Phytochemical characterization and biological activity of secondary metabolites from three Limonium species

Article abstract of DOI:10.1007/s00044-017-1973-z

The comparative phytochemical constituents of three Limonium species Limonium myrianthum, Limonium leptophyllum, and Limonium gmelinii afforded a new compound (2R,3S)-2,3,4-trihydroxy-2-methylbutyl gallate (1) and twenty known compounds (2–21). Each species displayed different profiles in their phytochemical constituents. The isolated compounds (1–21) were evaluated for antifungal, antimalarial, and antitrypanosomal activities. Compound 1 showed good activity against chloroquine-resistant and chloroquine-sensitive strains of malaria, while compound 5 displayed moderate antimalarial activity. Compound 14 showed a significant activity against Trypanosoma brucei.

Full text of DOI:10.1007/s00044-017-1973-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1973-z
ORIGINAL RESEARCH
Phytochemical characterization and biological activity of
secondary metabolites from three Limonium species
1
,2 ●
1,3 ●
4,5 ●
Anastassiya V. Gadetskaya
Shaymaa M. Mohamed
Amer H. Tarawneh
1
,3 ●
1 ●
6 ●
2 ●
Nesma M. Mohamed
Guoyi Ma Boris N. Ponomarev Galiya E. Zhusupova
Charles L. Cantrell Stephen J. Cutler Samir A. Ross
7 ●
4 ●
1,4
Received: 23 October 2016 / Accepted: 26 June 2017
©
Springer Science+Business Media, LLC 2017
●
●
●
Abstract The comparative phytochemical constituents of
three Limonium species Limonium myrianthum, Limonium
leptophyllum, and Limonium gmelinii afforded a new
compound (2R,3S)-2,3,4-trihydroxy-2-methylbutyl gallate
Keywords Plumbaginaceae Limonium Antifungal
●
Antimalarial Antitrypanosomal
(
1) and twenty known compounds (2–21). Each species
displayed different profiles in their phytochemical con-
stituents. The isolated compounds (1–21) were evaluated
for antifungal, antimalarial, and antitrypanosomal activities.
Compound 1 showed good activity against chloroquine-
resistant and chloroquine-sensitive strains of malaria, while
compound 5 displayed moderate antimalarial activity.
Compound 14 showed a significant activity against Trypa-
nosoma brucei.
Introduction
Plant tissues typically contain a diverse complement of
secondary metabolites (Keinänen et al. 1999 and
Julkunen–Tiitto 1989) that provide protection against var-
ious biotic and abiotic hazards (Berenbaum 1995 and Wink
2003). Chemical similarities are commonly used to infer
phylogenetic relationships among plant taxa (Downie and
Palmer 1994). The genus Limonium Mill (Plumbaginaceae)
includes over 300 species world-wide, of which 18 are
found in Kazakhstan (Zhusupova 2007). Many of these
Limonium species have been widely used in folk medicine
as rich sources of bioactive compounds (Lin and Chou
Electronic supplementary material The online version of this article
(
doi:10.1007/s00044-017-1973-z) contains supplementary material,
which is available to authorized users.
2
1
000; Tang et al. 2014; Yang et al. 1987; Liebezeit et al.
999 and Rastogi et al. 2016). More recently, several spe-
*
Samir A. Ross
sross@olemiss.edu
cies of the Limonium genus have been shown to have
potential scientific, pharmacological, and medical uses
1
2
3
4
5
National Center for Natural Products Research, University of
Mississippi, University, MS 38677, USA
(Murray et al. 2004; Aniya et al. 2002; Yuh-Chi et al. 2002
School of Chemistry and Chemical Technology, Al-Farabi Kazakh
National University, Almaty 050040, Kazakhstan
and Kandil et al. 2000). For example, L. brasiliense has
anti-inflammatory and antibacterial properties (Murray et al.
