|Year : 2021 | Volume
| Issue : 1 | Page : 33-41
Chemical and biological studies on Moringa oleifera L. cultivated in Egypt
Mona A Mohamed1, Magda T Ibrahim2, Nahla S Abdel-Azim1, Mostafa M El-Missiry1
1 Department of Chemistry of Medicinal Plants, Division of Pharmaceutical and Drug Industries Research, National Research Centre, Egypt
2 Department of Pharmacognosy, Faculty of Pharmacy, Al-Azahar University (Girls), Cairo, Egypt
|Date of Submission||16-Jul-2020|
|Date of Decision||16-Jul-2020|
|Date of Acceptance||25-Oct-2020|
|Date of Web Publication||06-Jan-2021|
MSc Mona A Mohamed
Department of Chemistry of Medicinal Plants, Division of Pharmaceutical and Drug Industries Research, National Research Centre, El-Buhouth Street, Dokki, Cairo 12622
Source of Support: None, Conflict of Interest: None
Background Nowadays, Moringa oleifera is being cultivated in Egypt on a wide scale owing to its proven medicinal and economic benefits.
Objective The aim was to estimate quantitatively different phenolic acids and flavonoids in the M. oleifera L. leaves. Moreover, gas chromatography–mass spectrometry (GC/MS) analysis was carried out to evaluate seed oil obtained by three different methods of extraction. Different plant extracts were tested for their hepatoprotective, anticancer, and antibacterial activities.
Materials and methods Quantification of different phenolic acids and flavonoids in M. oleifera L. leaves was carried out using HPLC. GC/MS was used to determine fatty acid methyl esters of M. oleifera L. seed oil, extracted by three different methods (cold press, solvent extraction, and ultrasound-assisted extraction). Moreover, in-vitro investigations of hepatoprotective, anticancer, and antibacterial activities were done.
Results and conclusion HPLC profiling of leaves extract indicated that ellagic acid is the major phenolic acid (120.15 mg/g). Quercetin and rutin were recorded as major flavonoids. GC/MS of seed oil extracted by ultrasound-assisted extraction showed the presence of higher content of oleic acid comparable with other extraction techniques. The petroleum ether fraction of the leaves showed the most potent hepatoprotective and anticancer effects, whereas the ethanolic extract was active against the tested gram-positive and gram-negative microorganisms. Our findings confirm that M. oleifera L. cultivated in Egypt has unique phytochemical content (comparable with M. oleifera cultivated in other countries); consecutively, it has many potent biological activities. So, it is highly recommended to cultivate the plant species on a wide scale to make use of its constituents in pharmaceutical and nutraceutical industries.
Keywords: antibacterial, anticancer, flavonoids, phenolic acids, ellagic acid, hepatoprotective, HPLC, Moringa oleifera, seed oil, ultrasonic
|How to cite this article:|
Mohamed MA, Ibrahim MT, Abdel-Azim NS, El-Missiry MM. Chemical and biological studies on Moringa oleifera L. cultivated in Egypt. Egypt Pharmaceut J 2021;20:33-41
|How to cite this URL:|
Mohamed MA, Ibrahim MT, Abdel-Azim NS, El-Missiry MM. Chemical and biological studies on Moringa oleifera L. cultivated in Egypt. Egypt Pharmaceut J [serial online] 2021 [cited 2021 Jul 29];20:33-41. Available from: http://www.epj.eg.net/text.asp?2021/20/1/33/306300
| Introduction|| |
Moringa oleifera Lam. (Family: Moringaceae) commonly known as Drumstick tree has an impressive range of medicinal and nutritional uses. Different parts of the plant contain a profile of various important minerals and are a good source of proteins; vitamins A, C, and E; β-carotene; amino acids; and various polyphenolics. The extracts of the leaves, seeds, and roots of M. oleifera have been extensively studied for many potential uses, including wound healing, antitumor, antifertility, hypotensive, analgesic, antipyretic, antiepileptic, anti-inflammatory, antiulcer, antispasmodic, diuretic, hypocholesterolemic, antifungal, antibacterial, and antioxidant activities . M. oleifera is widely used in different countries in the traditional medicine, for example, in Malaysia, a poultice of young leaves is applied to the abdomen to expel tapeworms. Pods and flowers have also been used as anthelmintics. The leaves have been used in India as an antihypertensive; diuretic; antidiabetic; antianxiety agent; antidiarrheal; and to treat dysentery, colitis, and gonorrhea. Poultices made from the leaves have been used to treat glandular inflammation, headache, bronchitis, ear and eye infections, scurvy, and cataracts. In Nicaragua, India, Guatemala, and Senegal, poultices of young leaves are used to treat skin infections. Pods have been used to treat hepatitis and relieve joint pain. The roots of M. oleifera have been used in India as a laxative; to treat edema; to clean wounds and ulcers; to treat hiccups; to relieve pain associated with toothache and earache; and to treat asthma, gout, rheumatism, kidney stones, and diseases of the liver and spleen. In addition, M. oleifera has been used to arouse patients from comas and stupor. The bark of roots and stems has been used to treat wounds and skin infections, scurvy, sore eyes, delirium, digestive complaints, and to prevent snake venom from disseminating in the bloodstream. In addition, the bark has been used as an aphrodisiac and abortifacient and to treat upper respiratory tract infections. Moringa gum has been used to relieve pain from headache and gastroenteritis. Natives of India and Senegal have used the gum to treat fever, dysentery, asthma, dental caries, syphilis, and rheumatism, as well as to induce abortion. Seeds have also been used to treat fever, tumors, warts, hysteria, scurvy, prostate, and bladder problems. In addition, seeds act as a tonic and purgative .
