Industrial Crops and Products 59 (2014) 210–215

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Industrial Crops and Products

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Antibacterial and antifungal activities of methanol extract and

phenolic compounds from Diospyros virginiana L.
Khaled Rashed a , Ana Ćirić b , Jasmina Glamočlija b , Marina Soković b,∗
a
Pharmacognosy Department, National Research Centre, Dokki, Giza, Egypt
b
Institute for Biological Research “Siniša Stanković”, University of Belgrade, Blvd. Despota Stefana 142, 11000 Belgrade, Serbia

a r t i c l e i n f o a b s t r a c t

Article history: This study was carried out with the objective to investigate antibacterial and antifungal activities of
Received 14 November 2013 Diospyros virginiana fruits (methanol 80%) extract and the phenolic compounds isolated from the extract.
Received in revised form 13 May 2014 The in vitro antibacterial and antifungal assays were done against eight bacterial strains and against eight
Accepted 14 May 2014
fungal species using microdilution method. Eight phenolic compounds, m-gallate, gallic acid, luteolin,
quercetin, myricetin, myricetin 3-O-˛-rhamnoside, myricetin 3-O-ˇ-glucoside, and myricetin 3-O-ˇ-
Keywords:
glucuronide were isolated and identified by different spectroscopic tools (UV, 1 H NMR, 13 C NMR, MS).
Diospyros virginiana
The results of the methanol extract of Diospyros virginiana fruits and its isolated phenolic compounds
Fruits
Phenolic compounds
showed significant antibacterial and antifungal effects against all the tested microorganisms. The best
Antibacterial results were obtained for flavonoid aglycones which showed strong antimicrobial activity against all the
Antifungal microorganisms tested. These results may help to discover new chemical classes of natural antimicrobial
substances that could serve as selective agents for infectious disease chemotherapy and control.
© 2014 Published by Elsevier B.V.

1. Introduction
Diospyros is a genus of over 700 species of deciduous and ever-
green trees, shrubs, and small bushes. Diospyros virginiana L. is
a persimmon species commonly called the American Persimmon
from Ebenaceae family. It is a deciduous tree native to China, and
the southeastern portion of the United States. The tree grows wild
but has been cultivated for its fruit and wood since prehistoric
times by Native Americans (Phillips, 1979). The peculiar charac-
teristics of its fruit have made the tree well known. The fruit is
a globular berry, with variation in the number of seeds, some-
times with eight and sometimes without any. The fruit had high
content of vitamin C and it contained anti-oxidant compounds
like vitamin-A, beta-carotene, lycopene, lutein, and cryptoxanthin.
Fresh fruits also contained healthy amounts of minerals like potas-
sium, manganese, copper, and phosphorus. The fruit had many
valuable B-complex vitamins such as folic acid, pyridoxine. The
unripe fruit is extremely astringent and it has been beneficial in var-
ious forms of disease of the bowels, chronic dysentery and uterine
∗ Corresponding author at: Department for Plant Physiology, Institute for Biolog-
ical Research “S. Stankovic”, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia.
Tel.: +381 11 207 84 19.
E-mail address: mris@ibiss.bg.ac.rs (M. Soković).
haemorrhage (Briand, 2005). The ripe fruit has been used medic-
inally as antiseptic and for the treatment of diphtheria, dropsy,
fevers, and thrush (Briand, 2005). The bark is antiseptic, and its infu-
sion was employed to cleanse, stimulant foul and indolent ulcers
(Briand, 2005). There were few reports about phytochemicals and
bio-activities from D. virginiana. Methanolic extract from the leaves
showed antimycobacterial effect (Nasr et al., 2014). Roots of the
plant and the compounds isolated from it showed antifungal effect
(Wang et al., 2011). Infections due to bacterial and fungal species
remain a serious therapeutic problem. Emerging resistance of these
species is seriously decreasing the number of effective antimi-
crobials. The industry has tended to reduce the use of chemical
preservatives in their products due to increasing pressure of con-
sumers or legal authorities, to either completely remove or to adopt
preservatives natural (Nychas, 1995). Medicinal plants and their
compounds are potentially useful sources of antimicrobial com-
pounds. A wide range of medicinal plant parts extract is used as
raw drugs and they had varied medicinal properties. The different
parts used include root, leaves, fruits, stems, flowers, and modified
plant organs. Numerous studies have been published on the antimi-
crobial activities of plant compounds against many different types
of microbes, including food-borne pathogens (Tassou et al., 2000;
Friedman et al., 2002; Mimica-Dukić et al., 2004; Rančić et al., 2005;
Glamočlija et al., 2006; Soković and Van Griensven, 2006; Kukić
et al., 2008). Due to these properties, ancient time spices and herbs

