increase in the number of people diagnosed with cancer in the Americas.⁶⁰

Currently, the main therapies used against cancer in medical oncology are surgery, hormonal therapy, radiation, immunotherapy, and chemotherapy. Despite their widespread use, these approaches have adverse effects because they affect cells that are in constant division, such as the bone marrow, lining of the mouth and intestines, and hair follicles. The type of side effect depends on the type of medication used, amount administered, and treatment time. The use of conventional approaches such as chemotherapy has resulted in very low survival rates for patients with advanced-stage cancer.⁶¹

3.3.1 In vitro antitumor evidence

With the aim of elucidating the potential antitumor capacity of various extracts from Moringa oleifera, Cuellar et al.⁶² conducted a study related to the effect of hydrolyzed extracts from the plant leaves on human colon cancer cells, HT-29 and HCT116, showing similar cytotoxic activity in both cell lines compared to untreated cells.⁶² According to the types of extracts evaluated in the study, it was observed that the aqueous extract, the methanolic extract, and the glucosinolate-rich hydrolysate showed the highest cytotoxic effect on the HCT116 human colon cancer cell line at concentrations of 20.1%, 21%, and 21.5%, respectively, unlike the HT-29 human colon cancer cell line, in which only the aqueous and methanolic extracts showed higher cytotoxicity at concentrations of 19.8% and 22%, respectively.⁶²

To determine the possible mechanism responsible for the observed tumor cytotoxicity associated with this plant, the apoptotic and antiproliferative activity of the aqueous extract of Moringa oleifera leaf in human melanoma cell lines A375, A2058, and normal human fibroblasts was assessed. This biological activity was evaluated through the implementation of the WST-1 cell proliferation assay and the Annexin V-associated apoptosis assay, showing that the aqueous extract causes growth inhibition due to apoptosis events in the target lines.⁶³ However, there was little effect on the growth of normal human fibroblasts, thus revealing the specificity of this compound.⁶³

Therefore, the aqueous extract of Moringa oleifera can be considered an alternative therapy for the treatment of skin cancer, while keeping in mind the importance of continuing in vitro and in vivo studies that provide scientific information about the biological activity of this plant. Several approaches associated with the study of the antitumor and chemopreventive capacities of Moringa oleifera are summarized in Table 2.

Attempting to explore the potential of the Moringa plant not only regarding its leaves but also other elements, Adebayo et al.⁶⁴ evaluated the cytotoxic activity and inhibition of cell proliferation of crude extracts and fractions of MO seeds on breast adenocarcinoma cells (MCF7) and normal breast cells (MCF10A) and found an insignificant effect of the crude ethanol extract on the proliferation of MCF7 cells.⁶⁴ In contrast, the dichloromethane fraction of the crude ethanol extract, the hexane fraction of Moringa oleifera seed, and the crude aqueous extract inhibited proliferation.⁶⁴ The observed cell inhibition effects in this study were most likely due to the presence of phenols. The hexane fraction of the ethanol extract exhibited the best effect, as it showed antitumor activity against MCF7 cells, while having limited cytotoxicity against normal breast cells.⁶⁴

To determine the properties of Moringa oleifera in other tumor cell lines, a study targeting breast cancer cells (MDA-MB-231) and colorectal cancer cells (HCT-8) was conducted with the aim of identifying phenotypic changes, effects on cell viability, apoptosis, and alteration of the cell cycle. The results did not show a significant decrease in cell populations when exposed to different parts of the plant; however, the seed extract decreased cell motility and colony formation in both cell lines, despite not directly affecting their survival.⁶⁵

Regarding to the whole plant studies Diab et al.⁹¹ conducted a research aimed to explore the in vitro antiproliferative potential of extracts from the entire Moringa oleifera plant on cell lines of lung cancer (A549), prostate (PC-3), breast (T47D and MCF-7), colon (HCT-16, Colo-205), and leukemia (THP-1, HL-60) using two methods: cytotoxicity assay and MTT assay. The results showed in vitro cytotoxic activity of the plant extract actively inhibiting the growth of cancer cells in the cell lines A549, PC3, MCF-7, and HCT⁹²; however, it showed a moderate effect on T47D, Colo-205, THP-1, HL-60, and K562 cells.⁹¹