2004), L. wrightii can be used to treat arthritis and fever
Pharmacognosy Department, Faculty of Pharmacy, Assiut
University, Assiut 71526, Egypt
(Aniya et al. 2002), L. tetragonum and L. sinense have
Department of BioMolecular Sciences, School of Pharmacy,
University of Mississippi, University, MS 38677, USA
antiviral properties (Yuh-Chi et al. 2002), and L. axillare
and L. californicum have antibacterial and cytotoxic activity
(Kandil et al. 2000). L. gmelinii, one of the Limonium
species present in abundance throughout Kazakhstan, has
been studied extensively for its potential pharmacological
properties (Kozhamkulova et al. 2010; Korul’kina et al.
2004 and Zhusupova 2006). Another Limonium species
Department of Chemistry, Tafila Technical University, P.O. Box
1
79, Tafila, Jordan
6
7
Institute of Plant Biology and Biotechnology, Almaty, Kazakhstan
Natural Products Utilization Research Unit, USDA-ARS,
University, MS 38677, USA

Med Chem Res
widely available in Kazakhstan, L. myrianthum, is an her-
baceous plant with small violet flowers found in saline
marshes throughout the country (Zhusupova 2007).
Previous phytochemical and biological studies on the
Limonium genus demonstrated the presence of different
classes of biologically active compounds, such as poly-
saccharides, tannins, alkaloids, flavonoids, terpenes, ali-
phatic compounds, amino acids, and minerals (Medini et al.
rhamnopyranoside (20) and myricetin 3-O-(4′′-O-galloyl)-
α-rhamnopyranoside (21) were isolated only from L. gme-
linii and were not found in the other investigated species.
Compounds 14 and 15 were also found to be common to
both L. myrianthum and L. gmelinii. In this study, all iso-
lated compounds were evaluated for their antimalarial,
antileishmanial, antimicrobial, and antitrypanosomal
activities.
2
014 and Gadetskaya et al. 2015).
As a part of our search for novel, plant-derived biological
agents, the phytochemical constituents of L. myrianthum
Schrenk) Kuntze, L.leptophyllum (Schrenk) O. Kuntze, and
Materials and methods
(
L. gmelinii (Willd.) Kuntze were characterized. Herein, we
report the isolation of a new natural compound from a L.
leptophyllum, compound 1 (Fig. 1). Also found in this
Limonium species are emodin (2), bergenin (3), and
mahuannin B (4), 7-O-galloyl-D-sedoheptulose (5), arbutin
Plant material and Extraction
The aerial part of the L. leptophyllum (Schrenk) O. Kuntze
plant was collected near Balkhash Lake during its flowering
stage on 24th July 2004, and identified by the Botanical
Garden at the Institute of Botany and Phytointroduction in
Almaty, Kazakhstan. The voucher specimen number of the
sampled plant is 6351/18. The aerial part was air-dried
followed by grinding in a Willey–Mill plant grinder. The
ground plant material (260 g) was extracted at room tem-
perature using methylene chloride (1.5 L) and ethanol (1.9
L), which provided 6.7 g and 10.5 g, of the crude extracts,
respectively. Further extraction with water (2.3 L) provided
16.2 g of extract after lyophilization.