http://dx.doi.org/10.1016/j.indcrop.2014.05.021
0926-6690/© 2014 Published by Elsevier B.V.
 K. Rashed et al. / Industrial Crops and Products 59 (2014) 210–215
have been used as antimicrobial agents. The present study inves-
tigated antibacterial and antifungal activities of methanol extract
and phenolic compounds isolated from D. virginiana fruits.
2. Materials and methods
2.1. Experimental
UV/Vis: Shimadzu UV–vis recording spectrophotometer model-
UV 240 (NRC, Egypt). Spectroscopic data: NMR–Varian (1 H
NMR, 400 MHz, 13 C NMR, 100 MHz). MS (Finnigan MAT SSQ
7000, 70 ev). Thin layer chromatography (TLC) F254 plates.
Sephadex LH-20 (Pharmacia Fine Chemicals). Solvent mixtures,
BAW (n-butanol:acetic acid:water 4:1:5 upper phase, 15% acetic
acid:water:glacial acetic acid: 85:15). Paper Chromatography (PC)
Whatman No. 1 (Whatman Led.Maid Stone, Kent, England) sheets
for qualitative detection of flavonoids and sugars were used.
2.2. Plant material
The fruits of D. virginiana were collected from the Agricultural
Research Centre, Giza, Egypt in October 2010 and the plant was
identified by Dr. Mohammed El-Gebaly, Department of Botany,
National Research Centre (NRC), and by Mrs. Tereeza Labib consult-
ant of plant taxonomy at the Ministry of Agriculture and director
of Orman botanical garden, Giza, Egypt. A voucher specimen is
deposited in the herbarium of Agricultural Research Centre, Giza,
Egypt.
2.3. Extraction and isolation of the bioactive compounds
The fruits (650 g) of D. virginiana were extracted with methanol
70% at room temperature (20–25 ◦ C) till exhaustion. The extract
was filtered and concentrated under reduced pressure and the
weight of dried crude extract obtained was 34 g. The extract (34 g)
was defatted with n-hexane and the sugars were precipitated by
ethyl alcohol absolute and the residue of the extract (22 g) was
subjected to silica gel column chromatography using an increasing
gradient of ethyl acetate (EtOAc) in methylene chloride (CH2 Cl2 )
up to 100%, followed by an increasing gradient of methanol up to
100%. Four fractions A–D were collected. Fraction A (4.75 g) was
obtained from CH2 Cl2 :EtOAc (60:40) and it was further subjected
to preparative paper chromatography using BAW (n-butanol:acetic
acid:water 4:1:5 upper phase) as eluent. Two violet bands under
short Ultraviolet (UV) light were detected and each band was
cutted off and was washed with methanol to give compounds 1
and 2 which were purified through sephadex LH-20 column using
methanol alcohol as eluent. Fraction B (3.45 g) was eluted with
EtOAc:CH2 Cl2 (90:10), and yielded compound 3 which was puri-
fied through sephadex LH-20 column using methanol alcohol as
eluent. Fraction C (2.85 g) was eluted with EtOAc/MeOH (95:5) and
yielded compounds 4 and 5 which were purified through Sephadex
LH-20 column using mixture of methanol/distilled water. Frac-
tion D (5.35 g) was eluted with EtOAc:MeOH (70:30) and afforded
compounds 6, 7 and 8 which also were subjected further column
chromatography using sephadex LH-20 using mixtures of MeOH
and distilled water.
2.4. Acid hydrolysis of flavonoid glycosides
Solutions of 5 mg of compounds 6, 7, and 8 in 5 ml 10% HCl was
heated for 5 h. The aglycones were extracted with ethyl acetate
and identified by co-TLC with authentic standards. The sugars in
the aqueous layer were identified by co-paper chromatography
211
(co-PC) with authentic markers on Whatman No. 1 sheets in solvent
system (n-BuOH-AcOH-H2 O 4:1:5 upper layer).
2.5. Antibacterial and antifungal assays
2.5.1. Determination of antibacterial activity
The following Gram (−) (Enterobacter cloacae human iso-
late, Escherichia coli (ATCC 35210), Pseudomonas aeruginosa (ATCC
27853), and Salmonella typhimurium (ATCC 13311), and Gram
(+) bacteria (Bacillus cereus clinical isolate, Listeria monocytogenes
(NCTC 7973), Micrococcus flavus ATCC (10240), and Staphylococcus
aureus (ATCC 6538) were used. The organisms were obtained from
Mycological Laboratory, Department of Plant Physiology, Institute
for Biological Research “Siniša Stanković”, University of Belgrade,
Serbia. The antibacterial assay was carried out by microdilution
method (Hanel and Raether, 1988; Espinel-Ingroff, 2001). The
bacterial suspensions were adjusted with sterile saline to a con-
centration of 1.0 × 105 CFU/ml. The inocula were prepared daily and
stored at 4 ◦ C until use. Dilutions of the inocula were cultured on
solid medium to verify the absence of contamination and to check
the validity of the inoculum. All experiments were performed in
duplicate and repeated thrice. The minimum inhibitory and bac-
tericidal concentrations (MICs and MBCs) were determined using
96-well microtitre plates. The bacterial suspension was adjusted
with sterile saline to a concentration of 1.0 × 105 CFU/ml. The tested
compounds and extract were added (1 and 10 mg/ml) in broth
Triptic Soy broth (TSB) medium (100 ␮l) with bacterial inoculum
(1.0 × 104 CFU per well) to achieve the wanted concentrations.
The microplates were incubated at rotary shaker (160 rpm) for
24 h at 37 ◦ C. The lowest concentrations without visible growth
(at the binocular microscope) were defined as concentrations that
completely inhibited bacterial growth (MICs). The MBCs were
determined by serial sub-cultivation of 2 ␮l into microtitre plates
containing 100 ␮l of broth per well and further incubation for 24 h.
The lowest concentration with no visible growth was defined as the
MBC, indicating 99.5% killing of the original inoculum. The optical
density of each well was measured at a wavelength of 655 nm by
microplate manager 4.0 (Bio-Rad Laboratories) and compared with
a blank and the positive control. The antibiotics streptomycin and
ampicillin were used as positive controls (1 mg/ml in sterile phys-
iological saline). All the experiments were performed in duplicate
and repeated thrice.
2.5.2. Determination of antifungal activity
The used fungi; Aspergillus fumigatus (ATCC 1022), Aspergillus
versicolor (ATCC 11730), Aspergillus ochraceus (ATCC 12066),
Aspergillus niger (ATCC 6275), Trichoderma viride (IAM 5061), Peni-
cillium funiculosum (ATCC 36839), Penicillium ochrochloron (ATCC
9112), and Penicillium var. cyclopium were obtained from Myco-
logical Laboratory, Department of Plant Physiology, Institute for
Biological Research “Siniša Stanković”, University of Belgrade,
Serbia. The micromycetes were maintained on malt agar and the
cultures were stored at 4 ◦ C and sub-cultured once a month (Booth,
1971). The antifungal assay was carried out by modified microdilu-
tion technique (Hanel and Raether, 1988; Espinel-Ingroff, 2001).
The fungal spores were washed from the surface of agar plates
with sterile 0.85% saline containing 0.1% Tween 80 (v/v). The spore
suspension was adjusted with sterile saline to a concentration of
approximately 1.0 × 105 in a final volume of 100 ␮l per well. The
inocula were stored at 4 ◦ C for further use. Dilutions of the inoculum
were cultured on solid malt agar to verify the absence of con-
tamination and to check the validity of the inoculum. Minimum
inhibitory concentration (MIC) determinations were performed by
a serial dilution technique using 96-well microtiter plates. The
tested compounds and extract were diluted in 5% of DMSO (1 mg/ml
and 10 mg/ml) and added in broth Malt medium (MA) with
212 K. Rashed et al. / Industrial Crops and Products 59 (2014) 210–215

