Journal of Reports in Pharmaceutical Sciences

ORIGINAL ARTICLES
Year
: 2020  |  Volume : 9  |  Issue : 2  |  Page : 221--236

Cytotoxic, antimicrobial activities, and phytochemical investigation of three peach cultivars and acerola leaves


Seham S El-Hawary1, Ola Mohamed Mousa2, Rana Ahmed El-Fitiany1, Rania A El Gedaily1,  
1 Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Gizah, Egypt
2 Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Gizah, Egypt; Department of Pharmacognosy, Faculty of Pharmacy, Ahram Canadian University, 6th of October City, Gizah, Egypt

Correspondence Address:
Dr. Rana Ahmed El-Fitiany
Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Gizah.
Egypt

Abstract

Background: Phytoconstituents of Prunus persica Linn. (Peach) and Malpighia glabra Linn. (Acerola) leaves were not thoroughly studied, although they are commonly incorporated in the food industry. Aim: Our aim is to explore metabolites and vitamins in three peach cultivars leaves; Desert red, Florida prince, Swelling and acerola. Material and Methods: Analysis was done using GC/MS (gas chromatography–mass spectrometry), HPLC (high-performance liquid chromatography), and spectrophotometry. Cytotoxicity was performed using MTT assay. Results: Total phenolic and flavonoid content varied from 79.54 to 121.51 μg gallic acid equivalent/mg dry weight and 31.05 to 39.77 μg quercetin equivalent/mg dry weight, respectively. Twenty-four flavonoids were identified; hesperidin was the major flavonoid in peach cultivars (3863.4 mg/100 g in Desert red, 2971 mg/100 g in Swelling, and 2624 mg/100g in Florida prince). Glucuronic acid (33.04%) and vitamin C (34 mg/100 g) were major in acerola. Thirty-four metabolites including supraene and sitosterol as well as 24 fatty-acid esters including linoleic and oleic acids were detected in the unsaponifiable and saponifiable matter, respectively. Antimicrobial activity against bacterial and fungal strains was screened in comparison with ampicillin and amphotericin B. All tested extracts significantly decreased cell viability against breast (MCF-7) and colon cell lines (HCT-116). M. glabra showed no significant difference from standard doxorubicin (0.1 μg/mL) which may suggest a strong anticancer activity against colon cell line. Conclusion: This study may highlight the magnitude of the leaves of these plants as rich sources of important metabolites and vitamin C.



How to cite this article:
El-Hawary SS, Mousa OM, El-Fitiany RA, El Gedaily RA. Cytotoxic, antimicrobial activities, and phytochemical investigation of three peach cultivars and acerola leaves.J Rep Pharma Sci 2020;9:221-236


How to cite this URL:
El-Hawary SS, Mousa OM, El-Fitiany RA, El Gedaily RA. Cytotoxic, antimicrobial activities, and phytochemical investigation of three peach cultivars and acerola leaves. J Rep Pharma Sci [serial online] 2020 [cited 2020 Dec 5 ];9:221-236
Available from: https://www.jrpsjournal.com/text.asp?2020/9/2/221/292489


Full Text

 Introduction



Fruits and vegetables have a critical value in our nutrition and the human life. Due to the increasing world population and the changing dietary habits, the claim for such important food components has significantly increased.[1] Phytochemicals (mainly polyphenols), some vitamins (A, C, E, and folates), and dietary fibers are responsible for the great health benefits achieved by consuming vegetables, fruits, and other foodstuffs.[2] The wastes of fruits and vegetables can be used for the extraction and isolation of potentially bioactive compounds which can be incorporated in food, cosmetics, pharmaceutical, and textile industries.[1] The leaves of edible fruits can be considered as valuable byproducts which are wasted during harvesting of the fruits. These leaves have magnetized the attention of many researchers in the past few years in their search for new sources of valuable metabolites. For example, though rich in bioactive polyphenols, tons of berry leaves (blueberry, blackberry, raspberry, lingonberry, blackcurrant, bilberry, and cranberry) are wasted during harvesting each year.[3] Also surprisingly on comparing the phenolics of edible fruits and their leaves of seven selected species, Malus domestica, Cydonia oblonga, Chaenomeles japonica, Ribes nigrum, Aronia melanocarpa, Vaccinium macrocarpon, and V. myrtillus, the leaves contained notably higher polyphenol compounds compared to the fruits.[4] Polyphenols were also found abundant in strawberry leaves.[5] For our study, the leaves of two edible fruits were selected. Prunus persica Linn. and Malpighia glabra Linn. are two important edible plants belonging to families Rosaceae (2830−3100 species) and Malpigiaceae (1300 species),[6] respectively.