The oil content of the plant seeds ranges from 35 to 41% and is a rich source of omega-9 fatty acids, for example, oleic acid; however, the seeds are underutilized as an oil source . In this study, a quantitative estimation of different phenolic acids and flavonoids was done. In addition, different plant extracts were tested for their hepatoprotective, anticancer, and antibacterial activities.
| Materials and methods|| |
Moringa oleifera L. leaves and seeds were picked from trees grown on sandy soil in El-Sharkia Governorate, Egypt, in June 2014. The plant was identified by Professor Dr Aboelfetoh Mohamed Abd-Alla, Egyptian Scientific Society of Moringa, National Research Centre, Giza, Egypt. The different plant parts were air-dried, ground into a fine powder, and kept for investigation.
Solvents and chemicals
Distilled water, absolute ethyl alcohol, hexane, methanol, petroleum ether, methylene chloride, ethyl acetate, butanol, HPLC grade methanol, tri-fluoroacetic acid, acetonitrile, and 3- [4,5-dimethylthiazol-2-yl]-2,5 dipheny tetrazolium bromide (MTT) were obtained from Sigma (St Louis, Missouri, USA).
Ten bacterial strains were used in the study; among these were seven gram-negative bacteria, namely, Escherichia coli, Coliform spp., Klebsiella spp., Tannerella forsythia, Prevotella intermedia, Pseudomonas spp., and Aggregatibacter actinomycetemcomitans and three gram-positive bacteria, namely, Staphylococcus aureus, Bacillus cereus, and Streptococcus-B-hemolytic.
Quantification of phenolic acids and flavonoids using HPLC
HPLC analysis was carried out using an Agilent 1260 series. The separation was carried out using the Eclipse Plus C18 column (4.6×250 mm, i.d. 5 µm). The mobile phase consisted of water (A) and 0.02% tri-floro-acetic acid in acetonitrile (B) at a flow rate of 1 ml/min. The mobile phase was programmed consecutively in a linear gradient as follows: 0 min (80% A), 0–5 min (80% A), 5–8 min (40% A), 8–12 min (50% A), 12–14 min (80% A), and 14–16 min (80% A). The multiwavelength detector was monitored at 280 nm. Injection volume was 10 µl. The column temperature was maintained at 35°C.
Ultrasonic processor (UP) (400 W, 24 kHz, Hielscher)
The system with an ultrasonic probe with a tip diameter of 20 mm (the horn tip position inside the extraction vessel was 1 cm under the solvent level) was used.
Extraction and fractionation
Extraction of plant leaves
Overall, 100 g of the powdered leaves of M. oleifera L. was extracted till exhaustion with 80% methanol, then filtered and evaporated under reduced pressure at 45°C to give 23 g (23%). The residue was suspended in distilled water and then the residual aqueous solution was left in the refrigerator overnight and filtered. Then, it was successively extracted with petroleum ether, methylene chloride, ethyl acetate, and butanol (3×200 ml each). The obtained fractions were evaporated under reduced pressure to obtain 23, 1.06, 0.94, 1.48, and 16.03 g, respectively. Another batch (100 g) of the powdered leaves was extracted till exhaustion with distilled water, filtered, and evaporated under reduced pressure to give about 17 g. Moreover, absolute ethanol extract of the leaves was prepared and kept for antibacterial evaluation.