HO 3.1. Structure elucidation of the phenolic compounds

O
Methyl gallate (1): 34 mg, white amorphous powder. UV max
HO (MeOH): 272. 1 H NMR (DMSO-d6 , 400 MHz): ı ppm 6.94 (2H, s, H-
OR 2, 6), 3.62 (3H, s, OCH3 ). 13 C NMR (DMSO-d6 , 100 MHz): ı 166.84
( COO), 146.38 (C-3, 5), 138.92 (C-4), 119.85 (C-1), 109. 18 (C-2,6),
HO 52.64 (OCH3 ).
Gallic acid (2): 23 mg, white amorphous powder. UV max
1: Methyl gallate (R = CH3)
(MeOH): 270. 1 H NMR (DMSO-d6 , 400 MHz): ı ppm 7.12 (2H, s,
2: Gallic acid (R = H) H-2,6). 13 C NMR (DMSO-d6 , 100 MHz): ı 166.94 ( COOH), 145.48
(C-3, 5), 137.82 (C-4), 121.46 (C-1), 109.62 (C-2, 6).
OH
Luteolin (3): 18 mg, yellow powder. 1 H NMR: ı ppm 12.94 (1H,
OH s, 5-OH), 7.42 (1H, d, J = 8 Hz, H-6 ), 7.38 (1H, d, J = 2 Hz, H-2 ), 6.85
(1H, d, J = 8 Hz, H-5 ), 6.65 (1H s, H-3), 6.44 (1H, d, J = 2 Hz, H-8), 6.15
HO O (1H, d, J = 2 Hz, H-6). EI-MS: m/z 286.
Quercetin (4): 15 mg, yellow powder. UV max (MeOH):
255, 267, 371; (NaOMe): 270, 320, 420; (AlCl3 ): 270, 455;
(AlCl3 /HCl): 264, 303sh, 315sh, 428; (NaOAc): 257, 274, 318, 383;
OH O (NaOAc/H3 BO3 ): 259, 387. EI-MS: m/z 302.
Myricetin (5): 12 mg, yellow powder. UV max (MeOH): 254,
3: Luteolin
272sh, 374; (NaOMe): 262sh, 285sh, 322sh, 423; (AlCl3 ): 271,
OH 316sh, 450; (AlCl3 /HCl): 266, 275sh, 308sh, 360sh, 428; (NaOAc):
269, 335(Dec.); (NaOAc/H3 BO3 ): 258, 304sh, 392. EI-MS: m/z 318.
OH Myricetin 3-O-␣-rhamnoside (6): 85 mg, yellow amorphous
powder. UV max (MeOH): 260, 296sh, 352; (NaOMe): 273, 321,
HO O 392; (AlCl3): 272, 312, 420; (AlCl3/HCl): 270, 310, 404; (NaOAc):
R1 270, 317, 364; (NaOAc/H3 BO3 ): 260, 303, 376. 1 H NMR (DMSO-d6 ,
400 MHz): ı ppm 6.89 (2H,s, H-2 , 6 ), 6.24 (1H, d, J = 2.5 Hz, H-6),
OR 6.37 (1H, d, J = 2.5 Hz, H-8), 5.28 (1H, s, H-1 ), 3.9–3.1 (m, remaining
OH
of sugar protons), 0.82 (CH3 -rhamnosyl, d, J = 6 Hz, H-6 ).
O
Myricetin 3-O-ˇ-glucoside (7): 36 mg, yellow amorphous pow-
4: Quercetin (R, R1=H) der. 1 H NMR (DMSO-d6 , 400 MHz,) ı ppm 7.16 (2H, s, H-2 , 6 ), 6.13
(1H, d, J = 2.5 Hz, H-6), ı 6.35 (1H,d, J = 2.5 Hz, H-8), 5.45 (1H, d,
5: Myricetin (R=H, R1=OH) J = 7.55, H-1 ), 3.9–3.2 (m, remaining of sugar protons). 13 C NMR
6: Myricetin 3-O-α-rhamnoside (R=rhamnose, R1=OH) (DMSO-d6 , 100 MHz): ı ppm 177.85 (C-4), 164.83 (C-7), 161.71 (C-
5), 156.81 (C-2), 156.71 (C-9), 146.49 (C-3 ), 145.87 (C-5 ), 137.97
7: Myricetin 3-O-β-glucoside (R=glucose, R1=OH) (C-4 ), 133.95 (C-3), 120.49 (C-1 ), 109 (C-2 ,6 ), 104.37 (C-10), 101.4
(C-1 ), 99.22 (C-6), 93.00 (C-8), 78.04 (C-5 ), 77.04 (C-3 ), 74.44
8: Myricetin 3-O-β-glucuronide (glucuronic acid, R1=OH) (C-2 ), 70.36(C-4 ) 61.52 (C-6 ).
Myricetin 3-O-ˇ-glucronoide (8): 28 mg, yellow amorphous
Fig. 1. Chemical structures of the phenolic compounds isolated from D. virginiana
fruits methanolic extract. powder. UV max (MeOH): 262, 298sh, 349; (NaOMe): 272, 324,