(
6), gallic acid-4-O-β-D-glucoside (7), myricetin (8), and
myricetin-3-O-L-rhamnopyranoside (9). Compounds 8 and
were found in all investigated species. Another ten known
9
compounds, 3,5,7,3′,4′,6′-hexahydroxyflavone (10), myr-
icetin-3-O-L-arabinoside (11), myricetin-3-O-β-D-gluco-
pyranoside (12), epigallocatechin-3-O-gallate (13),
myricetin 3-O-(6′′-galloylglucoside) (14), myricetin 3-O-(2′
′
-O-galloyl)-β-D-glucopyranoside (15), 1-galloyl-β-D-glu-
copyranoside (16), melitriose (17), β-sitosterol-3-glucoside
(
(
18), and β-sitosterol (19) were isolated from L. myrianthum
Fig. 1). While 11, 12, 14, 15, and 18 are reported for the
The L. myrianthum (Schrenk) Kuntze plant was collected
from South Kazakhstan in August 2010 during its flowering
stage and identified at the Biological Department at
first time from this species. Quercetin-3-O-α-L-
Fig. 1 Chemical structures of
isolated compounds (1–21)

Med Chem Res
Al-Farabi Kazakh National University. A voucher specimen
of the aerial part (6351) and the roots (359) of the plants
were deposited at the Herbarium of the Institute of Botany
and Phytointroduction in Almaty, Kazakhstan. Ground
plant materials from the aerial part (180 g) and roots (300
g) were separately and sequentially extracted by maceration
at room temperature using three solvents of different pola-
rities, hexane (0.6 L), acetone (0.9 L), and methanol
(5 g) column chromatography which was eluted with
dichloromethane-methanol 9:1 to give compound
2
(6.5 mg). B4 (60 mg) was subjected to SPE Silica gel
(5 g) column chromatography which was eluted with
dichloromethane-methanol 14:1 to produce compound 3
(20 mg). B5 (700 mg) was subjected to SPE RP-C18
(10 g) column chromatography which was eluted with
water-methanol gradient elution to produce compound 4
(30 mg).
(
1.0 L). The crude methanol extract from the aerial part of
the plant (1.57 g) and acetone (23.40 g) and methanol
38.42 g) extract from the roots were subjected to further
The acetone extract of L. myrianthum root (21.89 g) was
chromatographically fractionated on a silica gel column
stepwise from dichloromethane to methanol, yielding seven
fractions: dichloromethane (C1), 4:1 dichloromethane-
methanol (C2), 3:1 dichloromethane-methanol (C3), 7:3
dichloromethane-methanol (C4), 6:4 dichloromethane-
methanol (C5), 1:1 dichloromethane-methanol (C6), and
methanol (C7). C2 (220 mg) was subsequently chromato-
graphed on a silica gel column initially with hexane and
stepwise elution to ethyl acetate then to methanol yielding
59 subfractions (C2/1–59). The subfractions C2/23–24
(25.2 mg) were rechromatographed using Sephadex LH-20
eluted with methanol to furnish compound 19 (25 mg). The
subfractions C2/46-59 (599 mg) were rechromatographed
using SPE chromatography under vacuum eluted with
chloroform–methanol (5, 10, 15, 20, 30, 40, 50, 100%) to
furnish compound 18 (18.3 mg). C3 (3.1 g) was rechro-
matographed using High-Performance Flash Chromato-
graphy with silica gel cartridges (KP-SIL, 10 g, 40–63 μm,
60 Å, Biotage SNAP), eluted with 100% dichloromethane,
dichloromethane-methanol (10, 20, 40, 50%) and 100%
methanol yielding 38 subfractions (C3/1–38). Subfractions
C3/25–26 were identified as compound 9 (100 mg). The
combined subfractions C3/16-18 (910 mg) were rechro-
matographed on Sephadex LH- 20 eluted with methanol to
yield compound 15 (297 mg), compound 11 (66 mg) and
compound 13 (133 mg). C4 (1 g) was chromatographed on
Sephadex LH-20 eluted with methanol (100%) to yield
compound 10 (201 mg). C5 (1 g) was rechromatographed
by Sephadex LH-20 column elution methanol yielding
compound 17 (158 mg).
(
fractionation.
The aerial part of the L. gmelinii (Willd.) Kuntze plant
was collected during its flowering period (September 2012)
in the Almaty region of Kazakhstan and identified at the
Institute of Botany and Phytointroduction in Almaty,
Kazakhstan with the voucher specimen number 154958.
Air-dried ground plant material (900 g) was extracted with
methanol (3.1 L), which provided 58 g of the crude extract.
