Antibacterial Activity of a New Flavone Glycoside from the Stems of Holmskioldia sanguinea Retz.

Article abstract of DOI:10.14233/ajchem.2019.22026

A new flavone glycoside was isolated from ethanolic extract of stem parts of Holmskioldia sanguinea Retz. Its structure was characterized as 3,4′-dihydroxy-5,7-dimethoxyflavone-3-O-β-D-galactopyranosyl(1→4)-O-β-D-arabinopyranosyl-4′-O-α-L-rhamnopyranoside (1) by colour reactions, chemical degradation and spectroscopic analysis. Antibacterial activity of compound 1 was evaluated against various Gram positive and Gram negative bacteria showing a significant effects.

Full text of DOI:10.14233/ajchem.2019.22026

Asian Journal of Chemistry; Vol. 31, No. 8 (2019), 1815-1818
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2019.22026
Antibacterial Activity of a New Flavone Glycoside from the Stems of Holmskioldia sanguinea Retz.
*
R.N. YADAVA and JYOTSNA RAGHUVANSI
Department of Chemistry, Dr. H.S. Gour Vishwavidyalaya (A Central University), Sagar-470003, India
*Corresponding author: E-mail: rnyadava@rediffmail.com; sjyotsna0@gmail.com
Received: 25 February 2019;
Accepted: 15 April 2019;
Published online: 28 June 2019;
AJC-19458
A new flavone glycoside was isolated from ethanolic extract of stem parts of Holmskioldia sanguinea Retz. Its structure was characterized
as 3,4′-dihydroxy-5,7-dimethoxyflavone-3-O-β-D-galactopyranosyl(1→4)-O-β-D-arabinopyranosyl-4′-O-α-L-rhamnopyranoside (1) by
colour reactions, chemical degradation and spectroscopic analysis. Antibacterial activity of compound 1 was evaluated against various
Gram positive and Gram negative bacteria showing a significant effects.
Keywords: Holmskioldia sanguinea Retz., Flavone glycoside, Antibacterial activity, Verbenaceae.
INTRODUCTION
O
Holmskioldia sanguinea Retz. belongs to Verbenaceae
OH
family which is commonly known as Kapni or Rithoul in Hindi
O
OH
OH
[1,2]. It is distributed in Punjab eastward in hills ascending
upto 1500 m and subtropical and Himalayan regions from
Kumaon to Bhutan. It’s shrub is 10-30 feet in height. The crushed
fresh leaves and shoots are applied in rheumatism and rheu-
matoid arthritis. Its extracts of leaves and stembark are used
in the treatment of dysentery.Various pharmacological activities
like analgesic, anticancer, diuretic, anti-inflammatory, CNS
depressive and antimicrobial activities have been reported from
aerial parts of plant [3-8]. A large number of phytochemical
compounds isolated from plant have been reported by earlier
workers [9-11].
In the present work, the isolation and structural charac-
terization of a new compound from ethanolic extract of stems
parts of this plant which is reported as 3,4′-dihydroxy-5,7-
dimethoxyflavone-3-O-β-D-galactopyranosyl(1→4)-O-β-D-
arabinopyranosyl-4′-O-α-L-rhamnopyranoside (1, Fig. 1) which
showed a significant antibacterial activity.
H3CO
O
O
HO
O
OH
O
O
O
OH
OH
OCH3
HO
HO
Fig. 1. Structure of 3,4′-dihydroxy-5,7-dimethoxyflavone-3-O-β-D-
galactopyranosyl(1→4)-O-β-D-arabinopyranosyl-4′-O-α-L-
rhamnopyranoside (1)
1
spectrometer in MeOH. H NMR spectra were recorded on
Bruker DRX 300 MHz spectrometer in CDCl3 using TMS as
internal standard. 13C NMR spectra were recorded on Bruker
DRX 75 MHz spectrometer using CDCl3. The chemical shift
values are reported in ppm (δ) units and coupling constant (J)
in Hz. The FAB mass spectra were recorded on Jeol-SX (102)
mass spectrometer.
