Protein deficiency is a problem in the growth and development of children. Several studies reported that Moringa oleifera L. (MO) leaves contain protein, which can be used to balance the composition of amino acids and minerals in food. This study aimed to determine an extraction method for MO dry leaves, to obtain an optimal concentration of protein, and to identify the composition of amino acids, minerals, and bioactive compounds, as well as the antioxidant activity and cytotoxicity. An ultrasonic-assisted extraction (UAE) method using ethanol was used to extract protein from MO dry leaves at 40°C for 60 minutes. The protein contents obtained from this extraction were 45.5%, with the composition of the dominant amino acid phenylalanine at 22.25 g/kg and the non-essential amino acid, glutamic acid at 60.03 g/kg. The macro and micro minerals were kalium (1174.23 mg/100g) and selenium (149 mg/100g). The standardization test of extracted MO quality revealed a low aflatoxin level of microbial contamination as well as a low level of heavy metal contamination. The antioxidant activity and cytotoxic activity showed IC50 values of 41.04 ppm and 182.66 ppm, respectively. We concluded that MO extracted with the UAE method produces qualified food-grade protein in high concentrations.
Article
Impact of Ultrasonic Assisted Extraction on the Protein Yield from Moringa Oleifera Lam Leaves and Its Functional Characterization
https://doi.org/10.21203/rs.3.rs-2986309/v1
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Antioxidant
Cytotoxicity
Moringa oleifera
UAE
Ultrasound assisted extraction
Protein
Protein has a very important function and cannot be replaced by other nutrients, especially in maintaining cells and building body tissues. Protein deficiency will cause major problems for tissue development and cell metabolism. Protein deficiency in children, known as underweight and stunting conditions, gave implications on immune system, hepatic steatosis, decreased muscle mass, and cognitive disorders or delays in children's intellectual development.1
Various attempts have been made to improve protein deficiency conditions including the provision of food supplements.1 Protein sources are obtained from plants and animals sources; Animal protein has the advantage of high protein content, easy to digest but it is difficult to obtain and expensive for people living in poverty line.2
Moringa oleifera Lam (MO) plant which was reported to have protein content, is easy to grow in various seasons and is resistant to drought so it can be one of the best solutions as a source of plant protein. MO plant, particularly the leaves has been found to be rich in fiber, vitamins, minerals, protein, and other important phytochemicals. MO leaves have been extensively researched and found to have protein levels ranging from 23–27%.3,4
MO extracts are made from various parts of the plant, including the leaves, seeds, and bark.5 These extracts contain a high concentration of antioxidants, anti-inflammatory compounds, and other beneficial compounds with medicinal properties. They are frequently found in supplements, skincare, and other health and wellness products.6,7 Cold pressing, solvent extraction, and supercritical fluid extraction are all methods for obtaining the extract. Studies showed the highest protein content of MO leaves was reported in flour of leaves4, dry leaves4 and fresh leaves. 2,8,9,10,11
Vegetable proteins are generally bound in cells, therefore a proper extraction method is needed to open the cells and to pull out the protein.12 Conventional method such as maceration, is commonly used to extract bioactive or phytochemical compounds.13 The disadvantages of the maceration method include the need for more solvents/reagents where solvent replacement is carried out every day, the leftovers reagent is not environmentally friendly, the extraction time takes longer (3–5 days), the protein content is lower and the yield in the final extract is less.14,15
Currently, new extraction technologies such as ultrasound-assisted extraction (UAE) are getting more and more attention because of their shorter time and lower energy consumption, as well as higher extraction efficiency.12,16, Thus, in the present study, we aimed to apply UAE extraction method and characterize the yield of compounds from MO, and investigate its bioactivity in vitro.