Prunus persica L. or peach[7] contains a diversity of phytochemical compounds such as alkaloids, glycosides, flavonoids, carbohydrates, fixed oils, steroids, tannins, phenols, amino acids, and proteins.[8] Peach has many important biological activities: antidiabetic, antioxidant, antimicrobial, antitumor, antiallergic inflammatory, cholinesterase inhibitory, free radical scavenging, prokinetic, and polyphenol oxidase activities.[9] There are several cultivars of peach in Egypt; the most common cultivars are Florida prince (P. persica cv. Florida prince), Desert red (P. persica cv. Desert red), and Swelling (P. persica cv. Swelling).[10] According to the data acquired from Food and Agriculture Organization of the United Nations in 2016, 83 countries are cultivating peach worldwide; Egypt was ranked as the 11th country in peach production. Acerola is the common name assigned to the M. glabra L., also called “Barbados cherry” or “West Indian cherry.”[11] Some of the reported active constituents in acerola’s different organs are carotenoids,[12] fatty acids,[13] volatile constituents,[14] flavonoids,[15] and terpenes.[16] Acerola possess many reported significant pharmacological activities, for example, acetylcholinesterase inhibition,[17] antioxidant,[18],[19] antipyretic,[20] anti-inflammatory,[21] and antihyperglycemic.[22] It is worth mentioning that no data are available concerning the chemical composition, cytotoxicity, and antimicrobial activity of the leaves of M. glabra Linn. and the three P. persica Linn. cultivars under study. This drove the authors to deeply investigate the chemical profile of these edible plant leaves to highlight the importance of these great sources of bioactive metabolites wasted as harvesting byproducts. High-performance liquid chromatography (HPLC) analysis was used to determine their flavonoid, carbohydrate, and vitamin content. GC/MS was used to analyze their saponifiable and unsaponifiable matter. Quantification of flavonoids and phenolic content was performed by applying Folin–Ciocalteu and aluminum chloride methods, respectively.

 Materials and Methods



Plant material

Malpighia glabra L. leaves were collected from El-Orman Botanic Garden, Giza Governate, Egypt since May 2016 and it was kindly authenticated by the herbarium of El-Orman Botanic Garden. Samples of P. persica L. leaves of the three cultivars under investigation (cv. Florida prince, Desert red, and Swelling) were collected from Faculty of Agriculture, Cairo University and Manara-2 farm, Wadi El-Natrun, El-Behira Governate, Egypt in May 2015. They were kindly authenticated by Dr. Reem Samir Hamdy, associate professor of plant taxonomy, Department of Botany, Faculty of Science and Dr. Abdo Mohammed Abd El-Latef, associate professor in Fruit Orchards Department, Faculty of Agriculture, Cairo University, Egypt. Voucher specimens of M. glabra L. and P. persica Linn. cultivars were kept at the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Cairo University with serial numbers: 1.3.1.2019(1–4).

Preparation of plants extracts

Malpighia glabra L. leaves (450g) and P. persica L. leaves (2kg) of each of the three cultivars under investigation were separately air dried at 25°C and powdered in a household blender, then they were macerated in 80% ethanol for 1 week at room temperature, filtered, and the ethanol was evaporated using a rotary evaporator at 45°C. Maceration, filtration, and evaporation processes were repeated till exhaustion.

Phytochemical screening

The phytoconstituents of the peach cultivars and acerola leaves’ 80% ethanolic extracts were screened for the presence of flavonoids,[23] cardiac glycosides,[24] alkaloids,[25] anthraquinone glycosides,[26] carbohydrates,[27] saponins,[28] sterols and/or triterpenes,[29] tannins,[30] and volatile constituents.[31]

Quantification of the plant phytoconstituents

Spectrophotometric determination of total phenolic content

The total phenolic content of the 80% ethanolic extract of the leaves of the plants under investigation was estimated using UV–visible spectrophotometer (Shimadzu UV-1650 PC, Kyoto, Japan) using Folin–Ciocalteu method.[32] The absorbance was measured at 765 nm. A calibration curve of gallic acid (Sigma, St. Louis, MO, USA), ranging from 80 to 280 µg/mL, was constructed (R2 = 0.9856) [Figure 1]A, and total content (%) of phenolics was calculated as gallic acid equivalents using the regression equation of the calibration curve. All determinations were repeated three times.{Figure 1}

Spectrophotometric determination of flavonoid content

The total flavonoid content of the 80% ethanolic extract of the leaves of the plants under investigation was estimated using aluminum chloride colorimetric method.[33] The absorbance of the dilutions was measured at 415 nm expressed as quercetin equivalent (QE). A standard calibration curve (R2 = 0.9964) was established with different aliquots (5–100 µg/mL) of standard quercetin (Sigma) [Figure 1B]. Each sample was done in triplicate.