Plant material: The stems of the plant was collected locally
around Sagar region and identified by taxonomist, Department
of Botany, Dr. H.S. GourVishwavidyalaya, Sagar, India.A voucher
specimen (Bot/H/05/12/06) has been deposited in Natural Products
Laboratory, Department of Chemistry of this university.
EXPERIMENTAL
All the melting points were determined on a thermoelec-
trically melting point apparatus and are uncorrected. The IR
spectra were recorded at Shimadzu FT-IR 8400S in KBr disc.
UV spectra were determined on Shimadzu-120 double beam
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1816 Yadava et al.
Asian J. Chem.
Extraction and isolation:The air dried and powdered stems
OH
(5 Kg) of the plant were extracted with ethanol in Soxhlet
extractor for consecutive 7 days . The total ethanolic extract
was concenterated under reduced pressure and successively
partitioned with petroleum ether, chloroform, ethyl acetate,
acetone and methanol. The methanol soluble fraction of the
plant was concenterated under reduced pressure in the rotatory
evaporator to yield a light brown viscous mass which was
subjected to TLC examination over silica gel-G using nBAW
(4:1:5) as solvent and iodine as visualizing agent and showed
two spots on TLC. The compound was separated and purified
by column chromatography over silica gel using CHCl3:MeOH
(6:4) as eluent.The two compounds were referred as 1 and 2.
Compound 2 was obtained in small amount hence rejected
and further characterization of compound 1 was carried out.
The solvent was removed and crystallized from ether, which
yielded compound 1 (1.92 g).
H3CO
O
O
OH
OCH3
Fig. 2. Structure of compound 1A
the removal of aglycone was neutralized with BaCO3 and the
BaSO4 was filtered off. The filterate was concenterated under
reduced pressure and subjected to paper chromatography
examination using nBAW (4:1:5) as solvent system and aniline
hydrogen phthalate as spraying agent. The methylated sugars
were identified as , 2,3,4-tri-O-methyl-L-rhamnose (RG 1.01)
2,3,4,6-tetra-O-methyl-D-galactose (RG 0.78) and 2,3-di-
methoxy-D-arabinose (RG 0.64) .
Enzymatic hydrolysis of compound 1: Compound 1 (30
mg) was dissolved in MeOH (20 mL) and hydrolyzed with
equal volume of takadiastase enzyme.The contents were allowed
at room temperature for 3 days and filtered. The hydrolyzate
was concenterated and subjected to paper chromatography using
nBAW (4:1:5) as solvent system and aniline hydrogen phthalate
as spraying agent which showed the presence of L-rhamnose
(Rf 0.37) . The proaglycone was dissolved in MeOH (20 mL)
and further hydrolysed with almond emulsion enzyme at room
temperature yielded aglycone identified as 5,7-dimethoxy-3,4′-
dihydroxy flavone and sugars were identified as D-arabinose
(Rf 0.21) and D-galactose (Rf 0.16).
Spectral data of compound 1A: m.f. C17H14O6, m.p. 224-
225 ºC, [M+] m/z 314. Elemental analysis of C17H14O6 calcd.
(found) (%): C 65.04 (65.02), H 4.87 (4.89), O 30.54 (30.57).
UV (MeOH) λmax: 273, 323, 379 nm. IR (KBr, λmax, cm-1): 3450
(-OH), 1652, 1600, 1582, 1514. 1H NMR (300 MHz, CDCl3): δ
14.91 (1H, s, 3-OH), 5.2 (1H, s, 4′-OH), 3.78 (3H, s, C5-OCH3),
3.76 (3H, s, C7-OCH3), 6.07 (1H, s, H-6), 6.05 (1H, s, H-8), 7.19
(1H, d, J = 8.7 Hz, H-2′, H-6), 6.72 (1H, d, J = 8.7 Hz, H-3′, H-
5′). 13C NMR (75 MHz, CDCl3) 156.62 (C-2), 148.20 (C-3), 187.0
(C-4 ), 164.9 (C-5), 94.4 (C-6), 169.5 (C-7), 95.4 (C-8), 159.9 (C-
9), 103.1 (C-10), 56.0 (C-5, C-7- OMe), 126.5 (C-1′), 126.8 (C-
2′), 114.1 (C-3′), 158.0 (C-4′), 114.1 (C-5′), 126.8 (C-6′).