Sample Collection and Species Identification
MO leaves were collected in November 2021–2022, originating from the Timor Islands (Kupang city and Kupang regency), and Flores Island (Ende regency), East Nusa Tenggara (NTT), Indonesia. To ensure the species of MO leaves, we submitted the plants to Depokensis Herbarium (DEP), Biota Collection Room University of Indonesia (RKBUI), with certificate number 851/UN2.F3.II/PDP.02.00/2021 on December 1, 2021; and to the Bogoriense Herbarium, with certificate number B 168/II.6.2/DI.05.07/6/2022 on June 7, 2022. The identification process uses references and also comparisons with herbarium collections in the Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Indonesia by Mr. Alexander Tianara (Botanical and Zoological Specimen Curator of Biota Collection Room), and also by Mr. Alex Sumadijaya (Plant Systematist, Herbarium Bogoriense, RC for Biosystematics & Evolution), from Bogoriense Herbarium, Directorate of Scientific Collection Management, National Research and Innovation Agency (BRIN). The voucher specimen of MO was kept in Directorate of Scientific Collection Management, BRIN-Indonesia.
Extraction Method
A total of 2.8 kg of fresh MO leaves were washed, air-dried, and then dried for 24 hours in a 42°C oven. The weight of the dry leaves obtained was then calculated. The dry leaves were then ground into MO powder in a knife mill and weighed again. The finished MO powder was stored in an airtight container. All methods were carried out in accordance with the IUCN Policy Statement on Research Involving Species at Risk of Extinction.
Ultrasonic assisted extraction
MO extraction from dry leaves was carried out using a modified procedure described by Dadi et al.17 A total of 50.0 grams of MO leaf powder were extracted with ethanol 70% for 15, 45, and 60 minutes in a 1:10 (w/v) ratio using an ultrasonic bath with ultrasonic waves at a frequency of 40 kHz at 40°C. The filtrate was then filtered through Whatman No. 1 paper before being concentrated in a rotary evaporator. Dry extracts were obtained by 24-hour drying using a freeze-dried method. The dry extract was weighed and stored at 4°C in a light-protected package.
Characterization of MO ethanol extract
MO ethanol extract was analyzed for its protein and amino acid content. The extract was also tested for mycotoxin contamination, moisture content, ethanol content, total plate count, yeast mold, bacterial contamination, (Escherichia coli, Enterobacteriaceae, Salmonella sp., Shigella sp., Clostridium) and heavy metal contamination. Those tests are needed to ensure that it meets the standard qualification by the National Food and Drug Agency of Indonesia Regulation Standard Number 32 of 20192. All tests are carried out by SNI ISO/IEC 17025:2017-certified laboratories, PT. Saraswanti Indo Genetech, Jakarta, Indonesia (accredited by the National Accreditation Committee (KAN) with number LP-184-IDN).
Phytic Acid Content
Davies and Reid method was used to determine phytic acid content.18 A total of 1 gram of sample was suspended in 50 mL 0.5 M HNO3. This suspension was stirred using a magnetic stirrer for 2 hours at room temperature, then it was filtered. The obtained filtrate was used to determine the phytic acid content which was carried out in the following way: A mixture of 0.5 mL filtrate, 0.9 mL 0.5 M HNO3 and 1 mL 0.3 mM FeCl3 was added in a test tube. The test tube was closed and then soaked in boiling water for 20 minutes. After cooling down, 5 mL amyl alcohol and 1 mL 0.1 mM ammonium thiocyanate were added and followed with centrifugation at 1500 rpm for 10 minutes. After two layers were formed, the absorbance of the amyl alcohol layer was measured using a spectrophotometer at a wavelength of 465 nm with an amyl alcohol blank 15 minutes after the addition of ammonium thiocyanate.
Phytochemical Analysis
Positive mode of LC-MSMS was used to identify the phytochemical components of extracted MO leaves. The extract was dissolved in the appropriate solvent by injection of 1 µl water. The ESI mode was set to full scan m/z of 100–1200. The mobile phase was mixed with solvents H2O + 0.1% formic acid and acetonitrile + 0.1% formic acid. The analysis was done in LC-MSMS Waters™ Acquity UPLC-1 Class and XEVO G2-XS QTof (Shimadzu LCMS-8060), ACQUITY UPLC™ BEH C18 column (1.7 µhm 2.1 x 50 mm), and XEVO G2-XS TOF mass spectroscopy (Shimadzu LCMS-8060).
