University of Africa Journal of Sciences (U.A.J.S., Vol.

2, 77-91)

Characterization of Anti-microbial Compounds

Isolated from Mangifera indica L seed kernel
Intisar Salih Ahmed1, Mahgoub Shareif El Tohami2, Aisha Zoheir Almagboul3, Rob
Verpoorte1
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Abstract
Potential antimicrobial compounds from Mangifera indica L. seed
kernel were isolated by the bioassay guided fractionation process and
characterized by NMR and LC-MS. The extracts and isolated fractions
were evaluated for their antimicrobial activity against Bacillus
subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas
aeruginosa, and the fungal strains Aspergillus niger and Candida
albicans. Two phenolic compounds and two flavonoids were isolated
from ethyl acetate extract and their chemical structure was elucidated.
Key words: Mangifera indica L., seed kernel, antimicrobial activity.

Introduction
Mango is the vernacular name of Mangifera indica L.
(Anacardiaceae), a well-known tropical fruit. Most parts of the tree are
used medicinally for abscesses, tumour, snake bite, datura poisoning,
wounds, diarrhoea, indigestion, liver disorders, excessive urination,
anemia, tetanus, bronchitis and asthma. Aqueous extract of mango
bark (vimang) is used in Cuba by patients suffering from elevated
stress (Garrido et al., 2001).
1

Institute of Biology, Leiden University, Netherlands.

Faculty of Pharmacy, Omdurman Islamic University, Sudan.
3
Medicinal and Aromatic Plants Research Institute, Sudan.
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The flowers of Mangifera indica yielded alkyl gallates and some

essential oil were isolated from the leaves. The fruit pulp contains cis9, 15-octadecadieroic acid, vitamins A and C, -carotene and
xanthophylls.
Seven phenolic constituents; gallic acid, 3,4-dihydroxy benzoic acid,
gallic acid methyl ester, gallic acid propyl ester, mangiferin, (+)catechin, (-) epicatechin were isolated from stem bark extract.
Furthermore, mango fruit is promising source of phenolic compounds
that might be used as natural antioxidants, since it contains seven
quercetin O-glycosides, one kaempferol O-glycoside, four xanthone
C-glycosides
and
the
flavonol
hexoside
(www.mdidea.com/products/herbtract/ mangiferin/research.html).
Kraipinit et al.
(www.scisoc.or.th/stt/34/sec_i/paper/STT34_I_I0017.pdf) evaluated
the antibacterial activity of alcoholic extract of mango seed kernel
against Bacillus cereus, Salmonella typhi, Staphylococcus aureus and
Escherichia coli, and promising results were obtained.
Kabuki et al. (2000) tested the antibacterial activity of Mangifera
indica seeds extracts against Gram positive and Gram negative
bacteria. The seed kernel extracts was found to contain Phellandrene,
-pinene, ambolic acid, ascorbic acid, -carotene, gallic acid,
gallotannic acid, mangifelic acid, mangiferol peroxidase, phenyl
alanin and proline.
Ahmed et al. (2005) investigated the antimicrobial activity of
chloroform, methanol and aqueous extracts of Mangifera indica seeds
kernel against Bacillus subtilis, Staphylococcus aureus, Escherichia
coli, Proteus vulgaris, Pseudomonas aeruginosa, Aspergillus niger
and Candida albicans. These extracts showed high activity against all
tested organisms. The aqueous extract exhibited high activity against
both G+ and G- Proteus vulgaris, but it gave low activity against
Escherichia coli and was inactive against Pseudomonas aeruginosa.
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Extracts of diverse parts of Mangifera indica have been reported to

possess several biological and pharmacological properties (Barreto et
al., 2008).
The seed kernel extracts of three types of mango from Malaysia and
Thailand exhibited potential activity against Gram-positive and Gramnegative bacterial strains (Mirghani et al., 2009).
Sowmiya et al. (2009) indicated antibacterial activity for seed kernel
aqueous and ethanolic extracts. These extracts showed remarkable
activity against the uropathogens isolated from clinical samples of
Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus,
Klebisella pneumoniae, and Streptococcus pyogens.
The alcoholic extract of seeds exhibited anti-inflammatory activity in
acute, sub acute and chronic cases of inflammation (Shah et al., 2010).
Khammuang and Sarnthima (2011) studied the antioxidant and
antibacterial activities of four fresh mango seed extracts from Thai
varieties. Their strongest antioxidant property correlated with the total
phenol contents. All mango seed extracts showed interesting
antibacterial activity against both G+ and G- bacteria.