Antibacterial activity:Antibacterial activity of compound
1 was carried out by agar disc diffusion method [12,13]. Stock
solution of the compound was prepared in water, which was
further diluted to obtain desired concentrations (50, 100, 150,
200 µg/mL). Ofloxacin was used as standard antibacterial
agent. Inoculation of bacterial strains was done in nutrient broth
media and incubated for 6 h to maintain standard turbidity
(106 CFU/mL). Inoculum of bacterial strains (1 mL) was seeded
in Muller Hinton agar plate. Disc (6 mm) was dipped in different
concentrations (50, 100, 150, 200 µg/mL) of compound 1 and
allowed to dry and further impregnated on seeded agar plates.
The plates were incubated at 37 ºC for 24 h. The antibacterial
activity was done by measuring the diameter of the zone of
Spectral data of compound 1: m.f. C34H42O19, [M+] m/z
754, m.p. 266-267 ºC . Elemental analysis of C34H42O19 calcd.
(found) (%): C 53.84 (53.441), H 6.08 (6.03), O 40.07 (39.83).
UV (MeOH) λmax: 265, 312, 354 nm. IR (KBr, λmax, cm-1):
3397, 1658, 1600, 1558, 1512. 1H NMR (300 MHz, CDCl3):
δ 3.78 (3H, s, C5-OCH3), 3.76 (3H, s, C7-OCH3), 6.07 (1H, s, H-
6), 6.05 (1H, s, H-8), 7.19 (1H, d, J = 8.7 Hz, H-2′, H-6′), 6.72
(1H, d, J = 8.7 Hz, H-3′, H-5′), 5.88 (1H, d, J = 2.1 Hz, H-1′′),
4.18 (1H, dd, J = 7.2, 9.2 Hz, H-2′′), 3.85 (1H, t, J = 9.0 Hz, H-
3′′), 3.59 (1H, m, H-4′′), 3.21 (1H, m, H-5′′), 1.21 (3H, d, J = 5.8
Hz, CH3), 5.68 (1H, d, J = 7.1 Hz, H-1′′′), 3.73 (1H, dd, J = 7.8,
2.6 Hz, H-2′′′), 3.71 (1H, dd, J = 7.9, 2.3 Hz, H-3′′′), 3.65 (1H,
m, H-4′′′), 3.32 (1H, dd, J = 11.7, 2.1 Hz, H-5a′′′), 3.28 (1H, dd,
J = 11.5, 2.3 Hz, H-5b′′′), 5.03 (1H, d, J = 7.3 Hz, H-1′′′′), 3.66
(1H, dd, J = 7.6, 2.9 Hz, H-2′′′′), 3.49 (1H, dd, J = 7.7, 2.5 Hz,
H-3′′′′), 3.40 (1H, m, H-4′′′′), 3.36 (1H, m, H-5′′′′), 3.31 (1H,
dd, J = 12.4, 2.3 Hz, H-6a′′′′), 3.25 (1H, dd, J = 12.2, 2.5 Hz, H-
6b′′′′). 13C NMR (75 MHz, CDCl3) 156.62 (C-2), 148.20 (C-3),
187.0 (C-4), 164.9 (C-5), 94.4 (C-6), 169.5 (C-7), 95.4 (C-8),
159.9 (C-9), 103.1 (C-10), 56.0 (C-5, C-7- OMe), 126.5 (C-1′),
126.8 (C-2′), 114.1 (C-3′), 158.0 (C-4′), 114.1 (C-5′), 126.8 (C-
6′), 100.9 (C-1′′), 68.7 (C-2′′), 69.7 (C-3′′), 72.3 (C-4′′), 73.4
(C-5′′), 11.9 (CH3), 99.9 (C-1′′′), 67.5 (C-2′′′), 71.4 (C-3′′′), 68.6
(C-4′′′), 73.9 (C-5′′′), 97.3 (C-1′′′′), 75.1 (C-2′′′′), 70.0 (C-3′′′′),
69.1 (C-4′′′′), 71.1 (C-5′′′′), 62.5 (C-6′′′′).