Antioxidant Activity (DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) Test)
The possible antioxidant activities of MO extract were investigated using DPPH assay described by Desmarchelier19 and Rahman.20 Extracts at various concentrations (12.5–150 g/mL) were mixed with 2.4 mL of 0.1 mM DPPH. The solution mixture was vortexed and stored in a dark at room-temperature for 30 minutes. The absorbance was measured by spectrophotometry at 517 nm, with butylated hydroxytoluene (BHT) as a reference.
Cytotoxicity Test (3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide/MTT Test)
The cytotoxicity potential of MO extract was studied in HepG2 and MDCK cells using MTT test by adopting the procedure from Barhoi D21 and Abd Rabou AA.22 HepG2 and MDCK cells were cultured in a complete medium containing RPMI, 10% Fetal Bovine Serum, 1% of penicillin streptomycin, 1% amphotericin B in a 5% CO2 incubator. Briefly, 10000 cells of HepG2 or MDCK were grown in a 96 well plates, and were treated with various concentrations of MO extracts (from 3.0 to 500 µg/mL) for 24-hours. Afterward, the cells were reacted and incubated with MTT reagents. The absorbance of the viable cells was then measured using ELISA reader at a wavelength of 590 nm. The absorbance obtained was used to make a relationship curve between concentration and percentage of inhibition. From this curve, the concentrations of MO extracts that inhibits 50% of cell viability (IC50) was determined.
Optimal Results of MO Leaves Extract in Ethanol Extraction Using UAE Method
MO leaves used in this study were gathered in NTT from a variety of locations and seasons. The time of extractions with UAE method resulted in a different content of protein. Extraction of MO leaves from the Kupang Regency region with ethanol for 60 minutes yielded the most optimal results in protein content. With UAE method, 15 minutes of extraction resulted in a 44.2%, 45 minutes of extraction resulted in a 44,9%, our net yield of MO extracts from dried leaves were 0.71% with protein content ranging from 39.8 to 45.5% (Fig. 1). The extraction process using ethanol for 60 minutes was repeated 4 times to confirm the consistency of the method. Proximal analysis was used to compare the content between MO leaves powder and MO leaves extracts obtained from the optimum extraction of protein with ethanol for 60 minutes using the UAE method (Table 1)
| Sample Content | • MO leaves powder | MO leaves ethanol extract |
|---|---|---|
| Water, % Ash, % Carbohydrate, % Protein, % Crude Fat, % Crude Fiber, % | 5.35 13.82 34.15 28.76 7.49 10.94 | 4.63 10.20 36.86 45.50 1.10 1.81 |
Examination of MO Ethanol Extract Using Standard Test for Herbal Medicine
The extracted protein obtained from MO ethanol extract in 60 minutes UAE was examined using the standard tests for herbal medicine issued by the Indonesian National Agency for Food and Drug Control (PerKa BPOM no 32/2019). The results of the examination showed that our extracts fully met the criteria for herbal medicine (Table 2).
| No | Parameters | Unit | Content in MO leaves extract | Quality standards* |
|---|---|---|---|---|
| • 1 | • Aflatoxin G2 | • µg/kg | ND | ≤ 20 µg/kg |
| • 2 | • AflatoxinB2 | µg/kg | ND | ≤ 20 µg/kg |
| • 3 | • Aflatoxin G1 | µg/kg | ND | ≤ 20 µg/kg |
| • 4 | • AflatoxinB1 | µg/kg | ND | ≤ 20 µg/kg |
| • 5 | • Total aflatoxin | µg/kg | ND | ≤ 20 µg/kg |
| • 6 | • Water content | % | ≤ 10% | ≤ 10% |
| • 7 | • Ethanol content | µg/kg | < 1% | < 1% |
| • 8 | • Total plate content | colony/g | ≤105colony/g | ≤ 105colony/g |
| • 9 | • Yeast and mould | colony/g | ≤103colony/g | ≤ 103colony/g |
| • 10 | • Escherichia coli | colony/g | ≤10 colony/g | ≤ 10 colony/g |
| • 11 | • Enterobacteriaceae | colony/g | ≤ 103colony/g | ≤ 103colony/g |
| • 12 | • Salmonella sp. | /g | negative/g | negative/g |
| • 13 | • Shigella sp. | /g | negative | negative/g |
| • 14 | • Clostridium sp. | /g | negative | negative/g |
| • 15 | • Arsenic (As) | mg/kg | ND | ≤ 5 mg/kg |
| • 16 | • Mercury (Hg) | mg/kg | ND | ≤ 0,5 mg/kg |
| • 17 | • Cadmium (Cd) | mg/kg | ND | ≤ 0,3 mg/kg |
| • 18 | • Lead (Pb) | mg/kg | ND | ≤ 10 mg/kg |
| Note: ND = Not detected | ||||
| * Standard value was issued by the Indonesian National Agency for Food and Drug Control (PerKa BPOM no 32/2019). | ||||
Determination of Essential and Non-Essential Amino Acids from MO Leaves Extract
The MO ethanol extract was analyzed to determine the contents of essential and non-essential amino acids (Table 3). Eight types of essential amino acids were found, with the highest concentrations are phenylalanine, valine, threonine, and tryptophan. There were also eight types of detected non-essential amino acids, glutamic acid found as the highest concentrations, followed by aspartic, arginine and tyrosine.