Preparation of seed oil
The seed oil was prepared by three different methods as follows:
Solvent extraction (traditional method)
M. oleifera seed powder was prepared by grinding 10 g of the seeds in a clean coffee grinder for 30 s. The seed powder was soaked in 100 ml of n-hexane at room temperature for 72 h and filtered. The residue obtained was again extracted twice with 100 ml of n-hexane. The filtrates were collected and concentrated by using a rotary evaporator under vacuum at 40°C. The obtained oil was weighed (3.67 g) and stored at 4°C before analysis.
The device was operated at a frequency of 24 KHz and an ultrasonic input power of 400 W. Overall, 10 g of seed powder was mixed with 100 ml of n-hexane. Upon completion of extraction, the extract was removed from the vessel, filtered, and evaporated under vacuum. The extraction yield was 33.1 g and kept at 4°C before analysis.
Cold press extraction
M. oleifera mature seeds were dried in sun for 2–3 days. The dried seeds were sent to shell removing to unit, to get the kernels. The kernels were further dried for a week at the room temperature and handled carefully for cleanliness. Overall, 10 g of the seed was poured in the receiving funnel of the cold presser. After ten minutes, the oil was dripping from the outlet and collected in a container that was previously weighed. Overall, 2.53 g was obtained and kept at 4°C before analysis.
[Table 1] summarizes the extraction conditions and the oil yield for each experiment.
Samples of the oil obtained by seed hexane extraction, oil obtained by seed compression, total 80% methanolic, aqueous extracts, petroleum ether, methylene chloride, ethyl acetate, and butanol fractions (M1–M8 fractions, respectively) of M. oleifera L. leaves were investigated for their hepatoprotective and cytotoxic activities.
Preparation of FAMEs
One ml of each oil sample was subjected to the following methylation method: 2 ml N/2 alcoholic KOH was added to each sample in a test tube. The fatty acid methyl esters (FAMEs) were extracted by shaking with 4-ml hexane for 30 s. The hexane layer was separated and washed with distilled water, dehydrated over anhydrous sodium sulfate, and then subjected to gas chromatography–mass spectrometry (GC/MS) analysis.
In-vitro bioassay on human hepatocellular carcinoma (hepG2) cells
Cell propagation and maintenance
Human hepatocellular carcinoma (HepG2) cells were propagated in the proper conditions (at 37°C and 5% CO2), maintained in RPMI-1640 with 1% l-glutamine, and supplemented with 10% fetal calf serum for growth and 1% penicillin/streptomycin. When the cells are ∼80% confluent, they were subcultured using Trypsin-EDTA protocol . The second round of HepG2 cells sub-culturing was used for 96-well plates seeding, and after that, drug screening was done for testing the hepatoprotective and anticancer activities.
Hepatoprotective effect of Moringa oleifera L. samples
Carbon tetrachloride (CCl4)-induced hepatocytes injury was carried out according to Raj et al. . The screening of hepatoprotective activity was based on the protection of human liver-derived HepG2 cells against CCl4-induced damage  determined by estimating mitochondrial synthesis using the tetrazolium assay . Concentrations of the tested samples in the range of 0, 20, 40, 60, 80, and 100 μg/ml and standard drug Silymarin at 250 μg/ml which was proved as safe hepatoprotective dose of the positive reference (silymarin) were investigated (note: above this concentration, the extracts were toxic to the cells).
Cytotoxic effect of Moringa oleifera L. samples
The tetrazolium assay is based on the conversion of MTT into formazan crystals by living cells, determining mitochondrial activity . The potential cytotoxic effects of the studied samples (M1–M8) on the liver cancer HepG2 cell line in concentrations of 0, 20, 40, 60, 80, and 100 μg/ml were estimated by MTT assay. The cytotoxic activity (%) of the M. oleifera L. samples fractions was calculated and compared with the control.
The results were presented as the mean of three independent experiments and the SD. The IC50 was obtained using the four-parameter nonlinear regression on GraphPad prism.
The antibacterial assay
Fresh leaves’ water extract and the 100% ethanol extract were tested for their antibacterial activity. In-vitro antibacterial test was then carried out by disc diffusion method .
The discs were impregnated with 10 µl of the three different samples. Negative controls were prepared using the same solvents to dissolve the plant extracts. Imipenem (10 µg disc-1) was used as a positive control to determine the sensitivity of bacterial strain. Antimicrobial activity was evaluated by measuring the zones of inhibition against the tested bacteria. Each assay was carried out in triplicate.