Acid hydrolysis of compound 1: Compound 1 (150 mg)
was dissolved in 20 mL methanol and refluxed with 10 %
H2SO4 (10 mL) on water bath for 6.5 h. The contents so obtained
were concentrated and cooled, further residue was extracted
with diethyl ether. The ethereal layer was washed with water and
the residue was chromatographed over silica gel using a mixture
CHCl3 and MeOH as solvent to give aglycone 1A which was
identified as 5,7-dimethoxy-3,4′-dihydroxy flavone (Fig. 2).
Permethylation of compound 1: Compound 1 (35 mg)
was dissolved in 30 mL DMF and treated with methyl iodide
(5 mL) and Ag2O (15 mg) in a round bottom flask fitted with
air condenser and refluxed for 2 days. The reaction mixture
was filtered and washed with DMF. The filtrate was concen-
trated under reduced pressure and hydrolyzed with 10 % H2SO4
to give methylated aglycone identified as 5,7-dimethoxy-3,4′-
dihydroxy flavone. The aqueous hydrolyzate obtained after

Vol. 31, No. 8 (2019)
Antibacterial Activity of a New Flavone Glycoside from the Stems of Holmskioldia sanguinea Retz. 1817
inhibition (mm) formed around the disc.All experiments were
done in triplicates. Data was expressed as SD .
The aqueous hydrolyzate was neutralized with BaCO3 and
BaSO4 was filtered off. On concentrating the filtrate, it was
subjected to paper chromatography examination and showed
the presence of D-galactose (Rf 0.16), D-arabinose (Rf 0.21)
and L-rhamnose (Rf 0.37). Compound 1 on periodate oxidation
confirmed that all the sugars were present in pyranose form
[20]. Permethylation [21] of compound 1confirmed the position
of sugar moieties, followed by acid-hydrolysis, which yielded
methylated aglycone (1A) and methylated sugars. The methy-
lated aglycone was identified as 5,7-dimethoxy-3,4′-dihydroxy
flavone which confirmed glycosidation was involved at C-3-
OH and C-4′-OH positions of aglycone. The methylated sugars
were identified as 2,3,4,-tri-O-methyl-L-rhamnose (RG 1.01),
2,3,4,6-tetra-O-methyl-D-galactose (RG 0.78) and 2,3-di-
methoxy-D-arabinose (RG 0.64) by paper chromatography .
Therefore it was concluded that C-1′′′-OH of D-arabinose was
attached with OH group at C-3 position of aglycone, C-1′′′′-
OH of D-galactose was linked with C-4′′′-OH of D-arabinose
and C-1′′ of L-rhamnose was attached with OH group at C-4′
position of aglycone. Thus interglycosidic linkage (1→4) was
found between D-arabinose and D-galactose. Takadiastase
enzyme was used for enzymatic hydrolysis [22] of compound
1 which liberated L-rhamnose and 3,4′-dihydroxy-5,7-di-
methoxyflavone-3-O-β-D-galactopyranosyl(1→4)-O-β-D-
arabinopyranoside as proaglycone. Proaglycone on further
hydrolysis with almond emulsion enzyme liberated D-galactose
first followed by D-arabinose and aglycone. Thus, the compound
was identified as 3,4′-dihydroxy-5,7-dimethoxy-flavone-3-O-
β-D-galactopyranosyl(1→4)-O-β-D-arabinopyranosyl-4′-O-
α-L-rhamnopyranoside.
Minimum inhibitory concentration (MIC): MIC for the
compound 1 was determined by using broth dilution technique
[14]. The microbial suspension in nutrient broth media was
prepared and incubated for 24 h and 37 ºC and turbidity was
verified spectrophotometrically (1 × 108 CFU/mL) to find optical
density at 600 nm . In each test tube, sterile nutrient broth
media were added and extract was added in the first test tube
to get concentration of 100 µg/mL, from which serial dilution
(two-folds) were made to desired concentration (50-0.09 µg/
mL). Now the bacterial culture were added to each test tube
(0.5 mL) and incubated at 37 ºC for 24 h. The lowest concen-
tration of compound 1 preventing the visible growth of microbes
(no turbidity) were taken as MIC values. All MIC values were
taken in triplicates.