| Type of Amino Acid | Concentrations (g/kg) |
|---|---|
| Essential Amino Acid | • • |
| Histidine | 5.23 |
| Isoleucine | 4.99 |
| Leucine | 5.84 |
| Lysine | 3.00 |
| Phenylalanine | 22.25 |
| Threonine | 6.41 |
| Tryptophan | 2.04 |
| Valine | 13.09 |
| • • Non-Essential Amino Acid | |
| Glutamic Acid | 60.03 |
| Aspartic Acid | 40.33 |
| Arginine | 14.69 |
| Alanine | 13.50 |
| Serine | 8.12 |
| Proline | 7.07 |
| Glycine | 5.22 |
| Tyrosine | 3.07 |
Determination of Minerals and Flavonoids Content from MO Leaves Extract
The MO ethanol extract was analyzed to determine the content of the minerals (Table 4). Among the macro-elements, potassium was found to be the highest concentrations, followed by chloride and magnesium (Table 4). While the microelements, the most abundance was Selenium, followed by Copper, Zinc and Iron (Table 4).
| Mineral | Concentrations (mg/100 g) |
|---|---|
| Macro elements Chloride (Cl) | 734.07 |
| • Potassium (K) | 1174.23 |
| • Magnesium (Mg) | 251.07 |
| • Calcium (Ca) | 723.08 |
| • Microelements • Zinc (Zn) | 2.04 |
| • Iron (Fe) | 1.90 |
| • Copper (Cu) | 4.20 |
| • Selenium (Se) | 149 |
Analyzing the phytochemical content, major compounds found were flavonoids, namely kaempferol-3,7-diglucoside, kaempferol-3-O-rutinoside, kaempferol-7-O-α-L-rhamnoside and phenylpropionic acid (Table 5). To ensure the presence of phytic acid in MO which will not inhibit nutrient absorption, we analysed the level of phytic acid levels in MO leaves extracts using spectrophotometry and a phytic acid standard series. The analysis showed that the phytic acid content in our MO leaves extract was 0,13 g/g.
| • No. | • Compounds | • Chemical Formula | Chemical Structure | • Observed m/z | • Retention time (min) |
|---|---|---|---|---|---|
| • 1 | • Kaempferol-3,7-diglucoside | • C27H30O16 | • | • 611.1633 | 3.20 |
| • 2 | • Kaempferol-3-O-rutinoside | • C27H30O15 | • | • 595.1684 | 2.78 |
| • 3 | • Kaempferol-7-O-α-L-rhamnoside | • C21H20O10 | • | • 433.1143 | 3.25 |
| • 4 | • Phenyl propionic acid | • C9H11NO2 | • | • 166.0862 | 1.19 |
| • 5 | • Tryptophan | • C11H12N2O2 | • | • 205.0970 | 2.12 |
Antioxidant potential with low cytotoxicity of MO leaves extracts
The antioxidant activity of ethanol extracted MO leaves was shown with the IC50 41.04 ppm, with ascorbic acid used as the positive control of antioxidant agent with the IC50 of 15.8 ppm (Fig. 2). The concentrations of MO leaves ethanol extract needed to produce antioxidant activity were substantially lower than those required to cause cytotoxicity in HepG2 and MDCK cell lines (Fig. 3). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was used to investigate the cytotoxicity of MO leaves ethanol extract.