3.3.2 In vivo antitumor evidence

In an effort to identify the potential therapeutic role of Moringa oleifera in preclinical trials, Barhoi et al published in 2021 the results of a study evaluating the cytotoxic activity of this plant in BALB/c mice with mammary adenocarcinoma. The mice were administered aqueous leaf extract for a week, and after follow-up, a reduction in tumor weight and volume was observed with increased survival of the mice, without significant alterations in the liver, kidney, or hematological parameters after the treatment. In parallel, in vitro evaluations were carried out, showing the dose-and time-dependent cytotoxic activity of the plant in two cell lines: HEp-2 human laryngeal carcinoma cells and EAC (Ehrlich Ascites Carcinoma) mouse mammary adenocarcinoma cells. Regarding the mechanisms involved in the observed cytotoxic effects, flow cytometry analysis confirmed the significant induction of tumor cell apoptosis by changing the mitochondrial membrane potential in the EAC cell line.⁶⁶

Another in vivo model considered the use of Sprague Dawley rats in which breast cancer was induced through treatment with 7,12-dimethylbenz(a) anthracene (DMBA), in order to analyze whether the combination of ethanolic extract of Moringa leaves and ethanolic extract of papaya leaves (Carica papaya L)⁹³ could delay the appearance of tumor tissue. The results showed that there were different changes in body weight among the groups of rats for 12 weeks, and the appearance of tumor tissue was detected in the 7th week in the DMBA comparison group, while in the positive control group and the combined extract group of Moringa oleifera and Carica papaya leaves, the solid period began to be detected in the tenth week.⁹³ According to histopathological tests, the growth of tumor tissue was found in all test animals, concluding that the combination of ethanolic extract of Moringa and Carica leaves at a dose of 150 mg/kg delayed the formation of cancerous tissue⁹³; therefore, they can be considered as chemopreventive agents.

Similarly, Sadek et al. evaluated in vivo the chemoprophylactic efficacy of Moringa oleifera leaf ethanolic extract against hepatocellular carcinoma in male Wistar rats (40 male Wistar rats).⁹⁴ After determining the appearance and histological changes in the liver, they found that rats treated with Moringa oleifera leaf extract (MOLEE) showed a reduction in liver enlargement as well as in the bumps and morphological alterations that this organ had compared to the group of rats treated with diethylnitrosamine (DEN), indicating that MOLEE treatment generated a favorable change in hepatic architecture.⁹⁴

Continuing the trend regarding the study of ethanolic extracts, in 2020, the results of the effect of MOLEE on signaling pathways in response to DNA damage to protect hepatic tissue from apoptosis triggered by Cobalt Chloride (CoCl₂) in 40 male Sprague-Dawley rats were reported. The study showed that MOLEE reduced CoCl₂-induced hepatic genotoxicity through the transcriptional induction of DNA damage repair genes,⁹⁵ thus suggesting that MOLEE is a promising candidate for the food, nutraceutical, and pharmaceutical industries. Several studies approaches associated with the study of the antitumor and chemopreventive capacity of Moringa oleifera are shown in Table 3.

3.4 Biological activity of Moringa oleifera on infectious diseases

Antimicrobial resistance crises must be urgently managed. The Global Action Plan on Antimicrobial Resistance acknowledges and addresses the variability of resources available to countries to fight antimicrobial resistance and the development of new antimicrobials by the pharmaceutical industry.¹⁰⁴ This has led to several studies in the last decade focusing on the biological activity of phytochemicals from Moringa oleifera against different infectious agents.^105–107

