Potential substitution of the root with the leaf in the use of Moringa oleifera for antimicrobial, antidiabetic and antioxidant properties

SAJB-02277; No of Pages 7 South African Journal of Botany xxx (2019) xxx

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Potential substitution of the root with the leaf in the use of Moringa oleifera for antimicrobial, antidiabetic and antioxidant properties T. Tshabalala a,b, A.R. Ndhlala a,d, B. Ncube a,d, H.A. Abdelgadir c,d, J. Van Staden d,⁎ a

Agricultural Research Council (ARC), Vegetable and Ornamental Plants (VOP), Private Bag X923, Pretoria 0001, South Africa School of Agricultural, Earth and Environmental Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa Faculty of Animal Production and Pasture Sciences, Eldaien University, Eldaien, Sudan d Research Centre for Plant Growth and Development, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa b c

a r t i c l e

i n f o

Article history: Received 5 December 2018 Received in revised form 16 January 2019 Accepted 21 January 2019 Available online xxxx Edited by NE Madala Keywords: Flavonoids Medicinal plant Phytochemicals Tannins Total Phenolics

a b s t r a c t A study was conducted to assess variation in antioxidant, antimicrobial, antidiabetic and phytochemical properties between the leaves, and the main and lateral roots of Moringa (Moringa oleifera). Standard antioxidant models including the DPPH scavenging, ferric reducing power (FRAP) as well as α-glucosidase inhibitory activity were used to evaluate and compare their bioactivity. Antimicrobial efficacy was also tested against Gram-positive (Staphylococcus aureus; Bacillus subtilis) and Gram-negative (Escherichia coli) strains and the yeast-like fungus Candida albicans using the microdilution method. Acetone extracts of all plant parts exhibited good antibacterial activity (MIC b 1 mg/mL) against E. coli, B. subtilis and S. aureus, except for lateral root which exhibited weak activity against E. coli (MIC values N 1 mg/mL). However, all the plant part extracts exhibited low activity against C. albicans (MIC values b 1 mg/mL). Variation in the antioxidant activity was observed, with the main and lateral roots exhibiting better activity than the leaves. All the plant parts had better antioxidant activity than the reference compound ascorbic acid. Leaf extracts had significantly good antidiabetic activity as compared to the reference compound, acarbose. Variations were observed in the total phenolic, condensed tannins and flavonoid contents among the different plant parts tested. The leaf extracts exhibited the highest amount of total phenolics, while the lateral roots had higher amounts of condensed tannins and flavonoid contents. The roots can be used as a better source of antioxidants than the leaves. All the leaf extracts had significantly good antidiabetic and antimicrobial activity as compared to the roots. This study ascertains that these different plant parts of Moringa can be suitable candidates for antimicrobial, antioxidant and antidiabetic supplementations, particularly as it is already frequently used in animal and human diets. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Moringa (Moringa oleifera Lam.) of the family Moringaceae is a fast growing and drought tolerant plant widely cultivated in the tropical and subtropical areas (Anwar et al., 2007). The tree grows in a wide range of rainfall and soil conditions. Common names given to the tree include horse-radish tree because of the taste of its roots and is also known as drumstick tree because of the shape of the pods on the tree. Interestingly, Moringa is commonly known as a “miracle” tree, because all the plant parts of the tree have multi-purpose uses, including use as medicinal and functional foods (Ashfaq et al., 2012). The “miracle” tree has been reported to prevent a number of ailments (Anwar et al., 2007) and has the ability to purify water (Kumar and Gopal, 1999). ⁎ Corresponding author. E-mail address: [email protected] (J. Van Staden).

The leaves are particularly used for traditional medicine, and human and livestock nutrition (Popoola and Obembe, 2013). Moringa leaves are added to foods to increase food shelf life as the leaves are high in natural antioxidants (Siddhuraju and Becker, 2003). Antioxidants found in Moringa leaves include ascorbic acid, flavonoids and phenolics (Anwar et al., 2007). These antioxidant agents/compounds protect cells from free radicals and decrease the oxidative damage of molecular compounds such as lipids, proteins and nucleic acids. On the other hand, the roots (Fig. 1) of the Moringa tree have been reported to contain alkaloids, flavonoids, saponins, terpenoids, steroids and tannins (Raj et al., 2011). The root possesses antimicrobial activities against Gram-negative bacteria (Salmonella enteritica and Vibrio parahaemolyticus) (Dalukdeniya et al., 2016) and is also reported to have antiulcer activities (Choudhary et al., 2013), cure gout and contain a cardiac stimulant. However, there is still anecdotal evidence on the comparison of medicinal properties of different plant parts of Moringa, such as leaves, lateral and main roots. Additionally, there

https://doi.org/10.1016/j.sajb.2019.01.029 0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.

Please cite this article as: T. Tshabalala, A.R. Ndhlala, B. Ncube, et al., Potential substitution of the root with the leaf in the use of Moringa oleifera for antimicrobial, antidiabetic and antioxidant properties..., South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.01.029

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months from transplanting. Leaves were randomly sampled from the 10 plants and divided into 3 samples (n = 3). Both lateral and main roots were harvested and randomly divided into three samples (n = 3), respectively. 2.2. Chemicals and reagents 2,2-Diphenyl-1-picrylhydrazyl (DPPH), neomycin and α-glucosidase were purchased from Sigma–Aldrich (Sigma Chemical Co., Steinheim, Germany); butylated hydroxytoulene (BHT) and potassium ferricyanide from BDH Chemicals Ltd. (Poole, England, UK); trichloroacetic acid, ascorbic acid, ferric chloride (FeCl3) and methanol from Merck KGaA (Darmstadt, Germany). The rest of the chemicals used in this study were obtained locally (South Africa) and were of analytical grade. 2.3. Sample preparation

Fig. 1. Morphology of Moringa oleifera roots system, a white swollen tuberous main root with very sparse lateral roots.