High-performance liquid chromatography analysis for flavonoids

HPLC analysis of flavonoids (phenolics) was performed according to the method by Mattila et al.[34] using reversed-phase HPLC Agilent 1200 series (Agilent Technologies, Waldbronn, Germany) and ZORBAX ODS column 4.6 mm × 250 mm (Dupont Instrument, Wilmington, Delaware). Multiwavelength detector was set at 330 nm. Detailed analysis conditions are mentioned in Supplementary File (S1). All flavonoids were quantified using the external standard method. Quantification of samples and standards was based on peak area. Dilution of stock standards was done in methanol to give 2–20 µg/mL for the establishment of calibration curves.

High-performance liquid chromatography analysis for carbohydrates

HPLC analysis was done according to the method of Kiranmai et al.[35] The tested ethanolic extracts of the leaves were diluted to 1:10 (v/v) with deionized water and then filtered through a 0.22 µm filter membrane. An aliquot of 1.5 mL of each of these solutions was posited in vials for analysis. The analysis was performed using HPLC Agilent 1200 series (Agilent Technologies) using a Bio-Rad Aminex – carbohydrate HPX-87C column (300 mm × 7.8 mm). Detailed analysis conditions are mentioned in Supplementary File (S2). Sample detection was performed by comparing the retention time of analytes with standards. Quantification was based on peak area. Triplicate injections of seven different concentrations of each standard obtained by dilution in deionized water were performed. A calibration curve for each sugar was done by plotting the concentrations versus the peak area.

High-performance liquid chromatography analysis for vitamin C and E

Vitamin E and C quantification was performed according to the methods of Romeu-Nadal et al.[36] and Pyka et al.,[37] respectively, using reversed-phase HPLC Agilent 1200 series (Agilent Technologies). Multiwavelength detector was set at 254 and 292 nm for detection and quantification of vitamin C and vitamin E, respectively. The separation was carried out using ZORBAX ODS column 4.6 mm × 250 mm (Dupont Instrument). Detailed analysis conditions are mentioned in Supplementary File (S3). Ascorbic acid and α-tocopherol were recognized by comparing the retention time of the sample peak with that of the standard. A calibration curve of different standard concentrations ranging from 0.5 to 100 µg/mL was plotted and quantification was carried out using external standard method.

Gas chromatography–mass spectrometry analysis of unsaponifiable and saponifiable matters

Air-dried powdered leaves of M. glabra Linn. (250g.) and P. persica Linn. (171g) cultivars (Desert red, Swelling, and Florida prince) were, separately, defatted in n-hexane. The solvents were evaporated under vacuum to give 6.07, 2.75, 1.81, and 3.01g. residue, respectively. The unsaponifiable matters and the fatty acid methyl esters of the plants under investigation were prepared from the previously obtained hexane extracts.[38] The detection of saponifiable and unsaponifiable matters was carried out using gas chromatography coupled with mass spectroscopy (Shimadzu QP-5050 A, Japan) equipped with DB1-MS fused silica capillary column (30 m x 0.53 mm; film thickness 1.5 µm). Detailed analysis conditions are mentioned in Supplementary file (S4). All the standards were well resolved, and quantitative measures were obtained by correlating peak areas for all known compounds and relating them to standard curves of the standard compounds.