Twenty grams of concentrated extract were dissolved in
1
00 ml water and successively partitioned by liquid-liquid
extraction (3 times) with ethyl acetate (200 ml each). The
ethyl acetate extract was 3.2 g.
Isolation
The ethanolic extract of the aerial part of L. leptophyllum
(
4.5 g) was subjected to Diaion HP-20 column chromato-
graphy fractionation to give two main fractions, an aqueous
fraction (A) (2.14 g) and a methanolic fraction (B) (2.3 g).
The Diaion aqueous fraction (A) (2.14 g) was subjected to
MN-polyamide-SC-6 (85 g) column chromatography
which was eluted with water then with water-methanol
systems gradually decreasing in polarity to give seven
subfractions (A1–A7). A1 (130 mg) was subjected to
Sephadex LH-20 (25 g) column chromatography which
was eluted with methanol to give compound 6 (30 mg). A3
(
90 mg) was subjected to Sephadex LH-20 (15 g) column
chromatography which was eluted with methanol to pro-
duce compound 5 (12 mg). A4 (70 mg) was subjected to
Sephadex LH-20 (15 g) column chromatography which
was eluted with methanol to give compound 7 (5 mg). A6
The concentrated methanol extract obtained from the L.
myrianthum root (1.24 g) was fractionated on a Sephadex
LH-20 column eluted with methanol to yield 80 fractions
(D1-80). Fractions D13-19 yielded compound 16 (38 mg)
and fractions D26-28 yielded compound 8 (21 mg).
The concentrated methanol extract obtained from the
aerial part of L. myrianthum (1.57 g) was fractionated on
silica gel using ethyl acetate and methanol with increasing
solvent polarity yielding 91 fractions (E1–91). Fractions
E35–36 (24.1 mg) were combined and rechromatographed
to furnish compounds 12 and 14.
(
150 mg) was further purified using Sephadex LH-20 (25 g)
column chromatography which was eluted with methanol-
water (4:1) to give compounds 8 and 9 (60 mg). The Diaion
methanolic fraction (B) (2.3 g) was subjected to MN-
polyamide-SC-6 (100 g) column chromatography which
was eluted with water then with water-methanol systems
gradually decreasing in polarity to give five subfractions
(
B1–-B5). B1 (240 mg) was subjected to Sephadex LH-20
(
25 g) column chromatography which was eluted with
methanol to produce compound 1 (70 mg). B3 (120 mg)
was subjected to Solid Phase Extraction (SPE) Silica gel
The ethyl acetate extract of the aerial part of the
L. gmelinii plant (1 g) was chromatographed over a

Med Chem Res
polyamide column with chloroform, chloroform:ethanol,
ethanol, acetone with a gradient of water (30, 50, 70%), and
methanol. A total of 702 (F1-702) fractions were collected,
with subsequent combining and rechromatography. Com-
bined fractions F109-124 (26 mg) led to the identification
of compound 20. Compound 9 (72 mg) was found in
chromatographically identical fractions F133–152. Com-
pounds 21 and 15 were isolated from fractions F169–224,
with a total weight of 38 mg. Fractions F225–292 (18 mg),
Alkaline hydrolysis
Five miligram of compound 1 was dissolved in 10% alco-
holic KOH (2 ml) and left overnight at room temperature.
The reaction mixture was dried and the residue redissolved
in water and extracted with ethyl acetate (5 ml × 3). The
combined ethyl acetate fractions were dried to produce
2-methylbutane-1,2,3,4-tetraol (2 mg) (Fontana et al. 1999).
with the same R on the TLC, were identified as compound
In vitro antimicrobial assay
f
8
. Compound 14 (23 mg) was isolated from fractions
F365–435.