The leaves and roots (main and lateral roots) of Moringa were first oven dried for 48 h at 50 °C. This was followed by grinding the leaves and root samples into fine powders; ethanol, 50% aqueous methanol, acetone and water were used as extracting solvents in an ultrasonic bath for 1 h. The extracts were filtered through Whatman's No. 1 filter paper with the aid of a vacuum. Then a rotary pressure was used to concentrate the extracts in 50% of aqueous methanol, ethanol and acetone at 30 °C and a stream of air was used to completely dry the samples. Water extracts were freeze-dried. Acetone, ethanol and water extracts were used for antimicrobial assays while only water extracts were used for antidiabetic assay. For all the antioxidant assays, the dried extracts of 50% aqueous methanol were used. All phytochemical analyses were done from extracts prepared using 50% aqueous methanol without drying. 2.4. Bioassays

are high conservation concerns when the roots of the tree are harvested. Hence, there is a need to investigate the properties for the justification of use for different tree parts. In this study, we investigated the variations in the antioxidant activity using the DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical scavenging assay, ferric-reducing power (FRAP) and antidiabetic α-glucosidase inhibitory activity, as well as properties of the leaves and the main and lateral roots of Moringa. Additionally, colourimetric methods were used to investigate the variations of phenolic contents of the leaves and the main and lateral roots.

2. Materials and methods 2.1. General Moringa plants were cultivated at the Agricultural Research Council (ARC) experimental farm in Roodeplaat, Pretoria, South Africa (25°36′1.85″S; 28°21′54.78″E). Vegetation of the area is described as Savanna biome (Mucina and Rutherford, 2006) and as Sourish Mixed Bushveld (Acocks, 1988). Daily mean temperature varies from 11 °C to 27.2 °C with a minimum of 2.3 °C in winter and a maximum of 30 °C in summer. The experimental farm receives an average rainfall of 650 mm per annum, with most precipitation in summer. The farm is situated at an elevation of 1160 m a.s.l., on the Roodeplaat Igneous Complex belonging to the Post-Waterberg Formation (Panagos et al., 1998). Ten moringa plants were randomly selected from a field plot, with a spacing of 1 × 1 m. Plants were treated to routine maintenance such as weeding, cultivation, irrigation three times a week and no fertiliser was applied to the plants. The plants were harvested in December, two

2.4.1. Total phenolics, condensed tannins and flavonoids Following the method described by Makkar (1999), with slight modification by Ndhlala et al. (2007) the total amounts of phenolics were determined using the Folin–Ciocalteu (Folin C.) assay. The standard for this assay was gallic acid. The vanillin-HCl assay was used to determine flavonoids found in the roots and leaves of Moringa, this was done following Hagerman (2002) as slightly modified by Ndhlala et al. (2007). 2.4.2. Antifungal microdilution bioassay Anticandidal activity was determined by minimum inhibitory concentration (MIC), this was tested against the fungus Candida albicans using the microdilution assay (Eloff, 1998) but modified for the antifungal assay (Masoko et al., 2007). An antifungal amphotericin B was used as a positive control in this study. Acetone, ethanol and water were used as negative and solvent controls. 2.4.3. Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity Following the method described by Karioti et al. (2004) with some modifications, diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay was carried out. Methanolic extracts (50% v/v) from the leaves and roots (diluted in absolute methanol) were mixed with DPPH solution (750 μL, 50 μM in methanol) in a reaction mixture, under a dimmed light and incubated for 30 min at room temperature. This was done in a dark room and readings of the absorbance of the mixtures were read at 517 nm using a UV–vis spectrophotometer (Varian Cary 50, Varian Australia Pty. Ltd., Sydney, Australia). Methanol was used as a blank mixture. Ascorbic acid, a known antioxidant was used as a positive control. The experiment was repeated three times. The decolouration of the DPPH

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solution determined the free radical scavenging solution, using the formula: RSA ð%Þ ¼

   Abs517 nm Sample  100 1− Abs517 nm Neg Control

where: RSA is the Free Radical Scavenging Activity. Abs517 sample is the absorbance of the mixture containing the leaf, root extracts or the positive control solution. Abs517 Neg control is the absorbance of the negative control. The EC50 (effective concentration) values, representing the amount of extract required to decrease the absorbance of DPPH by 50% was calculated from the percentage radical scavenging activity. 2.4.4. Antibacterial microdilution assay Antibacterial activity determined by minimum inhibitory concentration (MIC), was tested against Bacillus subtilis, Escherichia coli and Staphylococcus aureus using the microdilution technique in 96-well (Greiner Bio-one GmbH, Frickenhausen, Germany) microtitre plates (Eloff, 1998) for water, acetone and ethanol extracts. A standard antibiotic, neomycin, was used as a positive control. Water, acetone and ethanol were used as the negative and solvent controls. The assay was repeated three times with two replications in each case. 2.4.5. Antidiabetic α-glucosidase inhibitory activity Antidiabetic activity of the M. oleifera leaf and root extracts was done following the method by Tao et al. (2013) as modified by Rengasamy et al. (2013). Firstly, an enzyme solution was made by dissolving α-glucosidase (0.1 Unit/mL) in 0.1 M potassium phosphate buffer (pH 6.8). The phosphate buffer was used as the negative control. The positive control was acarbose dissolved in dimethyl sulphoxide (DMSO). The following equation was used to calculate percentage inhibition: Percentage inhibition ð%Þ ¼