MO plants are cultivated all over the world. The plants are drought-resistant and has a tolerance of up to six months, as well as being resistant to winter. MO may grow in all types of soil in the tropics at temperatures ranging from 25 to 40 ℃, and at heights of up to 1000 m above sea level.23 In Indonesia, MO expanded throughout many island including Java, Sumatra, Kalimantan, Sulawesi, NTB, NTT, and others.
MO extracts have been shown to have high levels of nutritional and phytochemical content. It was known that MO leaves could provide 9 times more protein than yoghurt, 17 times more calcium than milk, 7 times more vitamin C than oranges, 10 times more vitamin A than carrots, 25 times more iron than spinach, and 15 times more potassium than bananas. 16,23 WHO had recommended MO leaves to be used as a nutritional supplement for children. Various studies in Indonesia has reported the ability of MO leaves to improve nutrition in stunting children24,25, to improve the diet pattern of teenage girls26, to complete the composition of weaning foods27,28, to increase the amount of breast milk and to reduce the malnutrition cases.27,29
In our study, the fresh MO leaves were harvested at different seasons, summer and rainy seasons, from several regions in NTT province, Indonesia; Kupang Regency, Timor Island; Kupang City, Timor Island; and Ende Regency, Flores Island. In NTT province, MO plants are commonly grown as land barrier, yard, or field fences and even for reforestation programs. The process of plant propagation is simply by multiplying cuttings or replanting MO seeds. Local communities consume MO leaves for daily food.30 Variation protein concentrations were found in those leaves ranging from 20–45%. The highest protein content (45,5%) of MO leaves was obtained from Kupang Regency collected in the beginning of the rainy season (November). This study results were supported with previous studies which reported that the differences in nutrient levels may be caused by differences in growing places and harvest times.31,32
The quantity of nutritional components and active phytochemicals in MO extracts are significantly affected by the plants source (leaves, tree trunk, roots, seeds, flowers) and method of extraction.33, 34. There are several extraction methods available such as maceration, reflux, percolation, Microwave-Assisted Extraction (MAE) and UAE. UAE method using different liquid solvents was reported to be the best method in producing high content of nutritional components and active phytochemicals.35,12,36 MO leaves were known to contain high protein, several studies had reported protein content in MO leaves ranging from 23–27%36, depending on part of plants. Previous studies had reported the protein yield from MO leaves extracts from many areas ranging from 6.7%-33,82%37,38,39,40,41 (fresh leaves), 24.2–29.8%42,43 (dried leaves), 27.1% (leaves flour) and 2.5% (seeds).44 Our extraction method, which were the UAE extraction with ethanol solvent for 60 minutes, had resulted in an optimal yield of protein obtained by ethanol extraction was 45.5%.
The UAE extraction method was commonly used to extract protein and several chemical components from the plants, such as flavonoids45, vitamins, polyphenols35, polysaccharides46 and other phytochemicals. It is known that the protein derived from MO flour has low solubility because the protein is bound in the cell, thus inhibiting protein absorption. The principal work of the UAE extraction method is as follows: High-power ultrasonic waves travel through the liquid solvent creates alternating high/low-pressure cycles, and the different pressure on the surface of the MO leaf cells produces a peeling/opening cell wall which effects in transferring proteins from the cells into the liquid solvent. This process will produce high protein content in the final product of extraction35.
According to Indonesian National Food and Drug Agency standards, there are several standard methods for extraction which the products can be used orally. We found that the MO extracts made using the UAE method met the requirements safety standards physically and chemically, as well as metal and microbiological contamination.
