1. INTRODUCTION:

Recently, the demand for olive leaf extract has increased for use in foodstuffs, Food Additives, and functional food materials¹, hence, important scientific and technical efforts have been made to determine the value of this agricultural waste². This demonstrates the great economic of this crop and the possible benefit to be derived from the utilization of any of its byproducts.

Olea europaea L. (Oleaceae) is considered one of the most important fruit In Mediterranean, areas there are approximately eight million hectares of cultivated olive trees (98% of the world crop). The Olive leaves are considered one of the byproducts of farming of the olive grove which they accumulate during pruning of the olive trees^3,4,5. Therefore, in the scientific community, the Olea europaea L. leaves have attracted growing interest. Recently, The scientific researchers paid attention of olive leaves for using as foodstuff, food additives and function food¹, According to the literature, olive and banana leaves is the most active in inhibiting polymerization of human chromosomes, hence the highest potential for inhibition of cancer cell formation^6,7^. The olive leaves restraining cell propagation in breast cancer cell lines (MCF-7, SKBR-3, JIMT-1)⁸, and human liver cancer cells (HepG2)⁹. A banana plant (Musa acuminate), is vastly being consumed across the world and used is known for many pharmacological activities. Recent studies have highlighted that olive leaves (OL) and banana leaves (BL) contain a large number of polyphenols which have several beneficial effects on health: anti-hypertensive¹⁰, anti-diabetic¹¹, anti-carcinogenic¹², anti-atherosclerotic, anti-inflammatory, antimicrobial, and antifungal activities¹³. The potential activity as antimicrobial action may be related to the compounds that can be used to inhibit the growth of forborne pathogens. Especially polyphenol oxidase which is used in the treatment of Parkinson’s disorder¹⁴

The objective of this study to examine and evaluate in vitro the chemical components of olive leaves and banana leaves in different solvent extracts and their antioxidant, antimicrobial and antitumor activities.

2. MATERIALS AND METHODS:

Chemicals and reagents:

ABTS˙⁺ (2, 2’-azinobis (3-ethylbenzothiazoline- 6-sulfonic acid)), Folin–Ciocalteau reagents, Gallic acid, Quercetin, DPPH˙ ( 2, 2-diphenyl-1-picrylhydrazyl), Ferrozine: (3-(2 - pyridyl)- 5, 6-bis- (4-phenylsulfonic acid)-1, 2, 4-triazine, BHT: Butyl Hydroxytoluene and, potassium ferricyanide, were purchased from Sigma Chemical Co. ( St. Louis, MO, USA).

Plant materials:

Leaves of banana variety Maghrabi were obtained from the local market at Giza, Egypt. And leaves of olive variety kalamata (Olea europea L.) were obtained from the Nubaria farm (National Research Centre, Giza, Egypt).

Preparation of sample:

The banana and olive leaves were washed and air-dried for ten days followed by drying in an oven at 40ºC for three days until the stability of the weight, ground than stored in -20 ºC until extraction and analysis.

Preparation of banana and olive leaves extracts:

The dried samples of banana and olive leaves (10 g) were submersed separately in 100 ml of distilled water, 80% methanol, 80% ethanol, and 80% acetone for 24 h at room temperature using a shaker. Each mixture was filtered through Whatman No. 1 filter paper and the extraction step was repeated three times. The filtrate was then concentrated to dryness at 40 °C in a rotary evaporator. The crude extracts were stored in a refrigerator until analysis.

Proximate analysis:

The moisture, ash, crude protein, total lipid, total carbohydrates, and macro-microelements were determined according to AOAC¹⁵.