Materials and Methods

Plant material
Mangifera indica L. fruit was purchased from super market and was
authenticated by Yahia Suleiman- Medicinal and Aromatic Plants
Research Institute- Sudan, and a voucher sample was deposited at
Herbarium of this institute.
Extraction and liquid fractionation
Powdered air-dried kernel of Mangifera indica (500 g) was extracted
by methanol and sonicated at room temperature for 1 hr, and the
solution was filtered. The procedure was repeated two times and the
filtrates were centrifuged. The solvent was removed under reduced
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pressure. The dried extract was dissolved in 300 ml water and was
partitioned with hexane (3x300 ml), chloroform (3x300 ml) and ethyl
acetate (3x300 ml), respectively.
Phytochemical screening
Phytochemical tests were carried out on the methanolic extract of the
seed kernel of Mangifera indica using standard procedures (Harborne,
1973; Sofowora et al., 1982).
Determination of antimicrobial activity of the extract
The antimicrobial activity of crude extract was tested against Bacillus
subtilis NCTC; Staphylococcus aureus ATCC; Escherichia coli
ATCC; Pseudomonas aeruginosa ATCC; Aspergillus niger ATCC
and Candida albicans ATCC using the agar diffusion method
(Kavanagh, 1972; Collins et al., 1995).
Antibacterial and antifungal activity of reference drugs against
standard organisms
Four antibacterial reference drugs (Ampicillin, Benzyl penicillin,
Cloxacillin and Gentamicin) and two antifungal reference drugs
(Clotrimazole and Nystatin) were prepared in suspensions of four
concentrations (40, 20, 10 and 5 g/ ml for all of them except Nystatin
which was prepared in 500, 50, 25 and 12.5 g/ ml) using sterile
distilled water. They were tested against Bacillus subtilis;
Staphylococcus aureus; Escherichia coli; Pseudomonas aeruginosa;
Candida albicans and Aspergillus niger to evaluate their antibacterial
and antifungal activities, using cup plate diffusion method (Ahmed,
2007).
Determination of minimum inhibitory concentration
Various concentrations of active components from Mangifera indica
ranging between 4.0 and 6.0 g/ml were introduced into different test
tubes inoculated with an overnight culture of micro organisms diluted
to give a final concentration of 106 cells per ml. The tubes were
incubated at 37C for 24 h. The least concentration of component that
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did not permit any visible growth of the inoculated test organism in
broth culture was regarded as the minimum inhibitory concentration
(MIC) in each case (Collins et al., 1995).
Column and thin layer chromatography
Column chromatography was performed with silica gel 60 mesh in a
5 x 58 cm column and Diaion HP-20 in a 2.5 x 40 cm column.
Preparative and analytical TLC were performed using aluminum TLC
plates of 20 x 20 cm silica gel 60 F 254.
Isolation of compounds
The ethyl acetate extract of Mangifera indica seed kernel was
chromatographed on a silica gel column and eluted stepwise with a
gradient (CHCl 3 -MeOH 1:0 0:1) to give fractions, which were
further tested for their antimicrobial activities. Fractionation and
isolation were monitored by TLC with visualization under UV
( max 254 and 365 nm), using the chloroform/ ethyl acetate solvent
system. The plates were sprayed with anisaldehyde reagent followed
by heating. According to their activity and TLC patterns further
purification with preparative TLC using a chloroform/ethyl acetate
(7:3) solvent system was carried out and resulted in compounds; gallic
acid, gallic acid ethyl ester, hydroxy-xanthone glucoside and quercetin
sulphate.
Structure elucidation
The structure of active compounds was elucidated by NMR and LCMS.
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Results and Discussion

The various Phytochemical tests of Mangifera indica seeds revealed
the presence of coumarins, terpenes, tannins and flavonoids.
The methanol extract of the seed kernel was found to be a potent
antibacterial and antifungal agents, since it gave inhibition zones
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ranging between (24 - 28 mm) at 50% concentration against Bacillus

subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas
aeruginosa, and 23 mm inhibition zone against Candida albicans. The
Gram positive bacteria were slightly more sensitive than Gram
negative one. Also, the extract possessed high antifungal activity
against Candida albicans; meanwhile it was inactive to Aspergillus
niger. The result was similar to those reported by Ahmed et al. (2005),
who indicated the high activity of methanol extract of seeds against all
tested organisms. Such results confirmed the findings of Kraipinit et
al. (www.scisoc.or.th/stt/34/sec_i/paper/STT34_I_I0017.pdf), who
showed the high antimicrobial activity of alcohol extract of mango
seed kernel against Staphylococcus aureu and Escherichia coli;
Mirghani et al. (2009) and Khammuang and Sarnthima (2011)
reported the potent antibacterial activity of mango seed extracts
against both Gram positive and Gram negative bacteria.
The antibacterial activity of methanol extract against Staphylococcus
aureus, Escherichia coli, and Pseudomonas aeruginosa is higher than
40 g/ml Gentamicin. Its high antifungal activity against Candida
albicans is similar to 12.5 g/ml Nystatin. Also, its inhibition of the
Staphylococcus aureus growth is similar to that produced by 10 g/ml
Benzyl penicillin and 20 g/ml Cloxacillin.
Out of the three solvents used for liquid extraction, the ethyl acetate
extract showed high activity (25 -30 mm) for the bacteria and 26 mm
for Candida albicans (Table 1). The minimum inhibitory
concentration (MIC) of ethyl acetate extract ranged between 4.0 and
5.0 g/ml: Bacillus subtilis (MIC 4.5 g/ml), Staphylococcus aureus
(MIC 4.0 g /ml), Escherichia coli (MIC 4.5 g /ml) and
Pseudomonas aeruginosa (MIC 5.0 g/ml) and Candida albicans
(MIC 4.5 g /ml).
Many fractions have been isolated from ethyl acetate extract by silica
gel column chromatography using chloroform, methanol as the eluent
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and four active fractions (1, 2, 3 and 4) were collected purely by

preparative thin layer chromatography and were identified by
comparing their 1H NMR and LC-MS data with literature values
(www.bmrb.wisc.edu/metabolomics/query_metab.php).
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Table1 Antimicrobial activity of Mangifera indica seeds extracts and reference

drugs against standard organisms
Standard organisms used/MDIZ (mm)
Sample
B.s
S.a
E.c Ps.a As.n
C.albs
Ethyl acetate extract
28
30
29
25
12
26
Methanol extract
27
28
27
24
11
23
Fraction1
N.D
N.D N.D N.D N.D
N.D
Fraction 2
25
27
26
22
20
Fraction 3
20
22
20
18
13
16
Fraction 4
26
30
25
23
13
29
Gentamicin (40g/ml)
30
20
22
18
Benzyl penicillin(10g/ml) 28
Cloxacillin(20g/ml)
27
Nystatin (12.5g/ml)
23
B.s = Bacillus subtilis, S.a = Staphylococcus aureus, E.c = Escherichia coli,
Ps.a = Psedomonas aeruginosa, C.albs = Candida albicans, As.n = Aspergillus
niger.
N.D = Not done, MDIZ= mean diameter of inhibition zone (mm), Conc. used =
100 mg/ml at 0.1 ml/cup.

Interpretation of results:
MDIZ: >15 Sensitive, 14 15 Intermediate, < 14 Resistant, - No inhibition zone.

Structure Elucidation
Gallic acid (3, 4, 5-tri hydroxybenzoic acid)
Fraction1 was obtained from chloroform as white crystals; its melting
point 145-147C (lit 145-148C). The APCI-MS spectra of the
compound showed a molecular ion peak [M+H]+ at m/z 170 typical of
Gallic acid (molecular weight 169.119) and in agreement with its
molecular formula C 7 H 6 O 5 (Willighagen et al., 2006). The 1H NMR
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spectrum showed doublet at 7.03 ppm attributed for the proton of

aromatic ring (www.chemspider.com/Chemical-Structure.362.html).
A similar compound was isolated before by Kabuki et al. (2000) from
ethanolic extract of the seeds of Mangifera indica.
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COOH

OH

HO
OH

Gallic acid

Gallic acid ethyl ester (3, 4, 5-tri hydroxybenzoic acid ethyl ester)
It was obtained from fraction2 as white crystals melting at 151-153C
(lit 152-154C), with the molecular formula C 9 H 10 O 5 based on the
APCI-MS data which showed a molecular ion peak [M+H]+ at m/z
199 typical of Gallic acid ethyl ester (molecular weight 198.17)
(www.chemicalland21.com/lifesciences/foco/
ethylgallate.htm). The 1H NMR spectrum showed signal at 7.11 ppm
for the proton of the aromatic ring. The protons of methyl group
resonated at 1.25 ppm, whereas the signal at 2.28 ppm accounts for
the methylene proton attached to carbonyl of ester (Table 2). This
derivative of gallic acid is known for its anti-inflammatory and antioxidative properties (Kroes et al., 1991).This result confirmed the
previous findings of Khan (1989, 1992), who isolated the same
compound, beside other alkyl gallates and chromones from the
flowers and stem bark of Mangifera indica.
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Table 2 1H (500 MHz) Chemical shift of gallic acid