The high protein content in MO leaf extract had the potential to be a source of nutrition. Fresh MO leaves contained vitamins A, C, B6, calcium, potassium, iron, and other nutritional content28. Several studies reported that the nutritional content of Ca, Mg, Na, P, and K was 1.63–2.2%, 0.36–0.53%, 0.18–0.43%, 012–0.22%, and 0.67–0.76%, respectively. Variations in the levels of polyphenols and flavonoids were 0.24–0.34% and 192–209 ppm27, respectively. The total amino acid content in MO leaves had been reported to range from 74.5 to 172.7 mg/g.11 The part of the plant that contained the most amino acids was the leaves, which was twice as large as the flower. Amino acids in higher proportions were glutamic acid, arginine, and aspartic acid, while the lowest concentrations were methionine and tyrosine.11 Natsir et al. reported 15 amino acids, with the highest concentrations of essential and non-essential amino acids being leucine, glutamic acid, respectively.43 Another study by Sena et al. also reported 20 amino acids from MO leaves, with the highest proportions being aspartic acid, glutamic acid, leucine, arginine, and valine.14
The composition of amino acids, such as aspartic acid, glutamic acid, histidine, threonine, alanine, tyrosine, arginine, methionine, phenylalanine, and lysine, showed variation in the published data by several investigators.39,40 It was possible that variations in the amino acid composition of the leaves were influenced by protein quality and plant origin (cultivated or wild). Based on the results of our research, we found complete amino acids in our extract, namely essential amino acids consisting of phenylalanine, valine, threonine, and leucine, and non-essential amino acids being glutamic acid, aspartic acid, arginine, and alanine.
Several studies had reported the mineral content of MO leaves, Natsir et al. reported 13 essential minerals including Al, Ca, Co, Cr, Cu, Fe, K, Mg, Mn, Ni, P, Se, Zn. The highest minerals were selenium (1097.84 ppm) and chrome (919.99 ppm). Sodium (68.83 ppm) and magnesium (60.84 ppm) were reported as minerals with the lowest levels.47 Moyo et al. also reported Calcium (3.65%), potassium (1.5%) and phosphorus (0.30%) of the macro-elements. Fe of 490 mg/kg was the highest for microelements, followed by Se 3.63 mg/kg, Copper was the lowest at 8.25 mg/kg.38 Several other researchers reported mineral content with variations in levels, but almost all types of minerals were found in MO leaves40,48.
Our investigation found that the largest mineral content was potassium, chloride, and calcium. The ethanol extract contained the highest proportions of potassium (1,174 mg/100g), chloride (734 mg/100g), and calcium (723 mg/100g) and the highest proportions of selenium (149 mg/100g), copper (4,2 mg/100g) and zinc (2,04 mg/100g).
Previous studies had extensively documented anti-nutritional components from MO leaves, and it was known that MO leaves possessed modest amounts of anti-nutrients such as saponins, phytates, and tannins.45 These compounds were created by regular metabolism plants and interacted with the chemical composition or interfered with digestion and metabolic processes in the body through various pathways, resulting in results that were contradictory to healthy nutrition.22 Saponins, tannins, flavonoids, alkaloids, protease inhibitors, oxalates, phytates, hemagglutinins or lectins, cyanogenic glycosides, cardiac glycosides, coumarins, and gossypol were all well-known anti-nutritional compounds. The amphitic acid concentration of MO leaves were observed to be lower than those of other household vegetables. The reported phytic content was 22.3 mg/g of dried leaves.46 Our investigation indicated that, the amount of phytic acid in the ethanol extract was low (0.13g/g), which would be unlikely to act as anti-nutritional.
Aside from its nutritional value, our extract demonstrated antioxidant activity as measured by the DPPH test. Because it is simple, fast, easy, sensitive, and requires a small amount of sample, DPPH is one of the traditional and widely used in-vitro methods for testing antioxidant activity.19,47 Another benefit of this approach is that it necessitates the use of stable DPPH compounds as well as comparator compounds such as vitamins C, A, and E. The IC50 value is used as a metric in this approach to measure the concentration of antioxidant chemicals that may stop 50% oxidation. The stronger the antioxidant activity, the lower the IC50 value.48
There were several studies reporting the antioxidant activity of MO leaves, with the active compounds responsible thought to be polyphenols, flavonoids, phenolics, and carotenoids. Several extraction methods that had been used to extract antioxidant compounds include the UAE method21,49,50, subcritical water extraction (SWE), supercritical fluid extraction (SFE)51, microwave assisted extraction (MAE).52 The antioxidant compounds were also found in MO leaves extracted by the UAE method in this study. Based on the results of the phytochemical test of the MO leaves extract, several compounds expected to be responsible for antioxidant activity, kaempferol-3,7-diglucoside, kaempferol-3-O-rutinoside and kaempferol-7-O-α-L-rhamnoside were found. These three compounds were derivatives of flavonoids which are having strong antioxidant activities.