3.4.1 In vitro antimicrobial properties

Within the wide range of characteristics exhibited by Moringa oleifera for the treatment of chronic diseases, the potential of extracts from different parts of the plant as antimicrobial agents has also been elucidated. Dzotam et al.¹⁰⁸ evaluated the antibacterial activity of the methanolic extract of M. oleifera leaves against sensitive and resistant strains of Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Providencia stuartii, as well as their synergistic effects with some antibiotics, finding growth inhibition in 13 (68.4%) of the 19 strains used.¹⁰⁸ Interestingly, it was also observed that the M. oleifera extract required one of the lowest minimum inhibitory concentrations compared to the other evaluated plants to counteract the growth of a multidrug-resistant strain.¹⁰⁸ Additionally, this extract showed synergistic effects with most of the antibiotics tested.¹⁰⁸

More recently and in line with Dzotam’s work, Umamaheswari et al. (2020) demonstrated the antimicrobial role of the plant, specifically against methicillin-resistant Staphylococcus aureus (MRSA) by evaluating the antimicrobial and antibiofilm potential of extracts from four plants, including Moringa oleifera. The ethanolic extract obtained from M. O leaves showed the lowest minimum inhibitory concentration (MIC) (250 μg/mL) and minimum bactericidal concentration (MBC) (500 μg/mL) among the evaluated plants.¹⁰⁹ The antimicrobial potential was attributed to terpenoids and flavonoids, which are the main groups of phytoconstituents with antimicrobial properties that are naturally present in plants. Furthermore, M. oleifera extracts showed–30-60% inhibition of biofilm formation, and other findings revealed that the extracts were not toxic and had minimal hemolytic activity.¹⁰⁹ Table 4 summarizes some in vitro experimental approaches focused on demonstrating the anti-infectious capacity of different parts of the Moringa oleifera plant.

Regarding the antiviral potential of the plant, the activity of different leaf extracts against the infectivity of the lentiviral vector HIV-1 has been studied. It has been found that all the tested extracts, including methanol (MM), aqueous (AM), and petroleum ether (EM) extracts were active against the vector, inhibiting early events of viral replication in HeLa cells in a concentration-dependent manner, with IC₅₀ values of 7.17 μg AM/mL, 7.72 μg MM/mL, and 7.59 μg EM/mL.¹¹⁰ Another significant finding is related to the selectivity index of M. oleifera extracts, as they showed sufficiently high selectivity towards the lentivirus, indicating that the observed antiviral activities were not due to cytotoxicity, but rather to the presence of phytochemical components such as saponins, alkaloids, glycosides, tannins, carbohydrates, flavonoids, resins, acidic compounds, and proteins.¹⁰⁶

It has also been demonstrated that the leaves of this plant possess antiparasitic characteristics, as shown by Cudjoe et al., ¹¹¹ who studied the short-term (72 h) and long-term (7 days) effects of aqueous leaf extracts from four selected plants collected from the western region of Ghana on the growth of NF54 (chloroquine-sensitive), CamWT_C580Y (artemisinin-sensitive), and IPC 4912 (artemisinin-resistant) strains of Plasmodium falciparum. Long-term treatment with derivatives of Moringa oleifera leaves at a dose of 100 μg/mL inhibited 80% of the development of asexual forms of the NF54 strain.¹¹¹

Such antiparasitic potential has been revealed not only in leaves but also in flowers. The anti-Trypanosoma cruzi activity and cytotoxicity in mammals of the aqueous extract of M. oleifera flowers, as well as the trypsin inhibitor protein isolated from this part, called MoFTI, have been evaluated. It was found that both the aqueous extract and MoFTI induced lysis of T. cruzi trypomastigotes with median lethal concentrations at 24 hours (CL₅₀/24 h) of 54.18 ± 6.62 μg/mL and 41.20 ± 4.28 μg/mL, respectively.¹²⁴ Both products showed low toxicity to murine peritoneal macrophages and Vero cells, with cytotoxic concentrations greater than 400 μg/mL at 50% (CC₅₀).¹²⁴ This study postulated that the flavonoids present in the extract were responsible for the observed cytotoxicity, as suggested by other studies.