In vitro antimicrobial screening

Determination of the antimicrobial activity

The ethanolic extracts of the plants under investigation were screened for antimicrobial activity by implementing a modified Kirby–Bauer disk-diffusion method[39] against bacterial strains Staphylococcus aureus (ATCC 12600), Bacillus subtilis (ATCC 6051), Escherichia coli (ATCC 11775), and Pseudomonas aeuroginosa (ATCC 10145), fungal strain (Aspergillus flavus [Link]), and yeast (Candida albicans [ATCC 7102]), which were available in the micro-analytical center, Faculty of Science, Cairo University, Egypt. Standard disks of ampicillin (antibacterial agent supplied from Bristol-Myers Squibb, Switzerland) and amphotericin B (antifungal agent supplied from Bristol-Myers Squibb, Switzerland) served as positive controls for antimicrobial activity, whereas filter disks impregnated with 10 µL of solvent (distilled water, chloroform from El-Gomhouria Company for Trading Chemicals and Medical Appliances, DMSO from Loba Chemie) were used as a negative control. The agar used is Meuller-Hinton agar for bacteria and Czapek’s Dox agar (sucrose-nitrate agar) for yeasts and fungi; they are rigorously tested for composition and pH. Determination of standard zones of inhibition was done for susceptible and resistant values. Blank paper disks (Schleicher and Schuel, Spain) with a diameter of 8.0 mm were impregnated with 10 µL of the tested concentration of the stock solutions of the plant extracts, which were dissolved in dimethyl sulfoxide. For the disk diffusion, slipping calipers of the National Committee for Clinical Laboratory Standards were used for the measurement of the zone diameters.[40] The test was performed in triplicates. Detailed analysis conditions are mentioned in Supplementary File (S5).

Determination of minimum inhibitory concentration

The minimum inhibitory concentrations (MICs) of the crude extracts under study were determined through performing the agar dilution method.[41] Stationary phase cultures of bacteria were prepared at 37°C and used to inoculate fresh 5.0 mL culture to an OD600 of 0.05. Incubation was done for the 5.0 mL cultures at 37°C until an OD600 of 0.10 was achieved from which standardized bacterial suspensions were prepared to a final cell density of 6 x 105 CFU/mL. Serial dilutions from the treatments (0–320 mg/mL) were prepared and mixed with 5.0 mL of the standardized bacteria suspension then added to the plates and incubated for 24h at 37°C. The colony-forming units (CFU) were counted for each dilution.

MTT assay

The cytotoxicity of the extracts under investigation was tested by performing MTT assay[42],[43] against human breast adenocarcinoma (MCF-7) and human colon adenocarcinoma (HCT-116) cell lines, which were acquired from American type culture collection (ATCC, Wesel, Germany) and grown in the tissue culture lab of the Egyptian company for vaccines, sera, and drugs production (Vacsera, Giza, Egypt). Heat-inactivated fetal bovine serum was supplied from Invitrogen, Carlsbad, California. MTT solution/well was bought from Sigma Aldrich, Missouri.

Absorbance was measured at 570 nm using Epoch-2C plate reader (Bio Tek, Vermont). The cell viability was expressed relative to the untreated control cells and the concentrations induced 50% growth inhibition (IC50) were calculated from the concentration-response curve using graph pad prism version 5 (GraphPad Software, California). The detailed analysis conditions are stated in Supplementary File (S6).

Statistical analysis

All the results were expressed as mean ± standard error of the mean (SEM). The inhibition zone diameters and cell viability were determined in triplicate for antimicrobial and cytotoxic activities, respectively, and the analysis of variance (ANOVA) was done, followed by Dunnett’s and Tukey’s multiple comparisons test for antimicrobial and cytotoxic activities, respectively, to determine the significance of difference among the studied groups. The statistical analyses were tested at 0.001 level of probability using the GraphPad Prism version 6 (GraphPad Software, San Diego, California).

 Results and Discussion



Extraction yield

The resulted dry extracts of M. glabra L. was 74.24g, whereas P. persica L. cultivars Florida prince, Desert red, and Swelling were 130.14, 242.38, and 75.91g, respectively. They were kept at 4°C for further phytochemical and biological studies.

Phytochemical screening

The results of the phytochemical screening showed the presence of carbohydrates and/or glycosides, flavonoids, sterols and/or triterpenes, tannins, and traces of volatile constituents in all tested plants. Alkaloids, saponins, anthraquinone, and cardiac glycosides were absent in all the extracts.

Determination of total phenolic and flavonoid content

The spectrophotometric analysis of the total phenolics revealed that P. persica L. cv. Swelling shows the highest total phenolic concentration (121.51 ± 0.001 µg of gallic acid equivalent/mg dry weight) followed by P. persica Linn. cv. Florida prince (118.74 ± 0.14 µg of gallic acid equivalent/mg dry weight) then M. glabra Linn. (110.52 ± 0.14 µg of gallic acid equivalent/mg dry weight) and P. persica Linn. cv. Desert red (79.54 ± 0.140 µg of gallic acid equivalent/mg dry weight).