392; (AlCl3 ): 272, 312, 428; (AlCl3 /HCl): 270, 310, 404; (NaOAc):
270, 318, 366; (NaOAc/H3 BO3 ): 260, 300, 374. 1 H NMR (CD3 OD,
inoculum. The microplates were incubated at rotary shaker
400 MHz): ı ppm 7.42 (2H, s, H-2 , 6 ), 6.45 (1H, d, J = 1.2 Hz, H-8),
(160 rpm) for 72 h at 28 ◦ C. The lowest concentrations without
6.22 (1H, d, J = 1.2 Hz, H-6), 5.47 (1H, d, J = 7.5 Hz, H-1 ). 13 C NMR
visible growth (at the binocular microscope) were defined as
(CD3 OD, 100 MHz)): ı 177.52 (C-4), 174.54 (C-6 ), 165.85 (C-7),
minimal inhibitory concentrations (MICs). The fungicidal concen-
162.68 (C-5), 158.42 (C-9), 148.26 (C-2), 146.94 (C-3 , 5 ), 137.55
trations (MFCs) were determined by serial subcultivation of 2 ␮l of
(C-3), 137.15 (C-4 ), 123.36 (C-1 ), 108.86 (C-2 ,6 ), 104.75 (C-10),
tested extracts dissolved in medium and inoculated for 72 h, into
104.28 (C-1 ), 99.58 (C-8), 94.64 (C-6), 78.26 (C-3 ), 78.48 (C-5 ),
microtiter plates containing 100 ␮l of broth per well and further
75.62 (C-2 ), 73.4 (C-4 ).
incubation 72 h at 28 ◦ C. The lowest concentration with no visible
Chromatographic separation of D. virginiana fruits methanol
growth was defined as MFC indicating 99.5% killing of the orig-
extract yielded eight phenolic compounds, m-gallate, gallic
inal inoculum. The fungicides bifonazole and ketoconazole were
acid, luteolin, quercetin, myricetin, myricetin 3-O-˛-rhamnoside,
used as positive controls (1–3500 ␮g/ml). All the experiments were
myricetin 3-O-ˇ-glucoside, and myricetin 3-O-ˇ-glucuronide. The
performed in duplicate and repeated thrice.
two hydrolysable tannins, methyl gallate, and gallic acid were
detected as two violet spots under short ultraviolet (UV) and
2.6. Statistical analysis also give specific colour reaction with potassium iodate (Paul
et al., 2002), and their chemical structures were established by
Results were expressed as means ± standard deviations (SD). comparison of their spectral data with those reported in the lit-
Statistical analysis was performed by the Student’s t-test. erature (Foo, 1993). Luteolin has shown a deep purple spot which
changed into yellow colour when exposed to ammonia vapour, and
3. Results and discussion gave fluorescence yellow after spraying with AlCl3 (Stephen et al.,
2003). Quercetin and myricetin aglycones showed yellow spots
Eight phenolic compounds were isolated and identified from D. and the two compounds yielded fluorescence yellow colour after
virginiana fruits methanol extract (Fig. 1) and their structures were spraying with AlCl3 (Lawrence et al., 2005). The three myricetin gly-
elucidated on the basis of UV, 1 H NMR, 13 C NMR and MS analyses. cosides, myricetin 3-O-˛-rhamnoside, myricetin 3-O-ˇ-glucoside,
 K. Rashed et al. / Industrial Crops and Products 59 (2014) 210–215 213

