New antifungal flavonoid glycoside from Vitex negundo

Article abstract of DOI:10.1016/j.bmcl.2006.09.051

Flavonoids are ubiquitous in photosynthesizing cells and are common part of human diet. For centuries, preparations containing these compounds as the principal physiologically active constituents have been used to treat human diseases. Increasingly, this class of natural products is becoming the subject of anti-infective research. Our bioactivity guided fractionation of ethanolic extract of leaves of Vitex negundo resulted in the isolation of new flavone glycoside (4) along with five known compounds 1-3, 5 and 6. All the isolated compounds were evaluated for their antimicrobial activities. The new flavone glycoside 4 and compound 5 were found to have significant antifungal activity against Trichophyton mentagrophytes and Cryptococcus neoformans at MIC 6.25 μg/ml.

Full text of DOI:10.1016/j.bmcl.2006.09.051

Bioorganic & Medicinal Chemistry Letters 17 (2007) 239–242
New antifungal flavonoid glycoside from Vitex negundoq
B. Sathiamoorthy,a Prasoon Gupta,a Manmeet Kumar,a Ashok K. Chaturvedi,b
P. K. Shuklab and Rakesh Mauryaa,*
aMedicinal and Process Chemistry Division, Central Drug Research Institute, Lucknow 226 001, India
bDivision of Fermentation Technology, Central Drug Research Institute, Lucknow 226 001, India
Received 4 July 2006; revised 15 September 2006; accepted 19 September 2006
Available online 5 October 2006
Abstract—Flavonoids are ubiquitous in photosynthesizing cells and are common part of human diet. For centuries, preparations
containing these compounds as the principal physiologically active constituents have been used to treat human diseases. Increasing-
ly, this class of natural products is becoming the subject of anti-infective research. Our bioactivity guided fractionation of ethanolic
extract of leaves of Vitex negundo resulted in the isolation of new flavone glycoside (4) along with five known compounds 1–3, 5 and
6. All the isolated compounds were evaluated for their antimicrobial activities. The new flavone glycoside 4 and compound 5 were
found to have significant antifungal activity against Trichophyton mentagrophytes and Cryptococcus neoformans at MIC 6.25 lg/ml.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Resistance to antimicrobial agents has become a global
problem since last three decades. About 2 million people
in US acquire bacterial and fungal infections each year,
of the 65% patients have resistance to at least one
drug.1,2 A similar cause of concern in other countries
including UK that leads to repeated use of antibiotic
and insufficient control of the disease.3 Second adequate
treatment of mycotic infections is difficult since fungi are
eukaryotic organisms with a structure and metabolism
that is similar to those of eukaryotic host. For this rea-
son, substantial researches in the field of anti-infectives
are now desperately needed to develop new prototype
antimicrobial agents to avert this situation.
Vitex negundo (Linn.) is a large shrub grown throughout
the India. It is one of the common plants used in tradi-
tional medicine and reported to have variety of biologi-
cal activities.5 The preparation of leaves used in
catarrhal fever and applied to sinuses and scrofulous
sores.6 Aqueous extract and oil of seeds possessed
anti-oxidant and anti-inflammatory property.7–9 Anti-
genotoxic effects were reported from the leaves of V.
negundo in combination with other constituents.10 Fla-
vonoid-rich fraction of the seeds showed anti-fertility ef-
fect in adult dogs.11 Plant also exhibited CNS depressant
activity12 and antihistamine release property.13 Flavo-
noids,14–16 iridoids,17–20 terpenes,21,22 and steroids23
are the major classes of compounds isolated from this
plant.
Development of new drugs from plants is not a new phe-
nomenon. Plants and plant derived agents have long his-
tory to clinical relevance as source of potential
chemotherapeutic agents.4 In our continuing efforts to
identify antifungal agents from plant sources, we have
studied antimicrobial activity of Vitex negundo (Leaves)
against a variety of microorganisms including Gram-po-
sitive and Gram-negative bacteria and fungi (Fig. 1).
In our study, we have found significant antifungal activ-
ity in ethanolic extract against Cryptococcus neoformans
and Trichophyton mentagrophytes. The extract of the
leaves and twigs was reported to show antibacterial
activity against Micrococcus pyogenes and Escherichia
coli.6 On bioassay guided fractionation of ethanolic ex-
tract, the activity was localized in n-butanol fraction.
Further work led to the isolation of compound (1–6)
including a new flavone glycoside (4). All the six com-
pounds were evaluated for antifungal and antibacterial
activity.
Keywords: Vitex negundo; Antimicrobial activity; Fluconazole.
q
CDRI communication no. 7053.
Corresponding author. Tel.: +91 522 2612411 18x4235; fax: +91 522
2623405/2623938/2629504; e-mail: mauryarakesh@rediffmail.com
*
Powdered leaves of the plant (5.0 kg) were extracted
with ethanol by percolation, affording (590 g) extract.
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.051