Furthermore, the analysis of flavonoid content showed that M. glabra Linn. contains the highest total flavonoid content (39.77 ± 0.01 µg of QE/mg dry weight), followed by P. persica Linn. cv. Florida prince (34.10 ± 0.06 µg of QE/mg dry weight) then P. persica Linn. cv. Swelling (33.18 ± 0.01 µg of QE/mg dry weight) and P. persica Linn. cv. Desert red (31.05 ± 0.01 µg of QE/mg dry weight) as shown in [Table 1]. No previous data were found concerning the total phenolic and flavonoid content of the leaves. Previous studies reported that the total phenolic and total flavonoid content in the pulps and peels of five Chinese peach cultivars were found in the range of 24.83–163.54 mg GAE/100g and 17.76–299.86 mg Rutin Equivalent/100g, respectively,[44] whereas the peel and pulp extracts from different varieties of peach from Pakistan showed an considerable amount of total phenolics and total flavonoids, ranging from 1209.3 to 1354.5, 711.7 to 881.3 mg GAE/100g and 599.7 to 785.5, 301.3 to 499.7 mg catechin equivalents (CE) /100g on a dry weight basis, respectively.[45] Cantin et al.[46] found out that the total phenolics of Spanish peach fruit among varied genotypes were in the range of 12.7–71.3 mg of GAE/100g and the total flavonoids content ranged from 1.8 to 30.9 mg of CE per 100g, with an average of 8.8 mg of CE per 100g. Also, previous reports regarding M. glabra L. showed that the contents of polyphenol in fruit extracts in India were 355.74 mg GAE/100g.[19] Moreover, the total phenolics of the fruits of M. glabra L. from Thailand was determined as 723.83 ± 36.94 mg GAE/100g and 195.36 ± 0.14 mg QE/100g.[47]{Table 1}

High-performance liquid chromatography analysis for flavonoids

HPLC analysis of the flavonoids in the tested leaf extracts led to the identification and quantification of a total of 24 flavonoids [Table 2]. Hesperidin was the most abundant flavonoid in the three peach cultivars (3863.4 mg/100g dry extract in Desert red, 2971 mg/100g dry extract in Swelling, and 2624.92 mg/100g dry extract in Florida prince); according to previous recorded preclinical studies and clinical trials, hesperidin is a bioflavonoid which showed several therapeutic effects in various diseases including neurological, psychiatric, cardiovascular disorders and others owing to its antihypertensive, anti-inflammatory, antioxidant, lipid-lowering, and insulin-sensitizing effects.[48] On the contrary, narengin (632 mg/100g dry extract) was the abundant flavonoid in acerola leaves. Luteolin 6-arbinose 8-glucose showed appreciable concentrations (3786, 2275, and 548 mg/100g dry extract) in Desert red and Florida prince cultivars, as well as acerola, respectively.{Table 2}

Also, rutin was detected in reasonable concentrations ranging from 48.68 to 127.14 mg/100g in all the tested extracts except Swelling cultivar of peach; it is worth mentioning that rutin was previously determined in peach kernel oil using HPLC analysis and it was found to be the major flavonoid in it beside (–)-epicatechin gallate.[49] Furthermore, quercetrin was identified and quantified in all the crude extracts under investigation and it was found in concentrations ranging from 17.69 to 300.72 mg/100g. It is noteworthy that quercetin 3-rhamnoside (quercetrin) was detected during the investigation of the phenolic compounds of 25 peach and nectarine fruit cultivars by HPLC−DAD−ESI-MS.[50] Other previous studies regarding HPLC analysis of flavonoids in peach different organs included the identification of multiflorin A in the methanolic extract of the leaves of the edible peach,[51] in addition to the determination of four kaempferol glycoside derivatives viz., multiflorin B, trifolin, afzelin, and astragalin from the peach flowers extracts.[52]

High-performance liquid chromatography analysis for carbohydrates

HPLC-RI resulted in the identification and quantification of 11 different sugars and sugar acids [Table 3]. Glucuronic acid was detected in a relatively higher percentage (33.04%) in acerola, whereas sorbitol was abundant in peach cultivars (ranging from 6.19% to 12.27%). Maltose, Lactose, Xylose, and Rhamnose were absent in all the tested extracts. From recent studies concerning peach kernels and fruits, sucrose, glucose, and fructose were detected as the most important sugars in peach kernels.[53] Also sucrose, glucose, fructose, sorbitol, malic acid, citric acid, and quinic acid were identified in the fruits of 106 peach cultivars from different breeding programs at Catalonia, Spain.[54]{Table 3}