myricetin 3-O-ˇ-glucuronide afforded a deep purple spot for each

compound under UV light and changed into yellow colour when

0.15 ± 0.05

0.15 ± 0.06

0.15 ± 0.03

0.15 ± 0.03
0.15 ± 0.00

0.30 ± 0.01
0.30 ± 0.05
0.50 ± 0.06

0.20 ± 0.05
0.15 ± 0.00
0.20 ± 0.06
0.10 ± 0.00

0.20 ± 0.00
0.1 ± 0.05

0.1 ± 0.05
0.1 ± 0.00
exposed to ammonia vapour, and yielded fluorescence yellow
after spraying with AlCl3 (Lawrence et al., 2005). The chemical

Amp

MBC
MIC
investigation of myricetin-O-glycosides were followed by paper
chromatography to identify the hydrolytic flavonoid-O-glycoside
products whether aglycone (myricetin) and sugar moieties, rham-

0.125 ± 0.015
0.25 ± 0.003
0.25 ± 0.003
0.05 ± 0.005

0.05 ± 0.006

0.05 ± 0.005

0.05 ± 0.006
0.15 ± 0.05
0.30 ± 0.03
0.10 ± 0.03
0.50 ± 0.06

0.10 ± 0.06

0.10 ± 0.03
0.50 ± 0.00
nose, glucose and glucuronic acid, respectively. The identification

0.1 ± 0.03

0.10 ± 0.0
of the isolated compounds was confirmed by co-chromatography

MBC
MIC
with authentic samples, UV and NMR spectroscopy and MS

Str
spectrometry, and the chemical structures were established by
comparison of their spectral data with those reported in literature

0.05 ± 0.000
(Harborne and Mabry, 1982; Markham, 1982).

0.20 ± 0.03
0.40 ± 0.06
0.60 ± 0.06
0.20 ± 0.03
0.40 ± 0.06
0.10 ± 0.03
0.30 ± 0.05
0.10 ± 0.05
0.40 ± 0.08
0.40 ± 0.05
0.50 ± 0.06

0.20 ± 0.03
0.20 ± 0.03
0.30 ± 0.06
0.05 ± 0.00
3.2. Antimicrobial activity

MBC
MIC
Ext.
Methanol extract of D. virginiana and the phenolic compounds

0.025 ± 0.003

0.025 ± 0.003
isolated from the extract were assayed in vitro for their antibacterial

0.005 ± 0.000
0.04 ± 0.006

0.05 ± 0.003
0.05 ± 0.006
0.05 ± 0.005

0.04 ± 0.003
0.05 ± 0.005

0.05 ± 0.005

0.04 ± 0.006
0.05 ± 0.006
0.04 ± 0.000

0.25 ± 0.03
0.40 ± 0.05

0.40 ± 0.01
and antifungal activities against gram positive and gram nega-
tive bacteria and microfungi. The results of antibacterial activity

MBC
MIC
are reported in Table 1. All the tested compounds, and extract

8
showed antibacterial activity against all the bacterial species.
Minimal inhibitory concentrations (MIC) of the compounds are

0.025 ± 0.003
0.04 ± 0.005
0.01 ± 0.006
0.01 ± 0.003
0.01 ± 0.006

0.01 ± 0.005
0.04 ± 0.006

0.04 ± 0.005

0.05 ± 0.006

0.04 ± 0.003
0.04 ± 0.000

0.02 ± 0.000

0.05 ± 0.000
ranged from 0.001 to 0.25 mg/ml, while bactericidal concentra-

0.25 ± 0.03
0.40 ± 0.05

0.40 ± 0.06
tion (MBC) is in range of 0.0025–0.4 mg/ml. The antibacterial

MBC
potential of these compounds could be expressed as follows: Com-

MIC
Minimum inhibitory (MIC) and bactericidal concentration (MBC) of methanol extract of D. virginiana and its phenolic compounds (mg/ml).