240
B. Sathiamoorthy et al. / Bioorg. Med. Chem. Lett. 17 (2007) 239–242
Figure 1. Chemical structure of isolated compounds 1–6.
Table 1. 1H and 13C data of compound 4 in DMSO-d6 (200 MHz)
Ethanolic extract (550 g) was fractionated into n-hexane
(180 g), CHCl3 (70 g) and n-butanol (240 g) soluble frac-
tions. The n-butanol soluble fraction was subjected to
column chromatography over flash silica gel (230–
400 mesh), eluted with a gradient of chloroform/metha-
nol (95:05) to methanol/water (95:05) to afford six frac-
tions (F-1 to F-6). Flash column chromatography of
fraction F-1 using CHCl3/MeOH (97:3) afforded com-
pound 1 (50 mg). Purification of F-2 using CHCl3/
MeOH (95:5) afforded compound 2 (350 mg) and with
CHCl3/MeOH (88:12) gave compound 3 (35 mg). Suc-
cessive purification of F-3 by reverse-phase flash chro-
matography, eluted with a gradient of MeOH/H2O
(30:70), afforded 4 (25 mg). Column chromatography
purification of F-4, using CHCl3/MeOH (80:20), affor-
ded 5 (500 mg). Repeated column chromatography of
fraction F-5, eluted with EtOAc/acetone/H2O
(80:18:2), yielded compound 6 (50 mg).
Position
dH (J in Hz)
dC
HMBCa
2
—
6.80 (s)
164.1
104.1
182.6
162.2
99.7
H-3, H-20, H-60
—
H-3
3
4
—
—
5
H-6
H-8
6
6.19 (d, J = 2.0)
—
7
165.0
94.8
H-6, H-8
H-6
H-8
8
6.45 (d, J = 2.0)
—
9
10
158.2
104.6
122.4
114.7
145.9
151.6
117.5
122.8
101.6
73.7
—
—
H-3, H-6, H-8
H-20, H-60, H-50
H-60
10
20
7.60 (d, J = 1.5)
—
—
30
H-100, H-50, H-20
H-20, H-60, H-50
H-600
40
500
6.97 (d, J = 8.2)
7.62 (dd, J = 8.2, 1.5)
5.21 (d, J = 6.2)
3.38 (m)
600
H-200, H-5000
H-300, H-5
H-1 , H-300, H-400
H-200, H-300, H-500
H-300, H-500
H-100, H-200, H-400
H-500, 600-OMe
—
100
200
300
3.30 (m)
3.42 (m)
76.1
72.2
Compound 4 was obtained as yellow colored solid. Po-
sitive Shinoda and Fiegel’s test indicated that compound
could be a flavonoid glycoside. The FAB-MS exhibited
molecular ion peak [M+H]+ at m/z 477 corresponding
to the molecular formula C22H20O12. IR absorption
bands at 3250, 1746, 1640, and 1498 cmꢀ1 indicated
the presence of hydroxyl group, ester moiety, conjugated
carbonyl, and aromatic ring, respectively. UV spectrum
showed kmax at 336, 329, 266, and 209 nm (MeOH). A
bathochromic shift (kmax 329, 266, 216 nm) of 7 nm of
the band II with NaOAc indicated the presence of free
7-OH group. Addition of NaOMe resulted in the bath-
ochromic shift (kmax 391, 324, 272 nm) of 55 nm of the
band I confirmed the presence of free 40-OH group.
The UV spectrum of 4 was unaffected by the addition
of NaOAc + H3BO3 (kmax 328, 266, 210 nm) which
clearly suggested the absence of ortho dihydroxyl
system.24
4
500
4.19 (d, J = 8.9)
—
3.67 (s)
75.9
600
170.0
52.9
—
600-OMe
5-OH
12.8 (s)
—
a HMBC experiment in DMSO-d6 (600 MHz).
J = 1.5 Hz), dC 114.7 for H-20, dH 6.97 (d, J = 8.2 Hz);
dC 117.5 for H-50 and dH 7.62 (dd, J = 8.2, 1.5 Hz) for
H-60. The other AX system at dH 6.19 (d, J = 2.0 Hz),
dC 99.7 and dH 6.45 (d, J = 2.0 Hz), dC 94.8 was assigned
to H-6 and H-8 protons, respectively. A singlet at dH
3.67 (dC 52.9) was assigned to methoxy group of the
esterified sugar. In case of 30, 40 dihydroxy flavone sys-
tem, studies revealed that dC value for 40 appeared
downfield24 than for 30. Hence dC 145.9, 151.6 were as-
signed to C-30 and C-40, respectively. The anomeric pro-
ton of the sugar appeared at dH 5.21 (d, J = 6.2 Hz; dC
101.6). The coupling constant of the anomeric proton
(J = 6.2 Hz) confirmed the b-linkage of the sugar.25
Using anomeric proton as starting point in 1H–1H
COSY spectrum other sugar protons were assigned,
respectively (Table 1). Downfield shift and splitting pat-
tern of H-5 sugar proton with absence of C-6 methylene
1H and 13C NMR (Table 1) spectrum showed the char-
acteristic signal for H-3 proton at dH 6.80 (s); dC 104.1.
A sharp singlet observed at dH 12.8 was assigned to the
C-5 chelated hydroxyl proton. The aromatic protons
were resolved as one ABX system at dH 7.60 (d,