The above is consistent with the findings from a study conducted by Nova et al. (2018) where treatment with MoFTI for 24 hours induced the lysis of T. cruzi tripomastigotes and did not affect the viability of human peripheral blood mononuclear cells (PBMCs).¹²³ The effects of MoFTI on the immune response of T. cruzi-infected human PBMCs showed that treatment with this compound at a dose of 87 μg/mL for 48 h stimulated the release of tumor necrosis factor (TNF) α and interleukin (IL) 10 by infected PBMCs, compared to those treated with the conventional drug for the acute phase of Chagas disease, benznidazole, which had no effect on cytokine modulation in infected and uninfected cells.¹²³ Thus, MoFTI is a potential trypanocidal and immunomodulatory agent in Chagas disease that can prevent its chronicity, so it should be taken into account and considered for future formulations developed by the pharmaceutical industry.

Similarly, Moringa oleifera also exhibits antiviral effects in substances obtained from its seeds, as demonstrated in studies on its antiviral properties against influenza A virus (IAV) using methanol extract and apparent regulation of transcription factor EB (TFEB). Initially, Moringa oleifera showed low cytotoxicity towards RAW264.7, and protected infected cells from cytopathic effects (CPE), inhibiting cell lysis by up to 93.7%.¹²⁶ Furthermore, the hemagglutination assay was positive, indicating that the viral load in the groups treated with the extract was significantly reduced, particularly at concentrations of 5 and 10 μM.¹²⁶ Additionally, this extract suppressed autophagy in H1N1-infected cells, and it was established that this suppression was dependent on the decrease in TFEB levels in the nucleus of treated cells. Similar to the observations related to the MoFTI protein, the methanol extract of the seed also showed immunomodulatory properties, reflected in the reduction in TNF-α, IL-6, IL-1β, and IFN-β levels, suggesting that this extract may decrease the levels of inflammatory cytokines induced by H1N1 infection by inhibiting TFEB.¹²⁶

Regarding the potential antiparasitic activity of Moringa oleifera seeds, Kandil et al.¹²⁷ evaluated the in vitro and in vivo effects of the methanol extract of plant seeds on Fasciola hepatica as an alternative treatment. They found that complete inhibition of development and total death of immature Fasciola hepatica occurred within 48-72 hours after treatment at concentrations of 10, 25, and 50 mg/mL.¹²⁷ In addition, the results of in vivo assays showed a 20% decrease in the mean egg count per gram of feces two days after treatment in infected rabbits treated with a daily oral dose of 150 mg/kg of body weight of the extract for 3 days. On the last day of treatment, the egg count decreased by 68%, and from the seventh day onwards, no eggs were detected in the feces of the animals.¹²⁷ Thus, Moringa oleifera exhibited promising and potent activity against fascioliasis.

3.4.2 Therapeutic properties evaluated in silico.

Approaches focused on demonstrating the antimicrobial characteristics of Moringa in preclinical assays are very scarce ( Table 5), but interestingly, studies based on computational tools were found, which were designed to elucidate the specific role of some molecules from M. oleifera extracts, all of which focused on the novel SARS-CoV-2 virus. These in silico studies evaluated active compounds from the plant to determine their inhibitory properties against the Mpro protease of SARS-CoV-2, a key protein involved in the pathogenesis of COVID-19, finding good docking with the protein, with kaempferol showing the highest inhibitory effect against SARS-CoV-2 Mpro. Quercetin, a compound present in moringa, showed significant assembly as well, indicating that the active phytoconstituents of this plant exhibit potential relative inhibitors against SARS-CoV-2 Mpro and pharmacological characteristics similar to hydroxychloroquine, making them possible antiviral candidates in the fight against COVID-19.^128–130 Other authors also mention that components such as niazirin, quercetin, moringin,¹³¹ apigenin, and ellagic acid,¹³² have an inhibitory role on the transmembrane serine protease 2 in SARS-CoV-2 infection and inhibition of two non-structural proteins 9/10 of SARS-CoV-2, respectively. Table 5 shows computational studies developed to demonstrate the anti-SARS-CoV-2 potential of Moringa oleifera.