RESULTS AND DISCUSSION
Compound 1 showed Molisch and Shinoda [15,16] tests
which confirmed the presence of flavonoidal glycoside. Its
UV (MeOH) absorbtion bands observed at 265, 312, 354 nm.
IR spectra showed the peaks at 3397 (-OH), 1653 (α,β-
unsaturated -C=O), 1600, 1581, 1512 corresponds to aromatic
ring system. In 1H NMR spectrum of compound 1, two singlets
at δ 6.07 (1H, s) and δ 6.05 (1H, s) were assigned for H-6 and
H-8 in ring A. Doublets at 7.19 (1H, d, J = 8.7 Hz) for H-2′
and H-6′ and at 6.72 (1H, d, J = 8.7 Hz) for H-3′ and H-5′ in
ring B.A singlet was assigned at δ 3.78, δ 3.76 (-OCH3) group
at C-5 and C-7 in ring A. The anomeric proton signals at δ
5.88 (1H, d, J = 2.1 Hz), δ 5.68 (1H, d, J = 7.1 Hz) and δ 5.03
(1H, d, J = 7.3 Hz) were assigned to H-1′′ of L-rhamnose, H-
1′′′ of D-arabinose and H-1′′′′ of D-galactose, respectively. Two
coupling constants at (J = 7.1 Hz) and (J = 7.3 Hz) confirmed
the β-configuration for the D-arabinose and D-galactose at
H-1′′′ and H-1′′′′ and (J = 2.1 Hz) corresponds to α-configura-
tion of L-rhamnose, respectively [17-19]. In the mass spectrum
of compound 1, characteristic ion peaks at m/z 754 [M+], 608
[M+-L-rhamnose], 446 [M+-D-galactose], 314 [M+-D-
arabinose, aglycone] were found by subsequent losses from
the molecular ion of each molecule of L-rhamnose, D-galactose
and D-arabinose hence indicating L-rhamnose to be linked at
C-4′ position of aglycone and D-arabinose and D-galactose
were linked to aglycone at C-3 position. Compound 1 on acidic
hydrolysis with 10 % ethanolic H2SO4 yielded compound (1A)
having m.f. C17H14O6, m.p. 224-225 ºC, [ M+] 314 (FABMS),
which was identified as 5,7-dimethoxy-3,4′-dihydroxy flavone.
Moreover, compound 1 showed significant antibacterial
activity, therefore it may be used as a potent antibacterial agent
against diseases caused by these bacteria (Table-1).
Conclusion
On the basis of physico-chemical tests and charac-
terization techniques, the structure of compound 1 was estab-
lished as 3,4′-dihydroxy-5,7-dimethoxyflavone-3-O-β-D-
galactopyranosyl(1→4)-O-β-D-arabinopyranosyl-4′-O-α-L-
rhamnopyranoside from the ethanolic extract of stems part of
the plant.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interests
regarding the publication of this article.
TABLE-1
ANTIBACTERIAL ACTIVITY OF COMPOUND 1
Zone of inhibition (mm)
Ofloxacin
10 g/mL
MIC
(mg/mL)
Organisms
µ
50
µ
g/mL
100
µ
g/mL
150
µ
g/mL
200 µg/mL
Bacillus subtilis
Actinomyces israelii
Proteus vulgaris
8.50 0.07*
6.50 0.02*
10.23 0.05*
10.27 0.08*
11.32 0.14**
9.25 0.16**
12.25 0.26**
12.35 0.23**
13.25 0.10**
11.25 0.14**
15.43 0.07*
16.01 0.07*
16.00 0.32**
15.50 0.28**
18.75 0.18**
19.75 0.20**
23.75 0.43**
27.50 0.30**
24.50 0.23**
23.01 0.15**
0.28
0.30
0.23
0.21
Salmonella typhimurium
Data are expressed as Mean SEM; Experiments were conducted in triplicates; P value indicates calculated probability; *p < 0.05, *< 0.01
considered as significant; *p indicates when p is less than 0.05 showed statistically significant values; **p indicates that when p is less than 0.01
showed statistically highly significant values

1818 Yadava et al.
Asian J. Chem.
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