Acontrol −Asample  100 Asample

where: Acontrol is the absorbance of the control. Asample is the absorbance of the sample. We determined the IC50 for each sample, which was the concentration of the inhibitor reduced by half. The experiment was repeated three times each with two replications. 2.4.6. Ferric-reducing antioxidant power (FRAP) assay Ferric-reducing power of the leaf and roots of the Moringa plants was carried out following the method of Lim et al. (2009). Briefly, extracts of the plant parts (50 μL) at 6.25 mg/mL and the positive control butylated hydroxytoluene (BHT) were added to a 96-well microtitre. 40 μL of potassium phosphate buffer (0.2 M, pH 7.2) and 40 μL of potassium ferricyanide (1% in phosphate buffer, w/v) were then added to the well and this was incubated for 20 min at 50 °C. Soon after incubation, 40 μL trichloroacetic acid (10% in phosphate buffer, w/v), 150 μL distilled water and 50 μL FeCl3 (0.1% in phosphate buffer, w/v) were added. A microtiter reader (Opsys MRTM, Dynex Technologies Inc., Palm City, FL, USA) was used to measure the absorbance at 630 nm of the Fe2+ produced from the reduction of the Fe3+/ferricyanide complex. 2.5. Statistical analysis One-way analysis of variance (ANOVA) was carried out on the data to determine the differences in means (± SE) of the phytochemical and antioxidant properties between the leaf and root of Moringa. The post hoc-Duncan's multiple range test was used to separate the bioactivity property means (±SE), with p-values less than 0.05 being statistically significant. All statistical analyses were carried out using the IBM

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Statistical Package for the Social Sciences (SPSS) v 25.0 for Windows (Chicago, IL, USA). 3. Results 3.1. Total phenolics, condensed tannin and flavonoid content The leaf of the Moringa plant had significantly (p ≤ .05) higher total phenolic compounds compared to both the main and lateral roots (Table 1). There were significantly (p ≤ .05) more flavonoids in the lateral roots than in the main roots and leaf (Table 1). Lateral roots of Moringa had the highest amount of condensed tannins of dry mass, which was approximately three times more than the condensed tannins found in the main roots and seven times more (p ≤ .05) than those found in the leaves (Table 1). 3.2. Antimicrobial activity The antibacterial and antifungal MIC values for the Moringa extracts of the leaves, lateral and main roots are presented in Table 2. Generally, the leaf samples exhibited better antibacterial activity compared to the root extracts against B. subtilis, E. coli and Staphylococcus aureus (MIC b 1 mg/mL) (Table 2). All the extracts indicated good activity against S. aureus with no activity difference among the plant parts. The extracts from the main and lateral roots demonstrated weak activity against B. subtilis and E. coli, except for the acetone extracts. All the extracts exhibited weak activity against the fungus C. albicans. 3.3. DPPH radical scavenging activity The EC50 values for the DPPH radical scavenging potential of the leaf, main and lateral roots of Moringa are shown in Table 3. The EC50 value of Ascorbic acid (Vitamin C) which was used as the positive control was 69.28 μg/mL. Ideally, the lower the EC50 value the greater the antioxidant potency of the tested extract, therefore the plant extracts with EC50 values less than or equal to the reference represented high antioxidant potential. In this study, all three plant parts (leaves, main and lateral roots) had a DPPH radical scavenging ability better than the reference compound (ascorbic acid, Vitamin C). The main and lateral roots had the highest activity of antioxidants (Table 3). The DPPH radical scavenging ability from the Moringa leaves was three times more than the reference compound, while the roots were 70 times more (Table 3). 3.4. Ferric-reducing power assay activity The ability of different concentrations of extracts found in Moringa leaves and roots in reducing Fe3+ complex to Fe2+, is shown in Fig. 2. As the concentration of all extracts from leaves and roots increased, the reducing power also increased. The antioxidants in the leaf extracts

Table 1 Total phenolic compounds, condensed tannins and flavonoids in the leaves, main and lateral roots of Moringa oleifera. Flavonoid and total phenolics values expressed as gallic acid equivalent (GAE), condensed tannins in leucocyanidin equivalent (LE) and catechin equivalents (CAT) per gram of dry weight (DW), respectively. Plant part

Leaves Main roots Lateral roots

Phytochemical Total phenolics (mg GAE/g DW)

Condensed tannins (mg LE/g DW)

Flavonoids (mg CAT/g DW)

0.97 ± 0.05a 0.11 ± 0.01b 0.19 ± 0.01b

0.06 ± 0.00a 0.15 ± 000b 0.44 ± 0.01c

0.09 ± 0.00a 0.16 ± 0.07a 0.36 ± 0.06b

Mean values (±SE) in the same column with different letters are significantly different (p ≤ .05; n = 3) due to Duncan's Multiple Range test.

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Table 2 Antimicrobial activities (Minimum Inhibitory Concentration – MIC mg/mL) of Moringa oleifera leaves and roots (main and lateral) extract on various micro-organisms as determined by the microdilution method. Micro-organism (MIC mg/mL)