3.5 Geographical data related to the study of Moringa oleifera in the last decade

The compilation of scientific material developed here has allowed not only to understand the research trends of several authors exploring the potential of M. oleifera in different clinical conditions but also to elucidate the geographical landscape of the sources of such publications. As expected, the majority of publications originated from Asia, since the plant is native to these regions, with India and Egypt being the two main countries where research on the biological activity of M. oleifera in human health is conducted, particularly in chronic diseases ( Figure 2A).

Figure 2. Geographical origin of publications on the biological activity of Moringa oleifera.

A) Studies on chronic diseases B) in vitro studies related to cancer C) in vivo studies related to cancer D) Studies in infectious diseases.

It is noteworthy that Moringa oleifera leaf extract (MOLE) is known for its various cytoprotective properties owing to its high levels of antioxidants, polyphenols, and flavonoids, which improve organ function by acting as regulators of damage and oxidative stress. An example of this is a study carried out in Egypt by Abou-Zeid et al.⁴⁷ and Fathy et al.,⁵⁷ where the group of rats treated with MOLE showed less impact than the untreated group in terms of increased serum markers such as ALT, AST, urea, and creatinine, and decreased levels of albumin and total proteins, indicating protection against hepatic/renal toxicity.

In Egypt, the neuroprotective activity of the ethanolic leaf extract described by Muhammed et al.⁵³ was reflected in a significant decrease in apoptotic-cerebral marker activity, such as caspase-3 and DNA fragmentation.⁵⁵ This resulted in nearly normal activity of the neurotransmitter acetylcholine as well as improved mitochondrial activity of NADH dehydrogenase and ATPase after treatment with the extract. These findings indicate that Moringa oleifera extract has an influence on the protection of mitochondrial function in neuronal cells, thus participating in the prevention of neurodegenerative diseases such as Parkinson’s disease caused by environmental factors.⁵⁵

India has the highest number of studies related to Moringa oleifera leaves. One of the research studies was proposed by Barhoi D, Upadhaya P, Barbhuiya S, Giri A, Giri S.⁶⁶ In 2021, this group developed an in vivo study on the anticancer potential of Moringa oleifera against ascetic and laryngeal cancers. The methodology involved two types of biological assays: the trypan blue dye exclusion assay and the MTT assay. The MTT assay revealed that treatment with Moringa oleifera induced dose- and time-dependent toxicity in both cancer types. This study highlights the potential of Moringa oleifera leaf treatment to induce apoptosis in cancer cells, indicating that this plant could be an excellent candidate for the development of new cancer therapies.

In the context of in vitro antitumor screening, India again stands out as the country with the highest number of studies, contributing 26% of the overall production ( Figure 2B). In 2010, Sreelatha reported the antiproliferative and apoptotic effects of the aqueous extract of Moringa oleifera leaves on a human tumor cell line (KB), using two types of biological assays, the cell proliferation assay and propidium iodide (PI) staining assay. The results showed that the aqueous extract significantly inhibited the proliferation of the studied tumor cell line and induced morphological changes (such as blistering of cell membrane) after 48 h of treatment, indicating apoptosis.⁷ Moreover, cell membrane shrinkage and loss of contact with other cells were observed. Similarly, propidium iodide staining revealed nuclear contraction, condensation, and DNA fragmentation in the cells,⁷ providing evidence of the significant biological effects of this extract, which could be prospectively used as a therapeutic and pharmacological product for treating certain neoplasms.