Compounds 1–21 were tested for antimicrobial activity
against a panel of microorganisms obtained from the
American Type Culture Collection (Manassas, VA, USA)
and included the fungi Candida albicans ATCC 90028,
Candida glabrata ATCC 90030, Candida krusei ATCC
(
2R,3S)-2,3,4-trihydroxy-2-methylbutyl 3,4,5-trihydroxy-
benzoate (1)
6
258, C. neoformans ATCC 90113, Aspergillus fumigatus
ATCC 204305, the bacteria Staphylococcus aureus ATCC
9213, methicillin-resistant S. aureus ATCC 33591
MRSA), Escherichia coli ATCC 35218, Pseudomonas
2
5
Oily colorless residue; [α] D–9.446 (c 0.01, MeOH); UV
1
(
MeOH) λmax 264.2 nm; H NMR (CD OD, 400 MHz,): δ
3
2
(
=
4
7.09 (2H, s, H-2, H-6), 4.16 (1H, d, J = 11.2 Hz, H-1′),
.23 (1H, d, J = 11.6 Hz, H-1′), 3.72 (1H, dd, J = 8, 3.2
Hz, H-3′), 3.85 (1H, dd, J = 11.2, 3.2 Hz, H-4′), 3.61 (1H,
m, H-4′), 1.20 (3H, s, H-5′); C NMR (CD OD, 100
MHz,): δ = 168.4 (>C=O, C-7), 146.2 (Ph(C), C-5, C-3),
39.7 (Ph(C), C-4), 121.3 (q-Ar(C), C-1), 110.2 (Ar(CH),
C-2, C-6), 75.8 (Sec-OH(CH), C-3′), 74.2 (t-OH(C), C-2′),
0.0 (CH O, C-1′), 63.6 (CH OH, C-4′), 19.7 (CH , C-5′);
aeruginosa ATCC 27853, and Mycobacterium intracellu-
lare ATCC 23068. The bioassays were performed as
described previously (Ma et al. 2004 and Bharate et al.
1
3
3
2
007). Ciprofloxacin and amphotericin B were used as
1
positive drug controls.
7
2
2
3
In vitro antimalarial assay
−
HRESIMS m/z: 287.0766 [M − H] (calcd. for C H O
1
2 16 8
2
88.0845).
Isolated compounds 1–21 were tested for antimalarial
activity, which was determined in vitro against chloroquine
sensitive (D6, Sierra Leone) and resistant (W2, Indo China)
strains of Plasmodium falciparum by a colorimetric assay
that determines the parasitic lactate dehydrogenase activity,
as described earlier (Hamid et al. 2004). Chloroquine was
used for positive drug control.
General experimental procedures
1
13
H and C NMR spectra were obtained on a Bruker model
AMX 400 NMR spectrometer with standard pulse sequen-
1
13
ces, operating at 400 MHz in H and 100 MHz in C. The
chemical shift values were reported in parts per million
units (p.p.m.) and trimethylsilane or known solvent shifts,
used as internal chemical shift references. Coupling con-
stants were recorded in Hertz (Hz). Standard pulse
sequences were used for COSY, HMQC, HMBC, TOCSY,
NOESY, and DEPT. High-resolution mass spectra were
measured on a Micromass Q-Tof Micro mass spectrometer
with a lock spray source. Column chromatography was
carried out on silica gel (70–230 mesh, Merck) and
Sephadex LH-20 (Mitsubishi Kagaku, Tokyo, Japan). TLC
In vitro antileishmanial assay
Compounds 1–21 were tested for antileishmanial activity.
Promastigote culture of Leishmanial donovani was grown
in RPMI medium with 10% fetal bovine serum (FBS) with
°
pH 7.4 at 26 C. Axenic amastigote culture of L. donovani
was grown in RPMI medium with 10% FBS with pH 5.5 at
37 °C in 5% CO incubator. The antileishmanial activity of
2
the compounds was tested in vitro against the promastigote,
axenic amastigote and macrophage internalized amastigote
forms of the L. donovani parasite. Promastigotes and axenic
amastigotes were based on alamar blue based florometric
growth analysis. Differentiated THP1 cells have been used
as macrophages in macrophage internalized amastigotes.