Chemical Analysis:

Total phenolic content:

The total phenolic (TP) was determined according to Folin Ciocalteu reagent assay using Gallic acid as standard¹⁶. A suitable aliquot (1ml) of banana and olive leaves extracts were added to 25 ml volumetric flask, containing 9ml of distilled water. One milliliter of Folin Ciocalteu`s phenol reagent was added to the mixture and shaken. After 5 min. 10ml of 7 % Na₂CO₃ solution was added to the mixture. The solution was diluted to 25ml with distilled water and mixed. After incubation for 90 min. at room temperature, the absorbance was determined at 750nm with a spectrophotometer (Unicum UV 300) against prepared reagent as blank. A total phenolic content in samples was expressed as mg Gallic acid equivalents (GAE)/g dry weight. All samples were analyzed in triplicates.

Total flavonoid content:

Total flavonoid (TF) was determined by the aluminum chloride method using quercetin as a standard¹⁷. One ml of banana and olive leaves extracts were added to 10 ml volumetric flask, containing 4ml of distilled water. To the flask 0.3ml 5% NaNO₂ was added and after 5 min 0.3ml 10% AlCl₃ was added. After a 6^th min, 2ml 1M NaOH was added and the total volume was made up to 10ml with distilled water. The solutions were mixed well and the absorbance was measured against prepared reagent blank at 510nm by using a spectrophotometer (Unicum UV 300). Total flavonoids in the sample were expressed as mg quercetin equivalents (QE)/g dry weight. Samples were analyzed in triplicates.

Total tannins content:

Total tannin (TT) was measured using the Folin-Ciocalteu reagent¹⁸. One ml of banana and olive leaves extracts were added to 7.5ml distilled water (dH₂O) then add 0.5ml of Folin reagent and 1ml of 35% sodium carbonate solution. The volume was made up for 10ml with distilled water and absorbance was measured against prepared reagent blank at 775nm by using a spectrophotometer (Unicom UV 300). Total tannins in the sample were expressed as mg tannic acid equivalent (TE)/g dry weight. All samples were analyzed in triplicates.

Identification of phenolic compounds by HPLC:

Phenolic compounds in banana and olive leave acetone extracts were identified using HPLC¹⁹. All chemicals and solvents used were HPLC spectral grade and obtained from Sigma (St. Louis, USA (and Merck –Shcuchrdt Munich, Germany). The HPLC system is Agilent 1100 series coupled with a DAD detector (G1315B) and (G1322A) DEGASSER. Sample injections of 5μl were made from an Agilent 1100 series auto-sampler. The chromatographic separations were performed on the ZORBAX-EclipseXDB-C18 column (4.6×250 mm, particle size 5 μm). A constant flow rate of 1ml/min was used with mobile phases: (A) 0.5% acetic acid in distilled water at pH 2.65; and solvent (B) 0.5% acetic acid in 99.5% acetonitrile. The elution gradient was linear starting with A and ending with B over 50 min, using a DAD detector set at wavelength 280nm. Phenolic compounds of banana and olive leave acetone extracts were identified by comparing their retention times with those of the standard mixture chromatogram. The concentration of an individual compound was calculated on the basis of peak area measurements and the results expressed as mg phenolic/100g dry weight.

Antioxidant Activity:

DPPH· Free radical scavenging activity:

Determination of DPPH^∙ (2, 2-diphenyl-1-picrylhydrazyl) free radical scavenging activity was measured spectrophotometrically²⁰. 0.1mM of DPPH^•in methyl alcohol was prepared and 0.5ml of this solution was added to 1ml of banana leaves and olive leaves extracts at different concentrations (25, 50, 75, 100 µg/ml). Methanol was used as a blank. The mixture was shaken vigorously and allowed to stand at room temperature (for 30 min.). Butyl Hydroxytoluene (BHT, Sigma) was used as a positive control; and negative control contained the entire reaction reagent except for the extracts. Then the absorbance was measured at 515 nm against a blank. The capacity to scavenge the DPPH^∙radical was calculated using the following equation:

DPPH^∙scavenging activity % = [(A_c – A_s (/ A_c] × 100

Where: (A_c) was the absorbance of the negative control reaction and (A_s) the absorbance in the presence of the plant extracts. The results were expressed as IC₅₀ (the concentration µg/ml of banana and olive leaves extracts that scavenge 50 % of DPPH^∙radical).