7
pound 5 > 4 > 3 > 1 > 2 > 8 > 7 > 6. Streptomycin showed MIC in range
of 0.05–0.25 mg/ml, and MBC of 0.1–0.5 mg/ml, while ampicillin
showed inhibitory effect at range of 0.1–0.3 mg/ml and bacteri-

0.05 ± 0.006
0.05 ± 0.006
0.06 ± 0.006
0.07 ± 0.005

0.06 ± 0.003
0.05 ± 0.005
0.07 ± 0.006

0.07 ± 0.006
0.04 ± 0.006
0.06 ± 0.006
0.04 ± 0.003
0.05 ± 0.006

0.06 ± 0.005
0.05 ± 0.000

0.06 ± 0.000

0.04 ± 0.000
cidal at range of 0.15–0.5 mg/ml. All the tested phenolic compounds
showed higher antibacterial activity than both antibiotics, except
MBC
MIC
against P. aeruginosa where MIC and MBC are slightly higher
6

than for ampicillin and streptomycin. The most sensitive bacterial 0.0025 ± 0.0003
species on these compounds are B. cereus and S. typhimurium while 0.005 ± 0.0006
0.005 ± 0.0005

0.005 ± 0.0006

0.015 ± 0.003

0.015 ± 0.006
0.025 ± 0.003
0.015 ± 0.006
0.020 ± 0.000
0.01 ± 0.005
0.01 ± 0.006
0.02 ± 0.005

0.02 ± 0.005
the most resistant one is P. aeruginosa. Methanol extract of D. vir-

0.15 ± 0.03
0.30 ± 0.06
giniana showed also a strong antibacterial activity with inhibitory 0.01 ± 0.00

concentration of 0.05–0.4 mg/ml, and bactericidal 0.2–0.6 mg/ml.

MBC
MIC

The extract proved lower antibacterial activity than the tested com-
5

pounds. The effect of extract was slightly lower or similar to both

0.0025 ± 0.0003
0.005 ± 0.0006
0.005 ± 0.0005

0.002 ± 0.0005
0.001 ± 0.0000

antibiotics. The results of antifungal activity of the tested com-

0.015 ± 0.006

0.015 ± 0.006
0.025 ± 0.003

0.015 ± 0.003
0.030 ± 0.006
0.01 ± 0.005
0.03 ± 0.003

0.02 ± 0.006
0.03 ± 0.003
0.20 ± 0.01
0.30 ± 0.06
pounds against eight fungi are presented in Table 2. It can be seen
that both the compounds, and extract showed antifungal effect.
MBC
MIC

MIC of the compounds is in range of 0.001–0.05 mg/ml and MFC

4

is in range of 0.005–0.06 mg/ml. Antifungal potential could be pre-

sented as follows: Compound 5 > 4 > 3 > 1 > 2 > 7 > 8 > 6. The majority
0.0015 ± 0.0003
0.0025 ± 0.0003
0.0025 ± 0.003

0.015 ± 0.006

0.015 ± 0.003
0.035 ± 0.005
0.015 ± 0.003

0.025 ± 0.003
0.035 ± 0.005

0.035 ± 0.003
0.030 ± 0.005

0.020 ± 0.006
0.005 ± 0.000

of the compounds showed the highest activity against A. fumigatus,

0.01 ± 0.006

0.20 ± 0.06
0.30 ± 0.05

while P. var. cyclopium was the most resistant species to the com-
pounds. The commercial antifungal agent, bifonazole, showed MIC
MBC
MIC

at range of 0.10–0.20 mg/ml and MFC at range of 0.20–0.25 mg/ml.

3

Ketoconazole showed fungistatic activity at 0.15–2.50 mg/ml and

fungicidal effect at 0.20–3.50 mg/ml. All the tested compounds
0.025 ± 0.003

0.25 ± 0.003
0.04 ± 0.005
0.01 ± 0.006

0.01 ± 0.005
0.04 ± 0.003
0.04 ± 0.006
0.05 ± 0.003
0.04 ± 0.005
0.05 ± 0.006

0.05 ± 0.005

0.01 ± 0.006
0.04 ± 0.006
0.04 ± 0.000

0.40 ± 0.06

0.40 ± 0.08

were more effective than both mycotics against all fungi. Methanol
extract showed MIC at range of 0.03–0.06 mg/ml and MFC at range
MBC
MIC

of 0.125–0.60 mg/ml. It can be seen that the extract showed lower

2

antifungal potential than all the tested compounds but it was more
effective than bifonazole and ketoconazole.
0.05 ± 0.0005
0.005 ± 0.000

0.005 ± 0.000
0.25 ± 0.005
0.04 ± 0.005
0.01 ± 0.006

0.04 ± 0.005

0.04 ± 0.006

0.04 ± 0.005

0.04 ± 0.005
0.04 ± 0.000
0.01 ± 0.000

0.01 ± 0.000

0.01 ± 0.000

Methanol extract of D. virginiana and its phenolic compounds

0.40 ± 0.05

0.40 ± 0.01

were tested for their antibacterial and antifungal activities. From

MBC

the obtained results, it could be noticed that all flavonoid aglycones

MIC
1

(myricetin, quercetin, and luteolin) showed better antibacte-

rial and antifungal activities than methyl galate and gallic acid
typhimurium
Escherichia coli
Staphylococcus
Bacillus cereus