B. Sathiamoorthy et al. / Bioorg. Med. Chem. Lett. 17 (2007) 239–242
Table 2. Antimicrobial activity of extract, fractions, and isolated compounds
241
Extract/fraction/pure compound
Minimum inhibitory concn (MIC) in lg/ml against
Fungi
Bacteria
3
1
2
4
5
6
7
8
9
10
11
Ethanolic extract
n-Hexane fraction
125
>500
250
250
>50
25
250
—
250
500
500
250
50
500
>500
>500
500
50
250
>500
>500
125
25
>500
>500
>500
>500
>50
>50
>50
>50
>50
>50
0.5
62.5
125
>500
62.5
12.5
25
>500
>500
>500
>500
>50
>50
>50
>50
>50
>50
2.0
125
>500
>500
250
25
>500
>500
>500
>500
50
>500
>500
>500
>500
25
Chloroform fraction
n-Butanol fraction
1*
2*
3*
4*
5*
6*
500
250
25
25
50
50
25
25
50
>50
>50
>50
50
25
>50
>50
>50
25
>50
>50
25
>50
>50
25
>50
>50
50
12.5
6.25
6.25
25
12.5
6.25
6.25
12.5
2.0
>50
>50
>50
>50
1.0
>50
25
25
25
>50
ND
50.0
25
50
2.0
Flu
Amp
ND
0.02
ND
0.09
ND
0.02
ND
0.09
1.0
ND
ND
ND
ND
ND
ND
1, Streptococcus faecalis; 2, Klebsiella pneumoniae; 3, Escherichia coli; 4, Pseudomonas aeruginosa; 5, Staphylococcus aureus; 6, Candida albicans; 7,
Cryptococcus neoformans; 8, Sporothrix schenckii; 9, Trichophyton mentagrophytes; 10, Aspergillus fumigatus; 11, Candida parapsilosis (ATCC-22019)
Flu, fluconazole; Amp, ampicillin; ND, not done; , pure compound.
*
of glucose indicated presence of carbonyl group at this
position. This observation was well supported with pres-
ence of carbonyl carbon at dC 170.0 in 13C NMR spec-
trum. The HMBC spectrum was utilized to identify
position of sugar, a long range correlation (Table 1) be-
tween H-100/C-30 confirmed the attachment of sugar at
C-30, other useful correlation between H-500/C-600,
-OCH3/C-600, H-20/C-30,40, and H-50/C-10,30 confirmed
the position of methoxyl and carbonyl group. Further
acid hydrolysis of 4 followed by co-TLC with authentic
samples glycone (methyl ester of glucuronic acid) and
aglycone (luteolin) was confirmed.26 Further D-configu-
In conclusion, we have isolated five known compounds
1–3, 5 and 6 and a new flavonoid glycoside 4 from V.
negundo through activity guided fractionation and dis-
covered the potent antifungal activity of isolated com-
pounds. The compounds 4 and 5 were found to be
most active at MIC 6.25 lg/ml among all the isolated
compounds. The further chemical transformation of
compounds 4 and 5 is under progress to improve their
biological profile.
Acknowledgments
ration of glycone was confirmed with optical rotation of
30
D
acetate derivative ½aꢁ +11.4ꢁ (CHCl3) with those
Authors are thankful to ICMR, New Delhi, for financial
assistance. We also thank Dr. Ashish Arora for the
HMBC spectral data and S. C. Tiwari for technical
assistance.
reported in the literature.27 Thus based on the foregoing
evidence, the structure of compound 4 was elucidated as
40, 5, 7-trihydroxy-30-O-b-D-glucuronic acid-600-methyl
ester, a new naturally occurring compound named vite-
gnoside. Other five known compounds, 50-hydroxy-30,
40, 3, 6, 7-pentamethoxyflavone (1)28, luteolin (2)24,
agnuside (3)17, negundoside (5)17,18, and iso-orientin
(6)29, were characterized by comparing their spectro-
scopic data with those reported in the literature.
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2 lg/ml against T. mentagrophytes. Compounds 1
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30
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