ABTS˙⁺ scavenging activity:

ABTS˙⁺ assay was generated by oxidation of ABTS˙⁺ with potassium persulphate²¹. ABTS˙⁺ was dissolved in deionized water to 7.4mM concentration, and potassium persulphate added to a concentration of 2.6mM. The working solution was then prepared by mixing the two stock solutions in equal quantities and allowing them to react for 12-16 h at room temperature in the dark. The solution was then diluted by mixing 1ml ABTS^•+solution with 60ml methanol to obtain an absorbance of 1.1 ± 0.02 at 73nm using the spectrophotometer. Fresh ABTS˙⁺solution was prepared for each assay. Banana leaves and olive leaves extracts (150µl) at different concentration (25, 50, 75, 100µg/ml) were allowed to react with 2850µl of the ABTS˙⁺solution for 2 h in the dark. Then the absorbance was taken at 734nm using the spectrophotometer. Results were expressed as in comparison with standard BHT. The activity to scavenge the ABTS˙⁺radical was calculated using the following equation:

ABTS˙⁺scavenging activity % = [(A₀ – A₁) /A₀] ×100

Where: A₀ is the ABTS˙⁺ absorbance of the control reaction and A₁ is the ABTS˙⁺ absorbance in the presence of the sample. The results were expressed as IC₅₀ (the concentration µg/ml of banana and olive leaves extracts that scavenge 50% of ABTS˙⁺radical).

Reducing Power:

The reducing power was assayed spectrophotometrically²². One ml of Banana and olive leaves extracts at different concentrations (25, 50, 75, 100µg/ml) were mixed with 2.5ml of phosphate buffer (50mM, pH 7.0) and 2.5ml of 1% potassium ferricyanide. The mixture was then incubated at 50°C for 20 min. After the addition of 2.5ml of trichloroacetic acid (10%) to the mixture, centrifugation at 3000rpm for 10 min was performed. Finally, 1.25ml from the supernatant was mixed with 1.25ml of distilled water and 0.25ml FeCl₃ solution (0.1%, w/v). The absorbance was measured at 700nm. BHT was used as a standard. The results were expressed as EC₅₀ (the concentration µg/ml of banana and olive leaves extracts that provided the reading of 0.5 absorbances at 700nm).

Ferrous Chelating Activity:

Chelating activity on ferrous ions was carried out colorimetrically²³. One ml of banana and olive leaves extracts or EDTA solution as a positive control at different concentrations (25, 50, 75, 100µg/ml) were spiked with 0.1ml of 2 mM FeCl₂- 4H₂O and 0.2ml of 5 mM ferrozine solution and 3.7ml methanol were mixed in a test tube and reacted for 10 min at room temperature then the absorbance was measured at 562nm. Mixture without extract was used as the control. A lower absorbance indicates a higher ferrous ion chelating capacity. The percentage of ferrous ion chelating ability was calculated using the following equation:

Chelating activity (Inhibition %) = [(A_c – A_s) / A_c] × 100

Where (A_c) was the absorbance of the control reaction and (A_s) the absorbance in the presence of the plant extracts. The results were expressed as IC₅₀ (the concentration µg/ml of the banana and olive leaves extracts that chelate 50 % of Fe²⁺ ions).

In-vitro antimicrobial assay of ethanol and acetone banana and olive leaves extracts:

Different bacteria strains of gram-positive (Bacillus subtilis NRRL B-94 & Staphylococcus aureus NRRL B-313) and, gram-negative (Escherichia coli NRRL B-3703 & Pseudomonas aeruginosa NRRL B-32), Aspergillus flutes NRC as mold, Saccharomyces cerevisiae, and Candida albicans NRRL 477 strains as yeast, were used. The measurement of growth inhibition was carried out with agar diffusion tests²⁴.