The macrophage internalized amastigote method was based
on parasites rescued and transformation assays (Jain et al.
2012). Pentamidine and amphotericin B were used as
(
silica gel 60 F254) was used to monitor fractions from
column chromatography. Visualization of the TLC plates
was achieved with a UV lamp (l = 254 and 365 nm) and
anisaldehyde/acid spray reagent (methanol-acetic acid-
anisaldehyde-sulfuric acid, 85:9:1:5). All chemicals used
were purchased from Sigma–Aldrich (St. Louis, MO,
USA). CD-spectra were measured on a JASCO J-715
spectrometer.

Med Chem Res
positive drug controls (Ma et al. 2004 and Hamid et al.
3-O-(2′′-O-galloyl)-β-D-glucopyranoside (15) (Akdemir
et al. 2001), melitriose (17) (Seibel et al. 2006), β-sitosterol-
2
004).
3-glucoside (18) (Chadwick et al. 2004), and β-sitosterol
In vitro antitrypanosomal assay
(19) (Zhang et al. 2006) (Fig. 1). Myricetin (8) (Medini
et al. 2014) and 1-galloyl-β-D-glucose (16) (Pawlowska
et al. 2006) were isolated from methanol extract of the roots
after fractionation over Sephadex LH-20. In addition, two
myricetin derivatives were isolated from the methanol
extract of the aerial part of the plant: myricetin-3-O-β-D-
glucopyranoside (12) (Scharbert et al. 2004) and myricetin
3-O-(6′′-galloylglucoside) (14) (Braca et al. 2003).
An ethanolic extract of the aerial part of L. leptophyllum
was fractionated using different chromatographic columns
to yield a new compound, (2R,3S)-2,3,4-trihydroxy-2-
methylbutyl gallate (1), along with emodin (2) (Guo et al.
2011), bergenin (3) (Nazir et al. 2011), mahuannin B (4)
(Calzada et al. 1999), 7-O-galloyl-D-sedoheptulose (5)
All isolated compounds (1–21) were tested for anti-
trypanosomal activity. Blood-stage forms of Trypanosoma
brucei brucei were grown in IMDM medium supplemented
with 10% FBS. The culture was maintained at 37 °C in a
5
% CO incubator. The antitrypanosomal screening assay
2
was based on Alamar blue fluorometric growth analysis at a
concentration range of 10–0.4 μg/ml. Active compounds
were further screened at a concentration range of
1
0–0.0032 μg/ml. Pentamidine and difluoromethylornithine
were used as positive drug controls (Manda et al. 2014).
IC50 and IC90 values were computed from a dose response
growth inhibition curve by XLfit version 5.2.2.
(Yokozawa et al. 2009), arbutin (6) (Miguel et al. 2014),
Conformational analysis and electronic circular
dichroism (ECD) calculation
and gallic acid-4-O-β-D-glucoside (7) (Pawlowska et al.
2006), myricetin (8) (Medini et al. 2014), and myricetin-3-
O-L-rhamnopyranoside (9) (Chung et al. 2004).
An ethyl acetate extract of the aerial part of L. gmelinii,
obtained after consistent liquid–liquid extraction with ethyl
acetate and water, was subjected to fractionation yielding
six compounds, 8, 9, 14, 15, along with quercetin-3-O-α-L-
rhamnopyranoside (20) (Kong et al. 2014) and myricetin 3-
O-(4″-O-galloyl)-α-rhamnopyranoside (21) (Lin and Chou
2000).