Listeria mono-

and other flavonoid glycosides (myricetin 3-O-ˇ-glucuronide,

Pseudomonas
aeruginosa
Enterobacter
Micrococcus

cytogenes

Salmonella

myricetin 3-O-ˇ-glucoside, myricetin 3-O-˛-rhamnoside). Among

cloacae
aureus
Bacteria

flavus

the compounds, the uppermost antibacterial, as well as the

Table 1

highest antifungal activity were observed for myricetin (5), fol-

lowed by quercetin (4) and luteolin (3). Antimicrobial activities
214 K. Rashed et al. / Industrial Crops and Products 59 (2014) 210–215

of those compounds were reported on different bacterial and

fungal strains different from our present study (Aljančić et al.,

0.15 ± 0.03
0.20 ± 0.03
0.50 ± 0.05
0.20 ± 0.03
0.50 ± 0.05

0.20 ± 0.06
0.20 ± 0.06
0.50 ± 0.05

0.20 ± 0.03
0.50 ± 0.06

0.20 ± 0.03
0.30 ± 0.06
1999; Tshikalange et al., 2005). Similar results were also pre-

2.5 ± 0.3
3.5 ± 0.5
1.0 ± 0.3
1.0 ± 0.3
viously published for some flavonoids (aglycones and flavonoid
Ketoc

MFC
MIC
glycosides) where luteolin showed relatively higher activities
than did other compounds and it was more effective against
fungi than against bacteria (Zhu et al., 2004; Kukić et al., 2008).
0.15 ± 0.03

0.15 ± 0.03

0.15 ± 0.03

0.15 ± 0.05

0.25 ± 0.05

0.25 ± 0.05
0.20 ± 0.06
0.10 ± 0.05
0.20 ± 0.03

0.20 ± 0.06

0.20 ± 0.03

0.20 ± 0.03
0.20 ± 0.06

0.20 ± 0.03

0.10 ± 0.05
0.20 ± 0.06
Among 38 flavonoids tested, only myricetin showed activity
against the gram-negative bacteria B. cepacia and K. pneumo-
MFC
MIC

niae (Bylka et al., 2004). Those authors reported that certain

Bif

features relating to flavonoid structure and antimicrobial activ-

ity can be identified. From 38 different flavonoids tested, the
0.125 ± 0.015

0.125 ± 0.015

0.125 ± 0.015

0.125 ± 0.015

0.125 ± 0.015
0.06 ± 0.005

0.06 ± 0.003

0.03 ± 0.003
0.60 ± 0.006
0.06 ± 0.005

0.06 ± 0.003

0.06 ± 0.003

0.06 ± 0.003

0.06 ± 0.005
active flavonoids were polyhydroxylated (myricetin, datiscetin,
0.60 ± 0.05

0.60 ± 0.05 quercetin, luteolin and kaempferol), except flavone which does
MFC

not contain any hydroxyl group. These active flavonoids had

MIC
Ext.

in common the obligatory C-4 keto group and hydroxyl group

substitutions at C-3, C-5 and C-7 and they have at least one
0.025 ± 0.005
0.035 ± 0.005
0.025 ± 0.005
0.035 ± 0.003
0.025 ± 0.003
0.035 ± 0.005
0.015 ± 0.005
0.035 ± 0.003

0.025 ± 0.005
0.035 ± 0.003
0.035 ± 0.003

0.035 ± 0.005
0.035 ± 0.000

hydroxyl group on ring B. The following order of potency was

0.02 ± 0.006

0.04 ± 0.008
0.05 ± 0.01

observed for the B-ring hydroxyl group configuration: myricetin

(3 ,4 ,5 -trihydroxy) > quercetin (3 ,4 -dihydroxy) > luteolin (3 ,4 -
MFC
MIC

dihydroxy). These observations indicate that the more hydrophilic

8

flavonols or flavones are better inhibitors than less hydrophilic

0.025 ± 0.005
0.035 ± 0.003

0.025 ± 0.005

0.015 ± 0.005

0.025 ± 0.005
0.035 ± 0.005

ones. These observations suggest that the hydroxyl group at C-3 is

0.02 ± 0.003

0.01 ± 0.006
0.03 ± 0.003
0.01 ± 0.006
0.03 ± 0.003

0.03 ± 0.003

0.03 ± 0.003
0.05 ± 0.005
0.03 ± 0.003
0.04 ± 0.006

required for activity. This requirement, however, is not necessary

for the flavone skeleton, as in the case of luteolin. The position of
MFC
MIC

the free hydroxyl groups in the molecule may also be important for
7

activity. We may note that with myricetin (3 ,4 ,5 -trihydroxy), the
Minimum inhibitory (MIC) and fungicidal concentration (MFC) of methanol extract of D. virginiana and its phenolic compounds (mg/ml).

tri-hydroxy substitution at position 3 , 4 , and 5 on B-ring is par-

0.03 ± 0.001
0.05 ± 0.005
0.04 ± 0.001
0.05 ± 0.005
0.04 ± 0.006
0.05 ± 0.003
0.04 ± 0.003
0.05 ± 0.006

0.06 ± 0.003
0.03 ± 0.003
0.05 ± 0.005
0.03 ± 0.006
0.04 ± 0.006
0.03 ± 0.006
0.05 ± 0.01