Procedure:

The bacterial strains were cultured in a nutrient broth media while the fungi and yeast strains were cultured in a malt broth media and yeast broth media, respectively. For bacteria and yeast, the broth media were incubated for 24 h. As for molds, the broth media were incubated for 48 h, with subsequent filtering of the culture through a thin layer of sterile sintered Glass G2 to remove mycelia fragments before the solution containing the spores was used for inoculation. For plate preparation, 1 ml Tween 20 and 500µl of inocula were added to 50ml of agar media 50°C and mixed by simple inversion. Wells of 6mm diameter were then mode in the solidified agar using proper sterile tubes. Plates were undisturbed for 30 min to allow diffusion of the sample (200, 400, and 600µg/l) into the agar, then incubation inverted at 30ºC for 48 h for bacteria and 72 h for fungi. The microbial growth inhibition zones, clear microbial free inhibition zones, were measured after incubation at 30ºC, beginning within 24 h for yeast, 24-48 h for bacteria and 48-72 h for fungi. Antimicrobial activities were calculated as a mean of three repetitions.

In-vitro cytotoxicity of banana and olive leaves acetone extract on the human cell line (HePG2 – MCF7)

Cytotoxic effect on human cell line (hepatocellular carcinoma HePG2 – Caucasian breast adenocarcinoma MCF7) Cell viability was assessed by the mitochondrial-dependent reduction of yellow MTT (3-(4,5-dimethylthiazol-2-yr)-2,5- diphenyl tetrazolium bromide) to purple formazan²⁵.

Procedure:

All the following procedures were done in a sterile area using a Laminar flow cabinet biosafety class II level (Baker, SG403INT, and Sanford, ME, USA). Cells were suspended in RPMI 1640 medium for HePG2 and - MCF7. The media are supplemented with 1% antibiotic-antimycotic mixture (10,000 U/ml Potassium Penicillin, 10,000 μg/ml Streptomycin Sulfate and 25μg/ml Amphotericin B), 1% L-glutamine and 10% fetal bovine serum and kept at 37°C under 5% CO₂. Cells were batch cultured for 10 days, then seeded at a concentration of 10x103 cells/well in fresh complete growth medium in 96-well microtiter plastic plates at 37°C for 24 h under 5% CO₂ using a water-jacketed Carbon dioxide incubator (Sheldon, TC2323, Cornelius, OR, USA). Media was aspirated, fresh medium (without serum) was added and cells were incubated either alone (negative control) or with different concentrations of banana and olive leaves extracts to give a final concentration of (100-50-25-12.5-6.25-3.125-1.56 and 0.78μg/ml). After 48 h of incubation, the medium was aspirated, 40μl MTT salt (2.5μg/ml) was added to each well and incubated for a further four hours at 37°C under 5% CO₂. To stop the reaction and dissolving the formed crystals, 200μl of 10% Sodium dodecyl sulfate (SDS) in deionized water was added to each well and incubated overnight at 37ºC. A positive control (Doxorubicin) 100μg/ml was used as a known cytotoxic natural agent who gives 100% lethality under the same conditions. The absorbance was then measured using a microplate multi-well reader (Bio-Rad Laboratories Inc., model 3350, Hercules, California, USA) at 595nm and a reference wavelength of 620nm. A statistical significance was tested between samples and negative control (cells with the vehicle) using an independent t-test by SPSS 11 program. DMSO is the vehicle used for the dissolution of banana and olive leaves extracts and its final concentration in the cells was less than 0.2%. The percentage of change in viability was calculated according to the formula: ((A _sample / A _control) -1) x 100

Where: A _sampleis the absorbance of treated cells with extract

A _control is the absorbance of untreated cells.

Statistical analysis:

Statistical analysis is carried out using SPSS computer program (version 8) combined with Costat statistical package data (ANOVA - complete randomize), where unshared letters are significant at P ≤ 0.05 according to Anonymous²⁶.