Minimum inhibitory concentration)
Minimum inhibitory concentration of three different samples, as mentioned earlier, was determined by 0.1 ml of samples added into the test tubes separately, containing 9 ml of a standardized suspension of tested bacteria.
The test tubes were incubated at 37°C for 24 h. Controls were used with the test organisms, using distilled water instead of the plant extract. The least concentration of the samples with no visible growth was taken as the minimum inhibitory concentration.
| Results and discussions|| |
Identification and quantification of different phenolic acids and flavonoids
Identification of phenolic acids and flavonoids from leaf extracts of M. oleifera L. was analyzed by HPLC, and obtained data of the retention time and concentrations are presented in [Table 2].
|Table 2 HPLC quantification of phenolic acids and flavonoids of different plant extracts and fractions|
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The obtained results showed that the butanol fraction contains the highest amounts of both gallic and chlorogenic acids (15.42 and 12.75 mg/g, respectively), whereas the ethyl acetate fraction contained the high concentrations of syringic and ellagic acids (9.28 and 120.15 mg/g, respectively). The flavanone, naringenin was detected in both ethyl acetate and butanol fractions (21.56 and 15.95 mg/g, respectively), which exceeds its content in its most abundant sources . The HPLC analysis showed that M. oleifera L. cultivated in Egypt could be considered as a great source of phenolic acids and flavonoids comparable with M. oleifera cultivated in other countries, as shown in [Table 3]. The present study showed that phenolics and flavonoids, especially ellagic acid, were present in a very high amount (120.15 mg/g) compared with the M. oleifera species cultivated in other countries. To the best of our knowledge, the highest reported concentration of ellagic acid was reported in the M. oleifera L. cultivated in Nigeria (52.95 mg/g) , whereas it is almost in double in that cultivated in Egypt.
|Table 3 Comparative HPLC study of major detected compounds in Moringa oleifera cultivated in Egypt and other countries.|
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Among the identified compounds, different phenolic acids and flavonoids with numerous biological activities were detected. These different compounds possess different biological effects, such as antioxidant activity (which may play a role in hepatoprotection activity) which was exerted by compounds such as gallic , chlorogenic , ellagic acids , rutin , and quercetin. Moreover, powerful anticancer activity was reported for chlorogenic , syringic , ellagic acids , naringenin , and quercetin. In addition, ellagic acid and naringenin  were reported to exert antimicrobial activity.
These results come in accordance with our biological findings which support that different plant extracts and fractions possess potent hepatoprotective, cytotoxic, and antibacterial activities.
GC/MS analysis of FAMEs of seed oil
The data of GC/MS analysis of FAME samples are compiled in [Table 4]. The previous results revealed that the seed oil obtained by ultrasound-assisted extraction (UAE) has palmitic acid (saturated fatty acid) and oleic acid (unsaturated fatty acid) as the major constituents in a percentage of 14.77 and 64.78, respectively. Both oleic and palmitic acids have many potent biological activities. This unique chemical composition of seed oil was only obtained by the UAE, as the oil obtained by cold compression has only palmitic acid as its major constituent in a percentage of 15.73%, whereas oleic acid was not detected and the seed oil obtained by solvent extraction has 13-Octadecenoic acid as its major constituent in a percentage of 51.37. However, both oleic and palmitic acid were absent, which give high curative and nutritive value for M. oleifera seed oil obtained by UAE technique. Oleic acid represents the principal constituent in seed oil obtained by the ultrasonic hexane extraction method (which is an innovative extraction technique). It presents in very high impressive percentage (64.78%), which gets close to the oleic acid content in olive oil (ranges from 56 to 84%), and the healthful properties of olive oil have been, until recently, exclusively attributed to its high monounsaturated fatty acid content, mostly in the form of oleic acid .
|Table 4 Comparative study of GC/MS analysis of FAMEs of seed oil obtained by three different methods of extraction|
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Different fatty acids were identified in M. oleifera L. seed oil with potent biological activities such as antioxidant activity, which was exerted by palmitic  and oleic acids ,, and also a hepatoprotective activity was reported for palmitoleic acid . Moreover, oleic acid was found to have potent anticancer activity. These results emphasize our biological findings that the plant seed oil exerts hepatoprotective and cytotoxic activities.
So, further biological studies should be done to make optimum use of M. oleifera L. seed oil obtained, especially by ultrasonic technique owing to its exceptional properties.