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ethyl ester in MeOD(ppm)

Position
ArCH 2
R

CH 3
R

Chemical shift
7.11(1H, d)
3.80(2H,d)
2.28(2H,dd)
1.59(3H, s)
0.91(3H,m)

COOC2H5

OH

HO
OH

Gallic acid ethyl ester

Hydroxy-xanthone-C-glucoside
The compound was obtained from fraction3 as a pale yellow crystals,
melts at 213-215 C, which showed a protonated molecular ion peak
[M+H]+ at m/z 374 in the positive ion APCI-MS. The 1H NMR
spectrum revealed signals at 6.41 ppm, 7.04 ppm, 7.23 ppm and 7.54
ppm attributed to the aromatic protons of the xanthone moiety. The
sugar protons resonate at 5.33 ppm and 3.70 ppm (Table 3). The
molecular formula was determined as C 19 H 18 O 8, which suggested a
hydroxy-xanthone-C-glucoside
(www.chemexper.com/search/cas/4773960.html).
This result is in good agreement with a previous phytochemical
investigation on M. indica which indicated a C-glucosyl xanthone as a
major constituent
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(www.mdidea.com/products/herbtract/mangiferin/research.html),
Wauthoz et al. (2007).
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Table 3 H (500 MHz) Chemical shift of hydroxy-xanthone-CP

glucoside in MeOD(ppm)

Position

Chemical shift

7.54(1H, s)

7.23(1H, s)

4,6

6.71(1H, d)

7,8,9

7.04(1H, m)

1`

5.35(1H, s)

Other sugar protons 3.20-3.70, m

OH
OH

H
H
O

1`

HO
H
H

OH

OH

Hydroxy-xanthone-C-glucoside

Quercetin-3-Sulphate(2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4oxo-4H-chromen-3-yl hydrogen sulfate)

Fraction4 crystallized from methanol as dark yellow crystals, melting
at 250- 253 C. The APCI-MS spectrum showed an intense molecular
ion peak [M+H]+ at m/z 383, indicating quercetin-3-sulphate
(molecular weight 381.99) which correspond to molecular formula
C 15 H 10 O 10 S and it was identified as a sulphate with significant
antimicrobial activity: specifically Klebsiella pneumoniae and
Mycobacterium tuberculosis (Habbu et al., 2009). The characteristics
of flavones 1H NMR spectra (Vega et al., 2007; Eldahshan et al.,
2008) were applied to this compound, in which the signals between
6.0-7.0 ppm were attributed to the protons adjacent to the hydroxyl
group in ring A and/or B. The signals above 7.0 ppm were attributed
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to the protons with meta position to hydroxyl group in ring B (Table

4). This result was relevant, and supported by Wu and Ming
(www.mdidea. com/products/herbtract/mangiferin/research.html),
who isolated quercetin from the leaves of Mangifera persiciformis.
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Table 4 1H (500 MHz) Chemical shift of Quercetin

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3-sulphate in MeOD(ppm)
Position

Chemical shift

6.29(1H, d)

6.44(1H, d)

2'

7.58(1H, s)

5'

6.54(1H,d)

6`

7.36(1H,m)
OH
OH

HO

O
O
O
OH

OH

Quercetin-3-sulphate

Conclusion
The methanol extract of Mangifera indica seeds, which contained
coumarins, terpenes, tannins and flavonoids showed high antibacterial
activity against Bacillus subtilis, Staphylococcus aureus, Escherichia
coli, Pseudomonas aeruginosa, and antifungal activity against
Candida albicans. Four phenolics and flavonoid compounds were
isolated and identified by 1H NMR and LC-MS.
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Acknowledgement
Intisar Salih Ahmed is grateful to Prof. Dr. R.Verpoorte/ Division of
Pharmacognosy/ Section of Metabolomics/ Institute of Biology/
Faculty of Sciences and Mathematics/ Leiden University/ Netherlands
for hosting and all facilities to perform this work.
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