Conformational analysis of compound 1 was performed
with Schrödinger Macromodel 9.9 (Schrödinger, LLC,
New York), employing the OPLS2005 (optimized
potential for liquid simulations) force field in methanol
(
http://www.schrodinger.com/productpage/14/11/). Two
conformers within a 2 kcal/mol energy window from the
global minimum were selected, based on Boltzmann
distribution calculated from Schrödinger software (http://
www.schrodinger.com/productpage/14/11/). The output
files for these conformers were prepared for Gaussian 09
calculation by Avogadro software. Geometrical optimi-
zation and energy were then calculated using the
B3LYP/6-31G method in the Gaussian 09 software.
Vibrational evaluation was done at the same level to
confirm minimal excitation energy (denoted by wave-
length in nm), rotatory strength dipole, and dipole length,
which were calculated in methanol by TD-DFT/ B3LYP/
Compound 1 was obtained as colorless oil. The HRE-
−
SIMS spectrum of 1 showed a molecular ion [M − H]
peak at m/z 287.0766, compatible with the molecular for-
1
mula C H O . The H NMR spectrum of 1 showed two-
1
2 13 8
proton singlets at δ 7.09 (s, 2H) assigned to meta-coupled
H
H-2 and H-6, respectively. Two protons at δ 4.16 (d, J =
H
11.2 Hz, 1H) and δ 4.23(d, J = 11.6 Hz, 1H) were ascri-
H
bed to oxygenated methylene H -1′. Two one-proton
2
doublets at δH 3.85 (dd, J = 11.2, 3.2 Hz, 1H) and 3.61
(1H, m) were assigned to oxygenated methylene H -4′ and a
2
6
-31G performed by the Gaussian 09 software
three-proton broad signal at δ 1.20 (s, 3H) attributed to the
H
package (http://avogadro.openmolecules.net/). ECD
curves were obtained in the SpecDis 1.62 program
tertiary C-5′ methyl protons. Compound 1 also showed one
proton signal (H-3′) at δ 3.72 (dd, J = 8, 3.1 Hz, 1H). The
H
1
3
(
Bruhn et al. 2013).
C NMR spectrum of 1 exhibited signals for ester carbon at
δC 168.4 (C-7) and another 6 aromatic carbon signals
including δ 121.3 (C-1), δ 110.2 (C-2, C-6), along with
C
C
Results and discussion
three phenolic groups with features characteristic of a gallic
acid moiety at δ 146.2 (C-3, C-5) and δ 139.7 (C-4). In
C
C
1
3
In this study, the acetone extract from the roots of L.
myrianthum was subjected to multiple chromatographic
fractionations over silica gel yielding 3,5,7,3′,4′,6′-hex-
ahydroxyflavone (10) (Shi et al. 2008), myricetin-3-O-L-
arabinoside (11) (Kadota et al. 1990), myricetin-3-O-L-
rhamnopyranoside (9) (Chung et al. 2004), epigalloca-
techin-3-O-gallate (13) (Yang et al. 2003), myricetin
addition, the C NMR spectrum of the compound 1
revealed signals characteristic of the polyol moiety: oxy-
genated methylene carbon at δ 70.0 (C-1′) and 63.6 (C-4′),
C
tertiary oxygenated carbon at δ 74.2 (C-2′) and secondary
C
oxygenated carbon at δ 75.8 (C-3′). HMBC correlations
C
allowed for the complete assignments (Fig. 2). The proton
at δ 7.09 (2H, s, H-2 and H-6) showed a strong correlation
H

Med Chem Res
with the carbons at δ 121.3 (C-1), δ 139.7 (C-4), δ 146.2
J = 11.6 Hz, 1 H) showed strong correlation with signals at
δ 19.7 (C-5′), δ 74.2 (C-2′), and δ 168.4 (C-7) (Table 1).
C
C
C
(
C-3 and C-5) and δ 168.4 (C-7). The two proton signals of
C
C
C
C
H -1′ at δ 4.16 (d, J = 11.2 Hz, 1H) and δH 4.23(d,
Accordingly, the structure of 1 was defined as 2,3,4-trihy-
droxy-2-methyl-butyl gallate.