High-performance liquid chromatography analysis for vitamin C and E

Vitamin C and E were characterized and quantified in the 80% ethanolic extract of the leaves of the plants under investigation using reversed-phase HPLC analysis. vitamin C and E were present in the tested extracts with reasonable concentrations. As the analysis results showed that all the examined extracts are rich in vitamin C and E at which their contents are ranging from 11.4 to 34 and from 0.01 to 0.14 mg/100g, respectively. Malpighia glabra Linn. contains the highest content of vitamin C (34.00288 mg/100g), whereas P. persica Linn. cv. Florida prince owns the highest content of vitamin E (0.1388673 mg/100g). On the contrary, P. persica Linn. cv. Swelling possesses the lowest concentration of both vitamins. The detailed results are shown in [Table 4]. The total amount of vitamin‐E‐active compounds in peach kernel oil from Turkey was previously estimated to be 62.9 mg/kg.[55]{Table 4}

Also previous studies regarding peach reported that the total vitamin C of western red nectarine of France (P. persica L. batsch) fruits is 5.34 ± 0.51 mg / 100 gram[56]; moreover, the Italian peach fresh fruit contains 7 mg vitamin C/100g and 3 mg vitamin C/100 in canned peach fruit.[57] Also the ranges of total vitamin C in peach were determined from California (in mg/100g of fresh weight) as follows: 5–14 (white-flesh nectarines), 6–8 (yellow-flesh nectarines), 6–9 (white-flesh peaches), and 4–13 (yellow-flesh peaches).[2] Therefore, our study exposed that the leaves of the Egyptian peach cultivars under study contain more vitamin C than those previously reported in the literature. The variation in vitamin C content detected may be attributed to variation in climatic conditions. Other previous studies concerning acerola fruits stated that Brazilian acerola fruits residues contain 170.73 ± 0.46 mg vitamin C/ 100g and 506.00 ± 11.00 mg vitamin C/ 100g in acerola edible portion of the fruit without residues.[58] Also São Paulo and Ceará aqueous fruit extracts contain 900.0 mg vitamin C/ 100g and 4,447.6 mg vitamin C/ 100g, respectively.[59] Moreover, it was mentioned that Italian acerola fresh fruit contains 1.677 mg vitamin C/ 100g,[57] whereas other report stated that western Mexico acerola contains 1000–4500 mg/100g of fruit.[60]

Gas chromatography–mass spectrometry analysis of unsaponifiable and saponifiable matters

From [Table 5], the percentage of the total identified constituents in the unsaponifiable matter represented 47.84%, 58.19%, 10.45%, and 28.92% of the total lipoidal content of the leaves of M. glabra Linn. and P. persica Linn. cultivars (Desert red, Swelling, and Florida prince), respectively. Sitosterol was detected in P. persica Linn. cv. Desert red at a concentration of 4.25%. Supraene (squalene) was the only triterpene found in M. glabra Linn. (11.78%), P. persica Linn. cv. Swelling (2.79%) and Florida prince (2.57%). It is important to mention that β-sitosterol was previously reported in peach leaves to be 1.12%.[61] Hentriacontane, heptacosane, bicyclohexanone, and cyclopentane were the major identified compounds in the unsaponifiable matter of the leaves of M. glabra Linn. and P. persica Linn. cultivars (Desert red, Swelling, and Florida prince) representing 20.43%, 20.78%, 4.25%, and 14.39%, respectively.{Table 5}

Also as shown in [Table 6], the total identified fatty acids represented 90.85% in M. glabra Linn. and 87.01%, 29.31%, and 67.11% in P. persica Linn. cultivars (Desert red, Swelling, and Florida prince), respectively. Oleic acid (0.7%) was detected only in M. glabra Linn., whereas Linolenic acid (9.75%) was identified only in P. persica Linn. cv. Swelling and linoleic acid (1.88%) was detected only in P. persica Linn. cv. Desert red. It is worth mentioning that oleic acid was previously determined in fruit residues including seeds and peels of M. glabra L. (23.2%).[58] Also it was determined in the total oil yield of seeds of three M. glabra Linn. genotypes ranging from 5 to 34%.[13]{Table 6}