3. RESULTS AND DISCUSSION:

3.1. Proximate Analysis:

The data of the chemical analysis of raw materials: banana and olive leaves are given in Table (1). The moisture content of raw materials was evaluated and was being 63.57 and 43.06% of fresh weight for banana and olive leaves respectively. The moisture content of banana leaves was less than that obtained by²⁷ (89.32%). The highest crude protein content was found in banana leaves (14.98% of dry weight) which were higher than that found by²⁸ (7.65%).

However, remarkable increases in the values of phytochemicals of olive leaves (80% methanol extract) hence, the value of total phenol was 85.50mg/g DW, flavonoid was 105.19mg/g DW and tannin was 51.03 mg/g DW. However, the values were higher than that in aqueous extract (TP was 42.02, TF was 31.22 and TT was 30.92mg/kg DW). The present results are in accordance with that of¹³^,41,42 for total phenol, flavonoid, and tannin of banana leaves. While the total phenols of banana leaves in methanol extract of the present study were 20.64mg/g DW which was higher than that obtained by⁴³ (their values ranged from 2.09 to 5.55 mg/g DW). Also, the results of olive leaves are in agreement with those determined by^44,45 for total phenol and flavonoid. The lower level of total phenol (ranged from 9.08 to 24.93 of mg/g DW) and flavonoid (6.23 to 21.47mg/g DW) reported by⁴⁶. the lower content of total tannin for olive leaves was recorded by^47,48.

3.2.2. Identification of phenolic compounds by HPLC:

Data presented in Table 4 shows that the concentration of individual phenolic compounds extracted by 80 % acetone extract.

Table 4. Quantification of the main phenolic compounds presents in acetone extracts (mg/kg crude extract) by HPLC.

Compounds	Banana leaves	Olive leaves
Gallic acid	-	343.19
Protocatechuic acid	109.37	7925.19
Gentisic acid	-	-
Catachine	686.33	688.74
Chlorogenic acid	314.40	-
Caffeic acid	650.83	840.98
Syringic acid	645.95	1378.95
Vanillic acid	1432.47	1175.45
Ferulic acid	1474.50	888.53
Sinapic acid	312.50	411.43
Coumarin	575.62	330.56
Rosmarinic acid	2712.83	216.80
Cinnamic acid	236.19	60.71
Chyrsin acid	145.28	5575.75
Ellagic acid	68.48	103.10
Tannic acid	-	5.69
Pyrogallol	3.41	-
Quercetin	68.25	70.16
Rutin	-	0.86
Acacetin	-	1.20
Oleuropein	-	247.97

The samples are identified and quantified by external standards being gallic, protocatechuic, gentisic, catechine, rosmarinic, chlorogenic, cinnamic, caffeic, syringic, ellagic, vanillic, tannic, ferulic and sinapic acids as well as coumarin, chyrsin, pyrogallol, quercetin, rutin, acacetin, and oleuropein. Results showed that all extracts contained different compositions and contents of phenolic compounds. It was observed from Table (4) that banana and olive leaves contain nearly a similar phenolic profile. In banana leaves acetone extract, vanillic, ferulic and Rosmarinic (1432.47, 1474.50 and 2712.83mg/kg crude extract are the major phenolic compounds) and pyrogallol was the minor phenolic compounds. Chlorogenic acid and pyrogallol were found only in banana leaves (314.40 and 3.41mg/kg crude extract, respectively). The results obtained are similar to those obtained by⁴⁹. Among the phenolic profiles Gallic, oleuropein, tannic, acacetin and rutin were detected only in olive leaves which were 343.19, 247.79, 5.69, 1.20 and 0.86mg/kg of crude extract, respectively. While, protocatechuic, chyrsin, syringic acid and vanillic acid were mostly found in olive leaves with values of 7925.19, 5575.75, 1373.95 and 1175.45mg/kg crude extracts. Gentisic acid was not detected in all extracts tested as represented in Table (4). Similar results were found by^48,50. The variety, environmental conditions, and agricultural practices greatly affect the structure and the concentration of phenolic compounds⁵¹. Nevertheless, the drying process may affect the components matrix structure and its bioactivity⁵². As previously reported that Oleuropein in olive leaf extract ranged from not detected to 539 mg/ Kg extract according to extraction conditions⁵³.