Main roots

Lateral roots

Neomycin Amphotericin B

Solvent Acetone Ethanol Water Acetone Ethanol Water Acetone Ethanol Water

Bs 0.78 0.78 1.56 0.78 1.56 1.56 0.78 1.56 1.56 1.6 × 10−3

Ec 0.78 0.78 0.78 0.78 1.56 3.12 1.56 1.56 3.12 0.8 × 10−3

Sa 0.78 0.39 0.78 0.78 0.39 0.78 0.78 0.78 0.78 1.6 × 10−3

Ca 3.13 3.13 3.13 6.25 3.13 6.25 6.25 6.25 6.25

had better reducing strength than those in main and lateral root extracts, respectively. 3.5. The antidiabetic activity The leaf extracts and the control (acarbose) had significantly higher (p ≤ .05) antidiabetic activity compared to both the main and lateral roots (Table 3). The leaves had significantly similar antidiabetic activity compared to the reference/positive control. In this study, this was considered to be good activity. There was more evidence of antidiabetic activity detected in the leaf extracts compared to the main and lateral root extracts (Fig. 3). However, the leaves and both root plant extracts performed below the acarbose which served as the control. 4. Discussion 4.1. Total phenolics, condensed tannin and flavonoid content Plants produce phenolics to act as a defence mechanism against herbivory by animals or micro-organisms (Bhattacharya et al., 2010). The results of this study corroborate with other studies, confirming that there are more total phenols in Moringa leaves than in the roots (Idowu and Oseni, 2015). Most of the Moringa nutrients are found in leaves (Leone et al., 2015a) and this entails that leaves are the most attractive for herbivory, hence a need for more defence mechanisms being located in the leaves. Phenolics are ubiquitous plant compounds having at least one benzene ring (C6) and with one or more hydroxyl groups (Bhattacharya et al., 2010). The phenolic hydroxyl groups are great reducing agents as they take part in a termination reaction which involves the hydrogen-donating antioxidants reacting with reactive oxygen and reactive nitrogen species, thereby reducing the cumulative generation of reactive oxygen species (Valentão et al., 2002). Congruent to this study, a somewhat similar flavonoid concentrations were reported by Ndhlala et al. (2014). The literature on Table 3 Antioxidant activity determined by the DPPH scavenging assay and antidiabetic activity as determined by the α-glucosidase inhibitory activity assay of extracts from the leaf, main and lateral root extracts of Moringa oleifera. Ascorbic acid (Vitamin C) was used as positive control for the DPPH assay while acarbose was the control for the antidiabetic assay. Mean values (±SE) in the same column with different letters are significantly different (p ≤ .05; n = 3) due to Duncan's Multiple Range test.

Leaves Main roots Lateral roots Ascorbic acid

0.15 0.10 0.05 0.00 0

1 2 3 Concentration (mg/ml)

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Fig. 2. Ferric reducing activity of the leaves, main and lateral root extracts of Moringa oleifera. BHT- butylated hydroxytoluene was used as positive control.

9.77 × 10−3

Bs- Bacillus subtilis; Ec- Escherichia coli; Sa- Staphylococcus aureus; Ca- Candida albicans. Values in bold are considered to be active (MIC b 1 mg/mL).

Plant part

Leaves Main Root Lateral Root BHT

0.20

DPPH activity (EC50) μg/ml a

20.54 ± 1.99 0.057 ± 0.03b 1.23 ± 0.02b 69.23 ± 1.14c

Antidiabetic activity (EC50) μg/ml a

40.7 ± 5.2 870.1 ± 12.0b 560.7 ± 7.4b 22.5 ± 0.3a

phytochemical analysis corroborates our findings showing that there is a better total flavonoid content in the roots than in leaves (Etejere et al., 2015; Idowu and Oseni, 2015; Vyas et al., 2015). Flavonoids are polyphenolic compounds consisting of a group of benzo-γ-pyrone structures and occur in almost every plant part, and they are responsible for antimicrobial, anticancer and antiaging activities (Kumar and Pandey, 2013). The flavonoids found in Moringa include myricetin, quercetin, kaempferol, isorhamnetin and rutin (Leone et al., 2015b). Saini et al. (2016) and Siddhuraju and Becker (2003) reports that the flavonoids, quercetin and kaempferol are predominantly found in the leaves and not in the roots. These flavonoids have potent antioxidant properties (Saini et al., 2016). Additionally, flavonoids like quercetin have been attributed as antioxidants that bring about a scavenging effect on ROS released from mitochondria, thereby protecting the beta cells and in turn keeping ailments such as hyperglycaemia controlled (Michalak, 2006; Al-Malki and El Rabey, 2015). The flavonoids found in the leaves in our study explain the better performance of the antioxidant activity from leaves compared to roots (Fig. 2). The variation in literature on the nutritional and bioactive compounds found in Moringa (Shih et al., 2011; Ndhlala et al., 2014; Leone et al., 2015a; Leone et al., 2015b) can be mainly attributed to the environmental factors including soil conditions or fertiliser treatments (Ncube et al., 2012; Dania et al., 2014), plant genetics and different extraction methods (Leone et al., 2015b), that affect the compound concentrations at any given time. The findings in our study do support the notion of knowing the nutritional and phenolic characteristics of Moringa cultivated locally, prior to them being incorporated into nutritional and pharmacological products. Condensed tannins, also known as proanthocyanidins, are defined as polymers of flavan-3-ols, classified into procyanidins and prodelphinidins which have two hydroxyl OH-groups and three OHgroups in the B-ring, respectively (Francisco et al., 2014). Hagerman et al. (1998) report that an increase in the number of condensed tannins results in higher antioxidant ability, because of the increase in hydroxyl groups taking part in the reduction of peroxyl radicals. The high DDPH scavenging activity of the roots reported in the current study (Table 3) 100 Antidiabetic activity (%)

Plant part Leaves

0.25 Ab 630 nm Fe2+

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Leaves Lateral root Main Root Acarbose

80 60 40 20 0 0.0

0.5 1.0 Sample concentration (mg/ml)

1.5

Fig. 3. Antidiabetic activity of the leaves, main and lateral roots of Moringa oleifera. Acarbose was used as a positive control.