Octadecatrienoic acid and octadecenoic acid methyl esters were the major identified compounds in the saponifiable matter of the leaves of M. glabra Linn. and P. persica Linn. cv. Florida prince representing 52.47% and 23.65%, respectively. Although pentadecanoic acid methyl ester was the major compound in P. persica Linn. cv. Desert red representing 36.36%, linolenic acid and octadecatrienoic acid methyl esters were the major components in P. persica Linn. cv. Swelling representing 9.75%. The fatty-acid components of kernel oils were determined from different Tunisian P. persica varieties including peach and nectarine at which the oleic acid (67.7%–75.0%) was found to be the predominant fatty acid, followed by linoleic (15.7%–22.1%) and palmitic (5.6%–6.3%) acids.[62] Furthermore, evaluation of the fatty-acid content was executed for the fleshes and peels of three P. persica cultivars at the Regional Centre of Agricultural Research in the Experimental Farm of Sidi Bouzid in Tunisia during two maturation stages; the results showed that palmitic (26.58–45.78%), oleic (2.23–31.33%), and linoleic acids (2.85%–10.14%) are the most copious fatty acids in P. persica cultivars.[63]

In vitro antimicrobial screening

Antibacterial activity

One-way ANOVA showed significant differences in the resulted antibacterial activities among the tested plant extracts on the Gram-positive bacteria (B. subtilis, S. aureus) (P < 0.0001) and on the Gram-negative bacteria (E. coli, P. aeuroginosa) (P < 0.0001). The average activities were reported as inhibition zone diameter in [Table 7a]nd [Figure 1C]. Ampicillin (standard antibacterial drug) displayed a strong antimicrobial activity against Gram-positive bacteria (B. subtilis, S. aureus) and Gram-negative bacteria (E. coli, P. aeuroginosa). The inhibitory activity of ampicillin against all the tested pathogens ranged from 21 to 26 mm/mg. Plant extracts where inhibition zone diameter values were 11.67–15.33 mm/mg are considered as having low activity compared to the reference drug ampicillin (P < 0.001). Malpighia glabra 80% ethanolic extract showed no antimicrobial activity against the Gram-positive (S. aureus) and Gram-negative bacteria (E. coli, P. aeuroginosa); this agrees with previous reports which stated that the fruits of M. glabra Linn. had no antimicrobial activity.[64],[65] Moreover, according to the performed MIC tests on the active extracts, it was noticed that P. persica Linn. cv. Florida prince 80% ethanolic extract was the most potent tested sample with a concentration of 96 mg/mL. In general, it was observed that the studied extracts are more potent against Gram-positive bacteria than Gram-negative bacteria; these results especially for peach cultivars are similar to that which was previously reported for the ethanolic extracted seeds of peach,[66] in addition to other studies which determined the antibacterial activity of the leaves’ ethanolic extract of peach against both Gram-positive and Gram-negative bacteria and the bark methanolic extract too.[67],[68]{Table 7}

Antifungal activity

One-way ANOVA showed significant differences in the antifungal activities among the groups of different tested plant extracts on the C. albicans (F [2, 6] = 61.23 P < 0.001). The average activity was reported as the inhibition zone diameter in [Table 7]. The average inhibitory activity of amphotericin B (antifungal standard drug) displayed a strong antifungal activity reported as 16 mm/mg sample and 19 mm/mg sample against A. flavus and C. albicans, respectively. It is noteworthy to know that all the tested plant extracts had neither antifungal activity against A. flavus nor on the C. albicans except Swelling 80% ethanolic extract where inhibition zone average diameter value was reported as 9 mm/mg sample, and therefore it is considered as having low activity against C. albicans as compared to the standard drug amphotericin B (P < 0.001). It is clear that the antifungal activities were very week, which is not in coordinance with a previous reported study, which showed that the antifungal activity was confirmed in the M. glabra Linn. but not in all parts; the most active organs were the leaves and bark.[69] Also, the antifungal activity was confirmed before in the methanolic crude extract of P. persica bark in a recent study as it showed a considerable antibacterial activity against Enterococcus faecalis and Klebsiella pneumonia.[70]