3.3. Antioxidant capacities of banana and olive leave extracts using DPPH˙, ABTS˙⁺, Reducing Power (RP) and Fe⁺² chelating:

The antioxidant activity of banana and olive leaves represented as IC₅₀ are shown in Table (5). The DPPH˙ radical scavenging activity of the leaves extracts was measured and compared with that of BHT to evaluate the in vitro antioxidant activity of banana and olive leaves extracts. Regarding the DPPH˙ assay, the antioxidant capacity (IC₅₀) is ranged from 17.97 to 44.50µg/ml. Olive leaves methanol extract had the highest DPPH˙ radical scavenging capacity (17.97µg/ml). While the lowest scavenging capacity (44.50µg/ml) was given in banana leaves aqueous extract. These results are in accordance with^54,55 for olive leaves. Also, the results of banana leaves were similar to those reported by^41,42. In the ABTS˙⁺scavenging activity, the values of IC₅₀ are varied significantly which ranged from 20.13 – 190.95 µg/ml. The highest value with olive leaves (20.13 µg/ml) was found in acetone extract, while the lowest value (190.95µg/ml) was found in olive leaves aqueous extract. Banana and olive leave extracts had the highest antioxidant activity by ABTS˙⁺ assay^42,44. The antioxidant activity of plant materials is significantly correlated well with the presence and the content of their phenolic compounds^56,57.

Table 6. Antimicrobial activity of ethanol and acetone extracts of banana and olive leaves

Samples	Extracts	Conc. µg/ml	Inhibition Zone (mm)
			Bacteria				Fungi	Yeast
			*B. subtils*	*St. aureus*	*E. coli*	*P. aeruginosa*	*A. fluves*	*S. cerevisiae*	*C. albicans*
Banana leaves	Ethanol 80%	200	00.00	00.00	00.00	00.00	00.00	00.00	00.00
		400	11.55	10.86	09.87	10.75	12.77	11.67	10.63
		600	17.70	16.54	16.16	16.78	17.54	15.66	15.79
	Acetone 80%	200	09.00	09.00	00.00	00.00	00.00	00.00	00.00
		400	16.96	15.73	13.23	14.33	12.23	12.80	13.69
		600	21.32	22.00	18.15	19.35	18.54	16.80	17.15
Olive leaves	Ethanol 80%	200	00.00	00.00	00.00	00.00	00.00	00.00	00.00
		400	13.50	13.55	11.73	10.53	10.20	11.47	11.47
		600	20.50	18.60	17.25	18.55	17.75	18.60	16.70
	Acetone 80%	200	09.00	08.50	00.00	00.00	00.00	08.10	09.34
		400	15.73	16.15	14.87	13.70	11.33	13.80	14.55
		600	20.75	21.20	18.73	19.46	17.85	17.87	19.86

The leave extracts of banana and olive exhibited the highest inhibitory effects which ranged from 16.54 to 22.00mm against gram-positive and 16.16 to 19.64mm against gram-negative and against fungus was 15.66 to 19.86mm at a concentration of 600µg/ml. These results are in agreement with that reported by⁶¹, who found that the average zone of inhibition of antibacterial was ranged from 6 to 22 mm at concentrations of 20 -200 µg/disc for banana leaves. Also, Ethanol and acetone extract of olive leaves showed inhibition zone 12.00mm against Escherichia coli and 7.00mm against Staphylococcus aureus. Additionally, olive leave extracts exhibited excellent antifungal activity against both Aspergillus terreus and Penicillium solitum³². The antimicrobial activity of tomato pomace extracts presented variable inhibition effects against pathogenic bacteria, and fungus⁶⁰. The previous study demonstrated that the banana and olive leaves acetone extracts showed moderate inhibition effect to strong activity against A. flutes, S. cerevisiae, and C. albicans.