The antioxidant activity (IC50 value) that we got was 41.04 mg/L for MO leaves ethanol extract with standard used was vitamin C which had IC50 of 1.58 mg/L. The DPPH and ABTS tests for the antioxidant activity of the MO leaves dichloromethane extract reported that the IC50 value was 1.60 ± 0.03 mg/ml in the DPPH test and IC50 = 1.02 ± 0.06 mg/ml in the ABTS test.49 Wang et al reported the IC50 value of the MO extract was 0.7440 mg/L, compared to OPC as a standard which had IC50 value of 0.0195 mg/L65.33
To investigate the likelihood of MO extract toxicity, we used the MTT test to assess the extract's cytotoxicity. The MTT Assay is a technique for determining a compound's cytotoxicity against cells. The reduction reaction of MTT to formazan crystals is the concept of the MTT test, which is based on the capacity of the cellular enzyme oxidoreductase to convert nicotinamide adenine dinucleotide (NADPH) into insoluble formazan with a purple color. Formazan will be created by live cells that have an active metabolism.21,22.
The MTT assay as one of the MO cytotoxicity test methods on various cells can also be used as an anticancer activity test. All parts of the MO plant have been well studied and proven for their anticancer activity including the leaves, seeds, bark, and roots. Anticancer research from MO plants, both in vivo and in vitro, has mostly focused on leaves extracts. MO leaves extract had been reported to had anticancer activity in liver cancer cells HepG250,51, breast (MCF-7)51,52, T47D53, MDA-MB-23154,colorectal (HCT-116)51,53, Colo-20553, Caco251, leukemia (THP-1)53, HL-6053, K56253, EAC50, prostate PC353,55, melanoma cells (A375)56,57, A205856,57, lung cancer A54953, HeLa58 cancer cells.
We have tested our sample on 2 cells namely HepG2 and MDCK cells and we found the IC50 values were 181.66 mg/L and 121.04 mg/L respectively. HepG2 cells are a hepatocarcinoma cell line that has been successfully cultivated. These cells have a high degree of morphological and functional differentiation. They are more stable and uniform than primary liver cells, making them suitable as a model for studying human protein dynamics in vitro. In vitro, HepG2 cells can produce CYP3A4 enzymes known to metabolize more than 50% of drugs.59,60Madin-Darby canine kidney (MDCK) cells are known to be widely used for drug elimination, permeability, and drug solubility tests in vitro, so it is hoped that MO extracts will have a low toxicity effect.61,62 Our study showed that the toxicity dose of ethanol extract MO was 6–7 times higher than the antioxidant dose in HepG2 cells and 8 times in MDCK cells.
Several previous studies reported that the IC50 value of MO leaves extract was 0.33 + 0.01 mg/mL51 (EAC cells), HepG2 (0.22 + 0.02 mg/mL), A549 13.2 ± 1.8 µg/mL, PC-3 (22.2 ± 4.9 µg/mL), T74D (33.5 ± 2.5 µg/mL), MCF-7 (26.4 ± 5.7 µg/mL), HCT-16 (28.8 ± 2.2 µg/mL), Colo-205 (49.7 ± 0.8 µg/mL), THP-1 (35.8 ± 1.7µg/mL), HL-60 (50.0 ± 1.0 µg/mL), and K562 (149.9 ± 1.7 µg/mL).53
Moringa oleifera leaf ethanol extract obtained by the ultrasound assisted extraction method at 40 ℃ for 60 minutes meets the extract's safety standards, has high nutritional value (protein, amino acid composition, balanced minerals, low acid content), and is a potent natural source of antioxidants with non-cytotoxic properties.