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can be explained by the high content of condensed tannins in the Moringa roots. 4.2. Antimicrobial activity Staphylococcus aureus, a Gram-positive bacterium normally found in the nasopharynx and the skin of humans, is known to cause skin infections, boils, abscesses (Singer and Talan, 2014) and lung infections leading to pneumonia (Nair and Niederman, 2011). Due to the incorrect use of antibiotics, the resistance of the bacteria has been reported to be on the increase, leading to a more resistant strain called methicillin-resistant Staphylococcus aureus (MRSA). The good activity shown by extracts from Moringa in this study may offer potential solutions against the MRSA (Liu et al., 2011). We recommend future studies should explore the antimicrobial potential of Moringa against MRSA. Candida albicans is normally a non-harmful fungus found on the skin and mucous membranes of the mouth, intestines and the vagina. The pathogen becomes harmful when the optimum conditions of the body change, which can be caused by overuse of antibiotics (Samonis et al., 1994), taking a diet high in carbohydrates and in immunocompromised individuals (Silva, 2010). Infections caused by C. albicans include chronic mucocutaneous candidiasis, gastrointestinal tract candidiasis, oesophageal candidiasis, respiratory tract candidiasis and vulvovaginal candidiasis. In the current study, none of the tested Moringa extracts showed any noteworthy activity against C. albicans. However, extracts from Moringa seeds have been reported to lower C. albicans activity (Rocha et al., 2014). 4.3. Antioxidant activity Free radicals are reactive molecular species that contain an unpaired electron, making them take part in redox reactions (Cheeseman and Slater, 1993). Free radicals are either internally generated in the human body, from mitochondria, peroxisomes and through phagocytosis or can be exogenously derived from X-rays, cigarette smoking and air pollutants (Bagchi and Puri, 1998). The role of antioxidant compounds is to combat the free radical mediated oxidation of other molecules, which lead to various degenerative disorders such as mutagenesis, type 2 diabetes, carcinogenesis and ageing (Singh and Singh, 2008). The 2, 2-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging activity assay represents the potential of antioxidant activity against free radicals in the human body. DPPH is a stable free radical which can change to a stable diamagnetic molecule when it accepts an electron (Bijaya and Bikash, 2013). The EC50 value of the DPPH radical scavenging activity assay represents the concentration of a test sample needed to decrease the initial concentration of DPPH by 50% (Atoui et al., 2005). The higher DPPH radical scavenging ability observed in the roots could be attributed to the condensed tannins found in the roots (Table 1) which contain hydroxyl groups (OH) which take part in antioxidant reactions (Hagerman et al., 1998). Vitamin C, which was used as a reference compound in this study, is an essential nutrient needed by humans for muscle tissue formation, to protect cellular damage, boost the immune system and is a potent water-soluble antioxidant (Padayatty et al., 2003). Studies have shown that Moringa can also provide seven times more Vitamin C than in orange fruits (Rockwood et al., 2013). Antioxidants occur naturally in foods and this study suggests that Moringa is potentially a better source of antioxidants, which can be incorporated into diet plans, skin products and anti-ageing products. The ferric-reducing antioxidant power (FRAP) assay activity measures the ability of the antioxidants (reductants) to reduce the Fe3 + complex of tripyridyltriazine (Fe(TPTZ)3+) to ferrous form Fe2+ complex (Fe(TPTZ)2+). This action by the antioxidants is characterised by higher absorbance values at λ 630 nm after the donation of electrons (Steenkamp et al., 2006). In the human body, this assay can be translated to the reduction of reactive oxygen species (ROS) by antioxidants to more stable products (Prior et al., 2005). ROS are bi-products of

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mitochondrial metabolism and energy production, they are radicals, ions or molecules that have an unpaired electron in their outermost shell of electrons, hence very reactive (Liou and Storz, 2010). These include superoxide anion (O2−), hydrogen peroxide (H2O2), and hydroxyl radical (HO•) (Ray et al., 2012). Failure of the antioxidants to deal with ROS culminates to oxidative stress which results in damage of DNA, protein, lipids (Jaiswal et al., 2013), and may lead to carcinogenesis (Liou and Storz, 2010), diabetes (Fakhruddin et al., 2017), hyperlipidaemia (Sangkitikomol et al., 2014), and ageing (Cui et al., 2012). The high level of phenolic compounds found in the Moringa leaves (Table 1) could be responsible for the reduction reactions as they contain free hydroxyl compounds which combat oxidation activity (Valentão et al., 2002). In the current study, the antioxidant activity in the leaves and roots was lower than that of the reference compound butylated hydroxytoluene (BHT) (Fig. 2). However, the antioxidant activity in Moringa has been reported to reduce oxidative stress and prevent degenerative disease such as diabetes through regular intake of leaves (Sangkitikomol et al., 2014). 4.4. Antidiabetic activity Diabetes mellitus is a common chronic disease, with an estimated 422 million people globally living with the disease (World Health Organization, 2016). The disease occurs either when the pancreas does not produce sufficient insulin (Type 1 diabetes) or when the body cannot efficiently use the insulin (Type 2 diabetes). Type 2 diabetes accounts for about 90% of the diabetes cases globally (World Health Organization, 2018). Type 2 diabetes is caused by a decreased insulin secretion resulting in increased blood glucose which takes part in glycolysis in the mitochondria of beta cells which then form reactive oxygen species (ROS) (Cerf, 2013). ROS results in oxidative stress, which leads to depletion of antioxidants in the body and promotes the generation of free radicals which cause apoptosis of pancreatic beta-cells and hyperglycaemia (Pi et al., 2010; Gandhi et al., 2012). Managing diabetes may involve inhibiting α-amylase and α-glucosidase enzymes which are responsible for the conversion of dietary starch to glucose (Casirola and Ferraris, 2006). Natural products from plants are preferred in the regulation of glucose levels, because of less complications arising from side effects (Ivorra et al., 1989).The results of this study corroborates with findings from other studies where the levels of phenolic compounds have been reported to be positively correlated with α-glucosidase (Molan and Mahdy, 2016). Although phenolic compounds form part of a suite of phytochemical compounds in a plant extract, it is tempting to speculate that the high amount of total phenolics recorded in the leaves in this study (Table 1), could, to some extent, account for higher antidiabetic and antioxidant activities of the leaf extract than those of the roots. Phenolic compounds in Moringa contribute to the antioxidant properties and these can combine with the ROS and prevent apoptosis of cells (Jaiswal et al., 2013; Al-Malki and El Rabey, 2015). 5. Conclusions A comparative antioxidant, antimicrobial, antidiabetic and phytochemical analysis of leaves, main and lateral roots of Moringa was carried out. There were significant (p ≤ .05) variations in the observed medicinal properties amongst the different studied Moringa plant parts (leaves, main and lateral roots). All the plant parts had better antioxidant activity than the reference compound ascorbic acid. The roots can be used as a better source of antioxidants than the leaves. Leaf extracts had significantly good antidiabetic activity when compared to the reference compound (acarbose). The leaf extracts exhibited the highest amount of total phenolics, while the lateral roots had higher amounts of condensed tannins and flavonoid contents. The different plant parts of Moringa can be used as a natural source to fight against infectious diseases caused by various microorganisms, which are rapidly