3.5. In-vitro cytotoxic effect:

Two cancer cell lines (MCF-7 breast, HepG2 hepatocellular carcinoma cells) were examined to define the cytotoxic effectiveness of banana and olive leave acetone extracts. Results were compared with Doxorubicin as a positive control. Results in Table 6 show the cytotoxic activity of banana and olive leaves extracts against two cancer cell lines. Banana leaves had the highest percentage of cytotoxicity against MCF-7 and HepG2 (48.60 and 33.50% respectively), followed by olive leaves (27.30 and 22.20% respectively). Results revealed that the potential activity of olive leaf extract as anti-proliferation against HepG2 cancer cell lines as recorded by ⁹.

Table 7. Cytotoxic effect of banana and olive leaves acetone extract (80%) at concentration (100µg/ml)

Samples	HePG-2 (%)	MCF-7 (% )
Banana leaves	33.50	48.60
Olive leaves	22.20	27.30
DMSO	1.00	1.00
Doxorubicin	100	100

HePG-2: Hepatocellular carcinoma cells

MCF- 7: Breast cell line

These results are in a good agreement with^50,67 for olive leaves. Additionally, peanut byproduct (peel and skin) might be beneficial as a potent antioxidant and anticancer agents and effectively employed as an ingredient in food applications⁵⁷. The tumor volume and weight reduced with treatment 150 and 225 mg/kg/day of olive leaves extract⁶⁸. The chlorophyll possesses therapeutic properties due to the ability to prevent cancer and is being used in cancer therapy⁶⁹.The oleuropein is the most abundant phenolic compound in olives. The oleuropein, a powerful antioxidant, and anti-cancer agent. Oleuropein swirled cancer cells, irreversibly prohibiting their replication, motility, and invasiveness; irreversibly in normal cells, these effects were reversible as noticed by⁷⁰. It has been found that colon carcinoma cell line and the anti-proliferative activity against two breast cancer cell lines, that is, MCF-7 and T-47D completely blocked the incursion of tumor cells and helped in tumor regression in presence of oleuropein and hydroxytyrosol⁷⁰ via arresting cell cycle progression, increasing cell mortality, and reducing the generation of reactive oxygen species (ROS)⁷¹. Banana extracts were tested for their ability to inhibit the growth of HePG2 and MCF-7 tumor cell lines (Table7). The extracts showing 33.50% and 48.60% inhibition of cell proliferation were considered to be moderate cytotoxic effect extracts. The acetone extract of banana leaves had a moderate cytotoxic effect on tested cancer cell lines. Our results are disagreement with results obtained by banana hexane peel extract wherein significant activities were observed towards cancer cell lines⁷². Generally, the biological activities of the fruits are slightly attributed to their wide range of necessary micronutrients and phytochemicals. These bioactive components may prohibit cancer meanwhile diverse modes of action including antioxidant activity, inhibition of cell proliferation, induction of apoptosis, Inhibition of cell invasion and subcellular signaling pathways^73,74.

4. CONCLUSION:

In-vitro antioxidant, antimicrobial and antitumor activities of olive and banana leaves. The plants displayed considerable antioxidant, antimicrobial and cytotoxicity effects in an in vitro model. These results obviously motivate the employment of olive and banana leaves as a vigorous, prospective source for medicinal and industrial use. Additional studies are prerequisite to get the components which are responsible for these activities.

5. CONFLICT OF INTERESTS:

The author did not declare any conflict of interest.

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Received on 04.07.2019 Modified on 12.08.2019

Research J. Pharm. And Tech 2020; 13(2):687-696.