Acknowledgements
We thank the Directorate of Research and Community Engagement Universitas Indonesia for providing publication grants in 2022. The authors acknowledge the facilities, scientific and technical support from Advanced Characterization Laboratories Serpong, National Research and Innovation Institute through E- Layanan Sains, National Research and Innovation Agency.
Contribution
NTK conceptualized the study, developed the proposal, and performed sample collection, laboratory work, data management, analysis, and manuscript writing. ML participated in conceptualized the study, developed the proposal, data analysis and manuscript writing. PEK and TS participated in result interpretation, study supervision, and manuscript editing. All authors read and approved the final manuscript.
Corresponding author
Correspondence to Taniawati Supali
Ethics declarations
Not applicable
Funding
This work was supported by Universitas Indonesia PUTI grants (PUTI NKB-1122/UN2.RST/HKP.05.00/2022).
Competing interests
The authors declare no competing interests.
Data availability
All data generated or analysed during this study are included in this published article [and its supplementary information files].
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Table 1. Proximate analysis of MO leaves powder vs ethanol extract
|
Sample Content |
|
MO leaves ethanol extract |
|
Water, % Ash, % Carbohydrate, % Protein, % Crude Fat, % Crude Fiber, % |
5.35 13.82 34.15 28.76 7.49 10.94 |
4.63 10.20 36.86 45.50 1.10 1.81 |
Table 2. Examination Results of MO leaves extract following herbal medicine standard
|
No |
Parameters |
Unit |
Content in MO leaves extract |
Quality standards* |
|
|
|
ND |
≤ 20 µg/kg |
|
|
µg/kg |
ND |
≤ 20 µg/kg |
|
|
µg/kg |
ND |
≤ 20 µg/kg |
|
|
µg/kg |
ND |
≤ 20 µg/kg |
|
|
µg/kg |
ND |
≤ 20 µg/kg |
|
|
% |
<10% |
<10% |
|
|
µg/kg |
<1% |
<1% |
|
|
colony/g |
≤105colony/g |
≤ 105colony/g |
|
|
colony/g |
≤103colony/g |
≤ 103colony/g |
|
|
colony/g |
≤10 colony/g |
≤ 10 colony/g |
|
|
colony/g |
≤ 103colony/g |
≤ 103colony/g |
|
|
/g |
negative/g |
negative/g |
|
|
/g |
negative |
negative/g |
|
|
/g |
negative |
negative/g |
|
|
mg/kg |
ND |
≤ 5 mg/kg |
|
|
mg/kg |
ND |
≤ 0,5 mg/kg |
|
|
mg/kg |
ND |
≤ 0,3 mg/kg |
|
|
mg/kg |
ND |
≤ 10 mg/kg |
Note: ND = Not detected
* Standard value was issued by the Indonesian National Agency for Food and Drug Control (PerKa BPOM no 32/2019).
Table 3. Analysis Results of Amino Acid Composition in MO Leaves Extract
|
Type of Amino Acid |
Concentrations (g/kg) |
|
Essential Amino Acid |
|
|
Histidine |
5.23 |
|
Isoleucine |
4.99 |
|
Leucine |
5.84 |
|
Lysine |
3.00 |
|
Phenylalanine |
22.25 |
|
Threonine |
6.41 |
|
Tryptophan |
2.04 |
|
Valine |
13.09 |
|
|
|
Glutamic Acid |
60.03 |
|
Aspartic Acid |
40.33 |
|
Arginine |
14.69 |
|
Alanine |
13.50 |
|
Serine |
8.12 |
|
Proline |
7.07 |
|
Glycine |
5.22 |
|
Tyrosine |
3.07 |
Table 4. Analysis Result of Mineral Content in MO Leaves Extract
|
Mineral |
Concentrations (mg/100 g) |
|
Macro elements Chloride (Cl) |
734.07 |
|
1174.23 |
|
251.07 |
|
723.08 |
|
2.04 |
|
1.90 |
|
4.20 |
|
149 |
Table 5. Phytochemical Compounds found in ethanol extracted MO Leaves.
No competing interests reported.