0.05 ± 0.01

ticularly important for the activity. All of these are conformed in

our research where luteolin showed lower activity than myricetin
MFC
MIC

and quercetin. The results obtained showed that total methanol

6

extract showed antimicrobial activity against all the microorgan-

isms tested, but lower than flavonoid compounds. Rauha et al.
0.0025 ± 0.00003

0.0025 ± 0.00005
0.0025 ± 0.0005

0.015 ± 0.0005
0.005 ± 0.0006

0.005 ± 0.0003
0.001 ± 0.0003

0.005 ± 0.0003

(2000) reported that the plant extract which contain flavonoids in

0.025 ± 0.005

0.015 ± 0.005
0.01 ± 0.006
0.01 ± 0.005

0.01 ± 0.006

0.02 ± 0.003
0.05 ± 0.005
0.01 ± 0.003

glycosidic form had lower antimicrobial potential than hydroxi-

lated or pure compounds. Hedin and Waage, (1986) also reported
MFC

that the antimicrobial activity of flavonoid glycosides is lower

MIC
5

than aglycones. The extracts that contained flavonoids had shown

antimicrobial activity only after hydrolysis with enzymes or acids
0.0025 ± 0.0005

0.0025 ± 0.0003

0.0025 ± 0.0005
0.005 ± 0.0003

0.005 ± 0.0006
0.001 ± 0.0005

0.005 ± 0.0003

(Luijenijk, 1995). Soković et al. (2000) reported that the ethanol

0.005 ± 0.001
0.02 ± 0.006
0.01 ± 0.005
0.02 ± 0.006
0.01 ± 0.005
0.02 ± 0.003

0.02 ± 0.006
0.01 ± 0.006
0.02 ± 0.005

extract hydrolyzed with ˇ-glucosidase showed a greater antifungal

activity against investigated micromycetes than the total extract
MFC
MIC

and that one hydrolyzed with HCl. Antimicrobial activity of the total
4

extract may be due to the presence of some aglycones, unstable

flavonoid glycosides, or some other bioactive secondary metabo-
0.0025 ± 0.0005
0.005 ± 0.0003
0.005 ± 0.0006

0.005 ± 0.0006
0.025 ± 0.005

0.035 ± 0.006

0.015 ± 0.005

lites. This is in agreement with the literature that the plant extracts
0.01 ± 0.006
0.02 ± 0.003
0.01 ± 0.006
0.03 ± 0.005
0.01 ± 0.003

0.02 ± 0.006

0.03 ± 0.003

0.03 ± 0.003
0.03 ± 0.000

generally contained flavonoids in glycosidic form. This may be the

reason why the plant extract did not produce as marked inhibition
MFC
MIC

as some fractionated extracts or as many of the pure compounds.

3

Definitely, flavonoid glycosides failed to show low activity against

the most microorganisms (Rauha et al., 2000).
0.025 ± 0.005
0.035 ± 0.005

0.035 ± 0.005

0.035 ± 0.005

0.015 ± 0.003

0.025 ± 0.005

0.015 ± 0.005
0.035 ± 0.005
0.01 ± 0.006

0.03 ± 0.003
0.04 ± 0.005

0.03 ± 0.006

0.03 ± 0.006
0.03 ± 0.006
0.05 ± 0.005
0.01 ± 0.000
MFC
MIC

4. Conclusion
2

This present study investigated antibacterial and antifungal

0.005 ± 0.0006

0.005 ± 0.0003

0.005 ± 0.0006
0.035 ± 0.005
0.025 ± 0.005

0.015 ± 0.003
0.005 ± 0.001
0.005 ± 0.000

0.005 ± 0.000
0.02 ± 0.006

0.03 ± 0.003

0.03 ± 0.006

0.03 ± 0.001

0.03 ± 0.006

0.03 ± 0.003

0.03 ± 0.006

activities of D. virginiana fruits methanol extract and its isolated

phenolic compounds. The tested compounds and extract showed
significant antifungal than antibacterial activity. Growth of tested
MFC
MIC

microorganisms responded differently to the methanol extract and

1

its phenolic compounds, which indicated that these drugs may have
ochrochloron

funiculosum

different modes of action or that the metabolism of some microor-

veruccosum

Trichoderma
fumigatus

ochraceus
versicolor

Penicillium

Penicillium

Penicillium
Aspergillus

Aspergillus

Aspergillus

Aspergillus

ganisms was able to overcome the effect of the tested compounds.

viride
niger

So that it could be the another reason for lower antimicrobial

Fungi
Table 2

activity of methanol extract than its phenolic compounds. The

results in this study proved considerable antimicrobial activity for
 K. Rashed et al. / Industrial Crops and Products 59 (2014) 210–215
D. virginiana fruits methanol extract, and its phenolic compounds.
This present study provided scientific evidence of traditional
medicinal uses of D. virginiana, and confirmed the concept that the
ethnobotanical approach for screening plants as potential sources
for bioactive substances could be successful.
Conflict of interest
There is no conflict of interest associated with the authors of this
paper.
Acknowledgement
The authors are grateful to Serbian Ministry of Education, Sci-
ence and Technological Development, grant number 173032 for
financial support.
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