MTT assay

We first examined the effects of the plant extracts which are under investigation on the viability of two human cancer cell lines: human breast cancer cell line (MCF-7) and human colon cancer cell line (HCT-116). We then compared them to doxorubicin standard. As shown in [Table 8], treatment with plant extracts of different concentrations significantly decreased the viability of all cell lines with IC50s ranging from 302 to >1000 µg/mL against colon cancer cell line and IC50s ranging from 249.5 to >813 µg/mL against breast cancer cell line; these results are agreed with a recent study that showed in vivo tumor growth inhibition and antimetastatic effects of the polyphenolics content of peach using a xenograft model and MDA-MB-435 breast cancer cells in a dose ranging from 0.8 to 1.6 mg/day.[71] It is known that the lower the IC50 value, the higher the potency of the tested sample. Therefore, the resulted values of IC50 of the tested extracts versus colon cancer cell line indicated that the arrangement of potency of the tested extracts will be as follows: Malpighia glabra 80% ethanolic extract > Swelling 80% ethanolic extract > Florida prince 80% ethanolic extract > Desert red 80% ethanolic extract, whereas regarding breast cancer cell line, the potency is as follows: Desert red 80% ethanolic extract > M. glabra 80% ethanolic extract > Swelling 80% ethanolic extract > Florida prince 80% ethanolic extract.{Table 8}

One-way ANOVA showed significant differences in the cytotoxicity among each group of the tested plant extracts on both HCT116 and MCF-7 cell lines (P < 0.0001). All the plant extracts with the lowest concentration (0.1 µg/mL) had significantly reduced the viability of HCT116 cell line at P < 0.001 [Table 9]. Tukey’s multiple comparisons test displayed that there was no significant difference between 0.1 µg/mL of the standard drug “doxorubicin” vs. 0.1 µg/mL of M. glabra 80% ethanolic extract (P < 0.0946) which may suggest a strong anticancer activity against HCT116 cell line. Malpighia glabra L. Brazilian leaves’ cytotoxicity was studied before it showed cell growth inhibition against breast and colon cell lines (64.4 and 16 cell growth inhibition percentage, respectively) with IC50 <1000,[72] whereas the Egyptian M. glabra leaves in our study showed cell growth inhibition with IC50 490 and 302 against breast and colon cell lines, respectively, which proves that our results suggest that the Egyptian M. glabra leaves are more potent than the Brazilian leaves.{Table 9}

Florida prince 80% ethanolic extract (P < 0.001), Desert red, and Swelling 80% ethanolic extracts (P < 0.01) and M. glabra 80% ethanolic extract (P < 0.05) at the lowest tested concentration (0.1µg/mL) had significantly reduced the viability of MCF-7 cell line [Table 10]. Tukey’s multiple comparisons test showed that there was no significant difference between 10 µg/mL of doxorubicin vs. 1000 µg/mL 80% ethanolic extract of Florida prince (P = 0.8701) on MCF-7 cell lines. This could suggest that the 80% ethanolic extract of Florida prince may show anticancer activity against breast cancer cell lines at high concentration.{Table 10}

 Conclusion



This work appeared to be the first detailed study on the chemical profile, antimicrobial, and cytotoxic activities of the leaves of the Egyptian cultivated M. glabra Linn. and P. persica Linn. cultivars (Desert red, Swelling, and Florida prince). This study clearly confirmed that the leaves under investigation are a tremendous source of nutritional and bioactive metabolites, as carbohydrates, flavonoids, phenolics, sterols, and vitamin C and E which may be responsible in part for their anticancer activity.[71],[73],[74] Also it could be concluded that their flavonoid and fatty-acid content may rationalize their antimicrobial activity.[75],[76],[77] In conclusion, the findings of this study indicated that the investigated plants are promising to continue the isolation of their bioactive compounds and to assess extra detailed biological studies.

Acknowledgement

Our sincere thanks go to Youssef Shalaby (teaching assistant in Pharmacology Department at Ahram Canadian University) for performing the antimicrobial and cytotoxicity statistical analysis, Dr. Mohamed El-Gebaly (plant classification specialist at Orman Botanical Garden, Egypt), and Prof. Dr. Mohamed EL-Khashab (professor in Faculty of Agriculture, Cairo University, Egypt) for their help in providing M. glabra Linn. leaves, as well as, Dr. Mohamed Alaa (Faculty of Agriculture, Cairo University, Egypt) for providing us the plant material of peach cultivars for free, Dr. Reem Samir Hamdy (associate professor of plant taxonomy, Department of Botany, Faculty of Science, Cairo University, Egypt) for her authentication of peach plant material and Dr. Abdo Mohammed Abd El-Latef (associate professor in Fruit Orchards Department, Faculty of Agriculture, Cairo University, Egypt) for his identification of peach cultivars.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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