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getting resistant to available drugs. However, the leaves had better antimicrobial activity than the roots. Overall, the analysis of Moringa various medicinal properties was done using wet chemistry methods. However, these methods are destructive, thus destroying the plants to analyse the chemical constituents, this is associated with high cost and consume a lot of time in ascertaining the medicinal properties of the plants. Alternative and complementary quick methods like the use of spectroscopy and hyperspectral remote sensing tools may need to be considered. For example, Bian et al. (2013) used spectroscopic data at the laboratory level to quantify the number of phenols in tea (Camellia sinensis) to determine its quality. There is a need for further research to see if this can be extended to some of the medicinal properties in plants such as Moringa. Additionally, there is a need to undertake studies in vivo to validate the in vitro findings.

Conflicts of interest There was no conflict of interest amongst the authors.

Acknowledgements This study was supported by the Department of Science and Technology, South Africa. References Acocks, J.P.H., 1988. Veld Types of South Africa. Department of Agriculture Technical Services, Pretoria, South Africa. Al-Malki, A.L., El Rabey, H.A., 2015. The antidiabetic effect of low doses of Moringa oleifera Lam. seeds on streptozotocin induced diabetes and diabetic nephropathy in male rats. BioMed Research International 1–13. Anwar, F., Latif, S., Ashraf, M., Gilani, A.H., 2007. Moringa oleifera: a food plant with multiple medicinal uses. Phytotherapy Research 21, 17–25. Ashfaq, M., Basra, S.M.A., Ashfaq, U., 2012. Moringa: a miracle plant of agroforestry. Journal of Agriculture and Social Sciences 8, 115–122. Atoui, A.K., Mansouri, A., Boskou, G., Kefalas, P., 2005. Tea and herbal infusions: their antioxidant activity and phenolic profile. Food Chemistry 89, 27–36. Bagchi, K., Puri, S., 1998. Free radicals and antioxidants in health and disease. Eastern Mediterranean Health Journal 4, 350–360. Bhattacharya, A., Sood, P., Citovsky, V., 2010. The roles of plant phenolics in defence and communication during Agrobacterium and Rhizobium infection. Molecular Plant Pathology 11, 705–719. Bian, M., Skidmore, A.K., Schlerf, M., Wang, T., Liu, Y., Zeng, R., Fei, T., 2013. Predicting foliar biochemistry of tea (Camellia sinensis) using reflectance spectra measured at powder, leaf and canopy levels. ISPRS Journal of Photogrammetry and Remote Sensing 78, 148–156. Bijaya, L.M., Bikash, B., 2013. Antioxidant capacity and phenolics content of some Nepalese medicinal plants. American Journal of Plant Sciences 4, 1660–1665. Casirola, D.M., Ferraris, R.P., 2006. Alpha-glucosidase inhibitors prevent diet-induced increases in intestinal sugar transport in diabetic mice. Metabolism 55, 832–841. Cerf, M.E., 2013. Beta cell dysfunction and insulin resistance. Frontiers in Endocrinology 4, 37. Cheeseman, K.H., Slater, T.F., 1993. An introduction to free radical biochemistry. British Medical Bulletin 49, 481–493. Choudhary, M.K., Bodakhe, S.H., Gupta, S.K., 2013. Assessment of the antiulcer potential of Moringa oleifera root-bark extract in rats. Journal of Acupuncture and Meridian Studies 6, 214–220. Cui, H., Kong, Y., Zhang, H., 2012. Oxidative stress, mitochondrial dysfunction, and aging. Journal of Signal Transduction 2012, 13. Dalukdeniya, D.A.C.K., De Silva, K.L.S.R., Rathnayaka, R.M.U.S.K., 2016. Antimicrobial activity of different extracts of leaves bark and roots of Moringa oleifera (Lam). International Journal of Current Microbiology and Applied Sciences 5, 687–691. Dania, S.O., Akpansubi, P., Eghagara, O.O., 2014. Comparative effects of different fertilizer sources on the growth and nutrient content of moringa (Moringa oleifera) seedling in a greenhouse trial. Advances in Agriculture 6 (6 pages). Eloff, J.N., 1998. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Medica 64, 711–713. Etejere, E.O., Olayinka, B.U., Lawal, L.O., 2015. Comparative studies of phytochemical constituents of leaf, bark and root of Moringa oleifera Lam. Jewel Journal of Scientific Research 3, 173–176. Fakhruddin, S., Alanazi, W., Jackson, K.E., 2017. Diabetes-induced reactive oxygen species: mechanism of their generation and role in renal injury. Journal of Diabetes Research 2017, 30.