Another interesting perspective in the present review involves the identification of regions from which in vivo research on the antitumor activity of Moringa oleifera has been conducted, which is not as abundant as in vitro approaches. In this regard, Egypt has led the production of studies in this area ( Figure 2C), specifically focusing on leaves, contributing to 31% of the total related production. This indicates that Egypt has been actively involved in the research and evaluation of the anticancer potential of Moringa oleifera in vivo. Representative of one of these works are the observations of Khalil (2020), related with the evaluation of the effect of MOLEE on signaling pathways in response to DNA damage to protect hepatic tissue from apoptosis triggered by CoCl2 in Sprague-Dawley rats. This study demonstrated that MOLEE reduces genotoxicity induced by CoCl2, as evidenced by the expression of DNA repair genes.⁹

Finally, concerning the biological activity of M. oleifera in infectious diseases, India was once again at the top of the list of articles in this field ( Figure 2D). We highlight a study originating from that country concerning the use of extracts from certain plants to synthesize metallic nanoparticles to support the generation of new nanomaterials with more potent and/or novel biological activities. Das et al.^121,122 by performing green synthesis of copper¹²³ and bismuth¹²⁴ nanoparticles from hydroalcoholic leaf extracts revealed considerable antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Enterococcus faecalis, and relatively stronger antifungal activity against Aspergillus niger, Aspergillus flavus, Candida albicans and Candida glabrata, especially a pro-metabolic and usable inhibitory activity against Candida spp.¹²⁰ These findings suggest that the synthesized copper and bismuth nanoparticles may be promising therapeutic candidates for the treatment of various bacterial and fungal infections, such as candidiasis. Owing to their encapsulation, these nanoparticles can be used in direct medical applications as antimicrobial agents. An ecological advantage of green nanoparticle synthesis is the production of non-toxic materials and the reduction of waste products compared to incinerated nanoparticles synthesized by conventional chemical-thermal methods.

3.6 Plan parts mentioned in studies related with the biological activity of Moringa oleifera in chronic diseases, cancer and infectious diseases

Although different parts of the plant have been studied under different conditions, in all experimental and preclinical approaches, the leaf is the most extensively studied raw material, mainly because of the relative ease of obtaining a viable product for testing. However, extracts from parts other than the leaf have also revealed effectiveness in the different research contexts in which they have been tested. Studies associated with chronic diseases and in vivo evaluations of cancer have led to the use of the leaves of this plant, with 73% and 71% of the investigations, respectively ( Figure 3A and 3C), followed by in vitro assays on cancer and studies of antimicrobial potential, with 60% and 56%, respectively, that is, more than half of the investigations ( Figure 3B and 3D). From this part of the plant, most scientific production has been generated, involving different types of extracts, among which aqueous, methanolic, and ethanolic extracts stand out. A small part of the work has studied the ethyl acetate extract; among them, the research carried out by Bamagous et al.⁴³ showed that this extract reverses diabetes markers such as serum levels of glucose and insulin, lipid profile, and liver damage, confirming the results of a histopathological study what was evaluated at the serum level.⁴³

Figure 3. Parts of Moringa oliefera used in studies related with the biological activity in A) Chronic diseases B) in vitro cancer studies C) in vivo cancer studies. D) Infectious diseases.

With regard to the study of cancer, it has already been mentioned that most studies carried out to demonstrate the antitumor role of Moringa oleifera contemplate the leaf as a target element. One of these in vitro approaches was conducted by Khalafalla et al.⁸⁹ in Egypt in 2010, where different leaf extracts were evaluated to observe the effects of the treatment on the viability of leukemia and hepatocarcinoma tumor cells using the MTT assay. The results showed that the average viability of the treated tumor cells was 25% lower than that of the control cells (human lymphoid cell line), which was 94.6%. Likewise, with regard to the viability of the HpG2 tumor cell line (liver cancer), they observed a significant decrease with respect to treatment with ethanolic extract.⁸⁹

Studies on the leaves of this plant have been devoted not only to revealing the ability of these extracts to attack tumor cells but also to show their harmlessness to normal cells. Thus, Suphachai in Thailand conducted research on the in vitro antiproliferative activity of two types of Moringa oleifera leaf extracts (methanol and biomethanol) on breast cancer (MCF-7 cell line), liver cancer (HepG2), and human fibroblast cells. It was found that concentrations up to 400 μg/ml of the two extracts did not induce toxicity in normal cells.⁹⁰ Regarding the two extracts evaluated it was also observed that biomethanol was the one that presented high antioxidant activity and potent antiproliferative activity in cancer cells.⁹⁰ This study reinforces evidence that Moringa oleifera has great potential in the field of medicine in relation to cancer chemotherapy and chemoprevention.