DOI: 10.5958/0974-360X.2020.00132.8

Sample	Moisture (g/100g fresh weight)	Component (g/100g dry weight basis)
Sample	Moisture (g/100g fresh weight)	Crude protein	Total lipid	Total carbohydrate	Ash
Banana leaves	63.57 ± 0.36	14.98 ± 0.34	10.37 ± 0.99	60.71 ± 0. 24	13.85 ± 0.69
Olive leaves	43.06 ± 0.24	10.71 ± 0.84	15.04 ± 0.85	66.07 ± 0.20	8.14 ± 0.13

Samples	K	Ca	Na	P	Mg
Banana leaves	3800 ± 43.01	1200± 12.28	2000 ± 20.24	1800± 60.03	9500 ± 50.17
Olive leaves	2100± 25.07	9800 ± 90.01	2900 ± 80.36	1400 ±50.27	6100 ± 20.17
Samples	Fe	Cu	Zn	Mn
Banana leaves	277.82 ± 0.94	7.97 ± 0.94	32.23 ± 4.23	256.61± 39.10
Olive leaves	242.03 ± 14.52	8.15 ± 0.54	29.16 ± 1.29	36.19 ± 18.69

Sample	Extracts	Phenolics (mg GAE/g DW)	Flavonoids (mg QE/g DW)	Tannins (mg TE/g DW)
Banana leaves	Aqueous	13.19^h ± 0.10	9.70^g ± 0.25	8.28^h ± 0.08
	Methanol 80%	20.64^g ± 0.07	20.50^f ± 0.15	13.41^f ± 0.06
	Ethanol 80%	21.81^f ± 0.05	21.39^e ± 0.06	12.19^g ± 0.01
	Acetone 80%	27.95^e ± 0.14	30.76^d ± 0.12	17.30^e ± 0.06
Olive leaves	Aqueous	42.02^d ± 0.48	31.22^d ± 0.11	30.92^d ± 0.28
	Methanol 80%	85.50^a ± 0.36	105.19^a ± 0.46	51.03^a ± 0.28
	Ethanol 80%	78.22^b ± 0.59	88.67^b ± 0.11	48.39^b ± 0.28
	Acetone 80%	56.98^c ± 0.47	68.00^c ± 0.40	39.19^c ± 0.17
LSD at 0.05		0.977	0.704	0.519

Samples	Extracts	DPPH^•IC₅₀ (µg / ml)	ABTS^•+ IC₅₀ (µg / ml)	Reducing Power EC₅₀ (µg / ml)	Fe²⁺-chelating IC₅₀ (µg / ml)
Banana leaves	Aqueous	44.50 ^a ± 0.20	122.31^c ± 0.54	290^a ± 2.65	1626.35^b ± 18.53
	Methanol 80%	33.16 ^b ± 4.95	121.60 ^c ± 1.91	235^b ± 1.94	759.52^d ± 5.23
	Ethanol 80%	40.63 ^a ± 1.70	134.62 ^b ± 1.58	233^b ± 11.63	422.94^e ± 10.87
	Acetone 80%	27.81 ^c ± 0.27	45.76 ^e ± 0.50	110^d ± 1.76	419.13^e ± 11.29
Olive leaves	Aqueous	41.64^a ± 1.01	190.95 ^a± 0.71	119^cd ± 7.16	1747.88^a ± 12.97
	Methanol 80%	17.97 ^d ± 0.25	94.90^d ± 0.43	90^e ± 1.14	367.56^f ± 8.82
	Ethanol 80%	28.09 ^bc ± 0.38	95.82^d ± 0.74	136^c ± 7.86	375.24^f± 3.37
	Acetone 80%	32.14 ^bc ± 0.22	20.13^f± 0.16	219^b ± 12.34	1323.15^c ± 7.42
BHT as standard		4.73 ^g ± 0.72	15.43^g ± 0.19	31.43^g ± 0.19	-
EDTA as standard		-	-	-	29.00^f ± 0.72
LSD at 0.05		5.16	2.69	29.09	19.47