Francisco, V., Costa, G., Neves, B.M., Cruz, M.T., Batista, M.T., 2014. Chapter 28 - Anti-inflammatory activity of polyphenols on dendritic cells. Polyphenols in Human Health and Disease. Academic Press, San Diego, pp. 373–392. Gandhi, R.G., Ignacimuthu, S., Paulraj, M.G., 2012. Hypoglycemic and β-cells regenerative effects of Aegle marmelos (L.) Corr. bark extract in streptozotocin-induced diabetic rats. Food and Chemical Toxicology 50, 1667–1674. Hagerman, A.E., 2002. Tannin Chemistry. Miami University Press, Miami, FL, USA. Hagerman, A.E., Riedl, K.M., Jones, G.A., Sovik, K.N., Ritchard, N.T., Hartzfeld, P.W., Riechel, T.L., 1998. High molecular weight plant polyphenolics (tannins) as biological antioxidants. Journal of Agricultural and Food Chemistry 46, 1887–1892. Idowu, K., Oseni, O.A., 2015. Compositional investigation of phytochemical and antioxidant properties of various parts of Moringa oleifera plant. European Journal of Basic and Applied Sciences 2, 1–10. Ivorra, M.D., Payá, M., Villar, A., 1989. A review of natural products and plants as potential antidiabetic drugs. Journal of Ethnopharmacology 27, 243–275. Jaiswal, D., Rai, P.K., Mehta, S., Chatterji, S., Shukla, S., Rai, D.K., Sharma, G., Sharma, B., khair, S., Watal, G., 2013. Role of Moringa oleifera in regulation of diabetes-induced oxidative stress. Asian Pacific Journal of Tropical Medicine 6, 426–432. Karioti, A., Hadjipavlou-Litina, D., Mensah, M.L.K., Fleischer, T.C., Saltsa, H., 2004. Composition and antioxidant activity of the essential oils of Xylopia aethiopica (Dun) A. Rich. (Annonaceae) leaves, stem bark, root bark, and fresh and dried fruits, growing in Ghana. Journal of Agricultural and Food Chemistry 52, 8094–8098. Kumar, S., Gopal, K., 1999. Screening of plant species for inhibition of bacterial population of raw water. Journal of Environmental Science and Health. Part A, Toxic/Hazardous Substances & Environmental Engineering 34, 975–987. Kumar, S., Pandey, A.K., 2013. Chemistry and biological activities of flavonoids: an overview. Scientific World Journal 2013, 162750. Leone, A., Fiorillo, G., Criscuoli, F., Ravasenghi, S., Santagostini, L., Fico, G., Spadafranca, A., Battezzati, A., Schiraldi, A., Pozzi, F., di Lello, S., Filippini, S., Bertoli, S., 2015a. Nutritional characterization and phenolic profiling of Moringa oleifera leaves grown in Chad, Sahrawi Refugee Camps, and Haiti. International Journal of Molecular Sciences 16, 18923. Leone, A., Spada, A., Battezzati, A., Schiraldi, A., Aristil, J., Bertoli, S., 2015b. Cultivation, genetic, ethnopharmacology, phytochemistry and pharmacology of Moringa oleifera leaves: an overview. International Journal of Molecular Sciences 16, 12791–12835. Lim, T.Y., Lim, Y.Y., Yule, C.M., 2009. Evaluation of antioxidant, antibacterial and anti-tyrosinase activities of four Macaranga species. Food Chemistry 114, 594–599. Liou, G.-Y., Storz, P., 2010. Reactive oxygen species in cancer. Free Radical Research 44. Liu, C., Bayer, A., Cosgrove, S.E., Daum, R.S., Fridkin, S.K., Gorwitz, R.J., Kaplan, S.L., Karchmer, A.W., Levine, D.P., Murray, B.E.e.a., 2011. Clinical practice guidelines by the infectious diseases society of america for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children: executive summary. Clinical Infectious Diseases 52, 285–292. Makkar, H.P.S., 1999. Quantification of Tannins in Tree Foliage: A Laboratory Manual for the FAO/IAEA Co-Ordinated Research Project on Use of Nuclear and Related Techniques to Develop Simple Tannin Assay for Predicting and Improving the Safety and Efficiency of Feeding Ruminants on the Tanniniferous Tree Foliage. Joint FAO/ IAEA Division of Nuclear Techniques in Food and Agriculture, Vienna, Austria. Masoko, P., Picard, J., Eloff, J.N., 2007. The antifungal activity of twenty-four southern Africa Combretum species (Combretaceae). South African Journal of Botany 73, 173–183. Michalak, A., 2006. Phenolic compounds and their antioxidant activity in plants growing under heavy metal stress. Polish Journal of Environmental Studies 15, 523–530. Molan, A.-L., Mahdy, A.S., 2016. Total phenolics, antioxidant activity and anti-diabetic capacities of selected Iraqi medicinal plants. American Journal of Life Science Researches 4, 47–59. Mucina, L., Rutherford, M.C., 2006. The Vegetation of South Africa, Lesotho and Swaziland. South African National Biodiversity Institute, Pretoria, South Africa. Nair, G.B., Niederman, M.S., 2011. Community-acquired pneumonia: an unfinished battle. Medical Clinics of North America 95, 1143–1161. Ncube, B., Finnie, J.F., Van Staden, J., 2012. Quality from the field: the impact of environmental factors as quality determinants in medicinal plants. South African Journal of Botany 82, 11–20. Ndhlala, A.R., Kasiyamhuru, A., Mupure, C., Chitindingu, K., Benhura, M.A., Muchuweti, M., 2007. Phenolic composition of Flacourtia indica, Opuntia megacantha and Sclerocarya birrea. Food Chemistry 103, 82–87. Ndhlala, A., Mulaudzi, R., Ncube, B., Abdelgadir, H., du Plooy, C., Van Staden, J., 2014. Antioxidant, antimicrobial and phytochemical variations in thirteen Moringa oleifera Lam. cultivars. Molecules 19, 10480. Padayatty, S.J., Katz, A., Wang, Y., Eck, P., Kwon, O., Lee, J.H., Chen, S., Corpe, C., Dutta, A., Dutta, S.K., Levine, M., 2003. Vitamin C as an antioxidant: evaluation of its role in disease prevention. Journal of the American College of Nutrition 22, 18–35. Panagos, M.D., Westfall, R.H., van Staden, J.M., Zacharias, P.J.K., 1998. The plant communities of the Roodeplaat Experimental Farm, Gauteng, South Africa and the importance of classification verification. South African Journal of Botany 64, 44–61. Pi, J., Zhang, Q., Fu, J., Woods, C.G., Hou, Y., Corkey, B.E., Collins, S., Andersen, M.E., 2010. ROS signaling, oxidative stress and Nrf2 in pancreatic beta-cell function. Toxicology and Applied Pharmacology 244, 77–83. Popoola, J.O., Obembe, O.O., 2013. Local knowledge, use pattern and geographical distribution of Moringa oleifera Lam. (Moringaceae) in Nigeria. Journal of Ethnopharmacology 150, 682–691. Prior, R.L., Wu, X., Schaich, K., 2005. Standardized methods for the determination of antioxidant capacity and phenolics in food and dietary supplements. Journal of Agricultural and Food Chemistry 53, 4290–4302.