As observed in vitro, in vivo approaches also use leaves as their main raw material for research ( Figure 3C). Among these, Cuellar et al.⁹⁷ focused on determining the physicochemical and nutraceutical properties of moringa leaves and their effects in a model of colorectal carcinogenesis in vivo. By testing Moringa oleifera leaf in male mice it could be evidenced that this extract decreased the activity of harmful fecal enzymes (β-glucosidase, β-glucuronidase, tryptophanase, and urease by up to 40%, 43%, 103%, and 266%, respectively), as well as tumor incidence in male CD1 mice.⁹⁶ These findings suggest that bioactive compounds in moringa, such as total dietary fiber and phenolic compounds, may have chemopreventive capacity. This is the first study on the suppressive effects of this plant leaf in an in vivo model of colorectal carcinogenesis.¹⁰⁵

The biological activity of the plant in infectious diseases, as in the other assays, is the most studied element ( Figure 3D). Ashraf et al.¹¹⁵ when comparing the cytotoxic and anti-influenza potential of ethanolic extract of Moringa oleifera leaves (EEOMOL) and amantadine, an anti-influenza drug, evidenced that this extract showed significantly better (P <0.05) anti-influenza activity (0.78 μg/mL to 100 ug/mL) than amantadine (12.5 ug/mL to 50 ug/mL) (112). Additionally, EEOMOL showed a higher cytotoxic concentration 50 (CC50) (100 μg/mL) than amantadine (50 μg/mL).¹¹⁵ Based on these observations, it can be inferred that the extract could be a breakthrough in combating future influenza outbreaks, especially those caused by the influenza A (H9) virus, which has shown resistance to amantadine due to mutations in the M2 gene.

3.7 Extract fraction and microorganisms described in Moringa oleifera research

Another observation derived from this work is related to the trend that microbiological studies have shown in the past 10 years regarding the type of pathogenic agents and extracts evaluated to reveal the antimicrobial role of the plant. Thus, aqueous and methanolic extracts were used in the highest number of studies ( Figure 4A), and bacteria were the most studied microorganisms ( Figure 4B).

Figure 4. Compendium of extracts implemented regarding the biological activity of Moringa oleifera on infectious diseases and pathogens studied.

A) Extracts B) Pathogens.

A noteworthy study in the line of antibacterial research was conducted by Ibrahim et al., ¹¹³ who evaluated the antibacterial activities of methanolic and aqueous extracts of Moringa oleifera leaves, with and without heat treatment at 45°C, 50°C, and 55°C for 30 and 60 min, to recreate the cooking and frying conditions to which the plant is subjected when used in the diet. The non-heat-treated extracts showed the ability to inhibit the growth of gram-positive bacteria (Staphylococcus aureus and Streptococcus agalactiae) and gram-negative bacteria (Salmonella typhi and Shigella boydii), with the methanolic extract showing a high inhibition zone at 150 mg/mL against S. aureus¹¹³; the aqueous extract did not show any inhibitory activity against S. agalactiae at a concentration of 125 mg/mL, which was attributed to the presence of low amounts of active constituents, indicating that the antibacterial activity depends on the concentration of the extract and the solvent used.¹¹³

In contrast, the antibacterial activity of the heat-treated extracts was affected. The methanolic extracts maintained their activity against all tested microorganisms after exposure for 60 min at 50°C, while aqueous extracts lost their activity against Shigella boydii but retained it against other microorganisms.¹¹³ Thus, Moringa oleifera could be proposed as an alternative for the control of infections caused by these four bacteria, considering that the thermal treatment of the plant reduces the antimicrobial activity of its phytoconstituents.

Ethics and consent

Ethical approval and consent were not required.