Evaluation of hepatoprotective activity
M1–M8 extracts and/or fractions were evaluated for their hepatoprotective activity, and the obtained results revealed the following: petroleum ether and methylene chloride fractions (M5 and M6, respectively) exhibited hepatoprotective activity at concentrations ranging from 40 to 100 µg/ml for M5 and at concentration 80 µg/ml for M6 fraction. The obtained results are summarized in [Table 5] and [Figure 1]. The results revealed low toxicity and high hepatoprotection activity for petroleum ether and methylene chloride fractions (M5 and M6, respectively). This means that the IC50 of these fractions was more than 100 µg/ml as the concentration that causes 50% cell death was more than 100 µg/ml, so it gives high safety margin for both plant fractions. Both fractions at different concentrations showed hepatoprotective activity exceeding the positive reference for hepatoprotection (silymarin).
|Table 5 Hepatoprotective effect of different concentrations of Moringa oleifera L. extracts and fractions|
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|Figure 1 Viability of monolayer of HepG2 cells after treatment with different concentrations of Moringa oleifera L. samples and 1% CCl4 in comparison with silymarin as control using MTT colorimetric assay.|
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These results come in accordance with the results obtained from a previous study , which also stated that n-hexane and dichloromethane fractions (of M. oleifera L. leaves, also cultivated in Egypt) have the most potent hepatoprotective effect among other fractions and extracts. So, future pharmacological and pharmaceutical studies should be conducted to make available drug formulations for liver protection.
Evaluation of cytotoxic activity
M1–M8 extracts and/or fractions were evaluated for their cytotoxic activity, and the obtained results revealed the following:
Petroleum ether fraction, total 80% methanolic extract, and methylene chloride fraction (M5, M3, and M6, respectively) exhibited very high cytotoxic activity represented by IC50 values (19.25, 21.09, and 21.52 µg/ml for M5, M3, and M6, respectively). The obtained results are summarized in [Table 6] and [Figure 2].
|Table 6 Cytotoxic effect of different concentrations of Moringa oleifera L. extracts and fractions on HepG2 cells|
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|Figure 2 Viability of monolayer of HepG2 cells after treatment with different concentrations of Moringa oleifera L. samples using MTT colorimetric assay.|
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Previously reported studies indicated M. oleifera L. leaf aqueous extract exhibits cytotoxic activity on BHK-21 and HepG2 cell lines, respectively ,. In our study, treatment with 80 µg/ml of M. oleifera leaf aqueous extract (M4) produced inhibition ratios get close to 80%, whereas in the previous reported study , this effect was produced by 200 µg/ml of M. oleifera leaf extract. Further studies are required to make optimum use of these valuable plant fractions as cytotoxic agents, especially the plant cultivated in Egypt.
The fresh leaves’ water extract and the 100% ethanol extract were tested for their antibacterial activity, and results are compiled in [Table 7].
|Table 7 The antibacterial activity of different Moringa oleifera L. extracts|
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The tested M. oleifera L. extracts showed antibacterial activity against Tannerella forsythia (a gram-negative bacteria) that exceeded positive control. Moreover, the 80% ethanolic extract showed antibacterial activity against Bacillus cereus (a gram-positive bacteria), which also exceeded the positive control (Imipenem). In addition, the 80% ethanolic extract of leaf of M. oleifera L. showed the maximum diameter of zone of inhibition against Staphylococcus aureus, that is, 26 mm, indicating a potent source of new antibiotic alternative.
This study showed that the extract of M. oleifera L. is active against the tested gram-positive and gram-negative microorganisms, and the results confirm the use of the plant in traditional medicine. Ethanol extract exhibited a higher degree of antimicrobial activity as compared with aqueous extract. However, further work is needed to isolate the secondary metabolites from the extract to test for specific antimicrobial activity. This in-vitro study demonstrated that folk medicine could be as effective as modem medicine to combat pathogenic microorganisms. M. oleifera L. represents an economical and safe alternative to treat infectious diseases, and it has many other uses. Its use is thus highly encouraged, and more work should be carried out to determine the antibacterial constituents that can be used for drug formulation.
| Conclusion|| |
M. oleifera seed oil is highly recommended owing to its proven health properties, which was emphasized by this FAMES GC/MS analysis, which revealed the presence of oleic acid in an imposing amount, especially in oil obtained by ultrasonic extraction technique. The biological evaluation (hepatoprotective, anticancer, and antibacterial activities) for the plant different extracts and fractions revealed positive test results for certain extracts and fractions, so further studies should be done to make optimum use of these medicinal plant as drug alternative. In conclusion, the oil of M. oleifera is interesting product for its nutritional composition and its biological activities. Its use could have a positive effect on the nutritional status and health of people of developing countries.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]