Please cite this article as: T. Tshabalala, A.R. Ndhlala, B. Ncube, et al., Potential substitution of the root with the leaf in the use of Moringa oleifera for antimicrobial, antidiabetic and antioxidant properties..., South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.01.029

T. Tshabalala et al. / South African Journal of Botany xxx (2019) xxx Raj, A.J., Gopalakrishnan, V.K., Yadav, S.A., Doraira, S., 2011. Antimicrobial activity of Moringa oleifera (Lam.) root extract. Journal of Pharmacy Research 4, 1426–1427. Ray, P.D., Huang, B.-W., Tsuji, Y., 2012. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cellular Signalling 24, 981–990. Rengasamy, K.R.R., Aderogba, M.A., Amoo, S.O., Stirk, W.A., Van Staden, J., 2013. Potential antiradical and alpha-glucosidase inhibitors from Ecklonia maxima (Osbeck) Papenfuss. Food Chemistry 141, 1412–1415. Rocha, M.F.G., Alencar, L.P.d., Brilhante, R.S.N., Sales, J.d.A., Ponte, Y.B.d., Rodrigues, P.H.d. A., Sampaio, C.M.d.S., Cordeiro, R.d.A., Castelo-Branco, D.d.S.C.M., Oliveira, F.C.d., Barbosa, F.G., Teixeira, C.E.C., Paiva, M.d.A.N., Bandeira, T.d.J.P.G., Moreira, J.L.B., Sidrim, J.J.C., 2014. Moringa oleifera inhibits growth of Candida spp. and Hortaea werneckii isolated from Macrobrachium amazonicum prawn farming with a wide margin of safety. Ciencia Rural 44, 2197–2203. Rockwood, J.L., Anderson, B.G., Casamatta, D.A., 2013. Potential uses of Moringa oleifera and an examination of antibiotic efficacy conferred by M. oleifera seed and leaf extracts using crude extraction techniques available to underserved indigenous populations. International Journal of Phytotherapy Research 3, 61–71. Saini, R.K., Sivanesan, I., Keum, Y.-S., 2016. Phytochemicals of Moringa oleifera: a review of their nutritional, therapeutic and industrial significance. 3 Biotech 6, 203. Samonis, G., Anastassiadou, H., Dassiou, M., Tselentis, Y., Bodey, G.P., 1994. Effects of broad-spectrum antibiotics on colonization of gastrointestinal tracts of mice by Candida albicans. Antimicrobial Agents Chemotherapy 38, 602–603. Sangkitikomol, W., Rocejanasaroj, A., Tencomnao, T., 2014. Effect of Moringa oleifera on advanced glycation end-product formation and lipid metabolism gene expression in HepG2 cells. Genetics and Molecular Research 13, 723–735. Shih, M.-C., Chang, C.-M., Kang, S.-M., Tsai, M.-L., 2011. Effect of different parts (leaf, stem and stalk) and seasons (summer and winter) on the chemical compositions and antioxidant activity of Moringa oleifera. International Journal of Molecular Sciences 12, 6077–6088.

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Siddhuraju, P., Becker, K., 2003. Antioxidant properties of various solvent extracts of total phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa oleifera Lam.) leaves. Journal of Agricultural and Food Chemistry 51, 2144–2155. Silva, R.F., 2010. Chapter 8: Fungal infections in immunocompromised patients. Jornal Brasilero de Pneumologia 36, 142–147. Singer, A.J., Talan, D.A., 2014. Management of skin abscesses in the era of methicillin-resistant Staphylococcus aureus. New England Journal of Medicine 370, 1039–1047. Singh, S., Singh, R.P., 2008. In vitro methods of assay of antioxidants: an overview. Food Reviews International 24, 392–415. Steenkamp, P.A., Harding, N.M., van Heerden, F.R., van Wyk, B.E., 2006. Identification of atractyloside by LC–ESI–MS in alleged herbal poisonings. Forensic Science International 163, 81–92. Tao, Y., Zhang, Y., Cheng, Y., Wang, Y., 2013. Rapid screening and identification of α-glucosidase inhibitors from mulberry leaves using enzyme-immobilized magnetic beads coupled with HPLC/MS and NMR. Biomedical Chromatography 27, 148–155. Valentão, P., Fernandes, E., Carvalho, F., Andrade, P.B., Seabra, R.M., Bastos, M.L., 2002. Studies on the antioxidant activity of Lippia citriodora infusion: scavenging effect on superoxide radical, hydroxyl radical and hypochlorous acid. Biological and Pharmaceutical Bulletin 25, 1324–1327. Vyas, S., Kachhwaha, S., Kothari, S.L., 2015. Comparative analysis of phenolic contents and total antioxidant capacity of Moringa oleifera Lam. Pharmacognosy Journal 7, 44–51. World Health Organization, 2016. Global Reports on Diabetes. World Health Organization, 2018. Global Health Estimates (GHE), 2000–2015 Estimates. http://www.who.int/healthinfo/global_burden_disease/estimates/en/.

Please cite this article as: T. Tshabalala, A.R. Ndhlala, B. Ncube, et al., Potential substitution of the root with the leaf in the use of Moringa oleifera for antimicrobial, antidiabetic and antioxidant properties..., South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.01.029