2
H
Hydrolysis of compound 1 led to the obtaining of 2-
methylbutane-1,2,3,4-tetraol. The optical rotation of the
alcohol part was measured at a concentration of 0.01 mg/ml
2
5
in methanol the [α]D value (−9.446) matches with
2R,3S)-2-methylbutane-1,2,3,4-tetraol mentioned in the
(
literature (Ghosh et al. 2012). The absolute configuration of
the C-2′ and C-3′ positions was further confirmed by
comparison of experimental ECD spectrum with calculated
values for the enantiomers. The 1D NOESY NMR experi-
ment showed no correlation between the methyl (C-5′) and
the proton resonating at δH 3.72 (H-3′) (supplementary
Figure 1), which indicates that the substitutes in positions 2′
and 3′ are either 2R,3S or 2S,3R enantiomers. The stereo-
chemistry of compound 1 was assigned as 2R,3S based on
the positive CD maxima at 245 and 298 nm (Fig. 3). The
B3LYP/6-31 G simulated ECD spectrum for 2R,3S enan-
tiomer, generated from 40 excited states using Gaussian
band shapes for the peaks, had peaks at 240 and 290 nm.
The calculated ECD of the 2R,3S enantiomer showed
excellent agreement with the experimental data. On the
basis of the above discussion, the structure of 1 has been
characterized as (2R,3S)-2,3,4-trihydroxy-2-methylbutyl
gallate, which represents a new gallic acid ester.
The chemical structures of the known compounds were
confirmed by comparison of their spectroscopic properties
with published data. The isolated compounds 1–21 (Fig. 1)
were evaluated for their antibacterial, antifungal, anti-
malarial, antitrypanosomal, and antileishmanial activities.
The antibacterial activity was tested against Staphylococcus
aureus, methicillin-resistant S. aureus (MRSA), Escher-
ichia coli, Pseudomonas aeruginosa, and Mycobacterium
Fig. 2 Chemical structure and the HMBC and COSY correlations of
the isolated new compound (1)
1
13
Table 1 400 MHz H-NMR and 100 MHz
compounds 1
C-NMR data of
a
No.
1(in CD
3
OD)
δ
H
(mult; J in Hz)
δc
1
2
3
4
5
6
7
–
121.3
110.2
146.2
139.7
146.2
110.2
168.4
7.09 (2H, s)
–
–
–
7.09 (2H, s)
–
1
2
3
4
5
a
′
′
′
′
′
4.16 (1H, d, J = 11.2), 4.23 (1H, d, J = 11.6)
70.0
74.2
75.8
63.6
19.7
–
3.72 (1H, dd, J = 8; 3.2)
3.85 (1H, dd, J = 11.2; 3.2), 3.61 (1H, m)
1.20 (3H, s)
The δ
H
C 3
and δ values were recorded using solvent signals (CD OD:
δ
H
3.31/δ
C
49.0 for 1) as references. Signal assignments were based on
1
1
the results of H– H COSY, HMQC, and HMBC experiments
Fig. 3 The experimental spectrum of the compound 1compared to the calculated ECD spectra of 2R,3S and 2S,3R - enantiomers

Med Chem Res
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tively. Structurally-related compound 5 showed moderate
activity with IC50 values of 3.947 and 3.657 µM, for D6
and W2, respectively. Compound 14 revealed significant
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1
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with IC50 values of 25.04, 16.41, and 25.13 µM respec-
tively. The IC50 value for the positive drug control α-
difluoromethylornithine was 13.07 µM.
Acknowledgements We are grateful to the Government of the
Republic of Kazakhstan and the National Center for Natural Products
Research (NCNPR), University of Mississippi, School of Pharmacy,
USA for financial support. Also we are grateful to Dr. Melissa Jacob,
Dr. Shabana Khan, and Dr. Babu Tekwani for performing anti-
bacterial, antifungal, antimalarial, and antileishmanial bioassays.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
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