HomeTrendingCurcuminoids from the Vietnamese Curcuma longa: Phytochemical analysis and biological activities

Curcuminoids from the Vietnamese Curcuma longa: Phytochemical analysis and biological activities

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Physical sciences
|
Chemistry
19
SEPTEMBER 2022
Volume 64 Number 3
Intr
oduction
Curcuma longa
L. (the Zingiberaceae family) is a
perennial herb, which is distributed throughout the world
and widely cultivated in Asian countries [1]. Its rhizomes
have features like oblong, ovate, pyriform, and are often
shorty branched [1, 2]. The powders derived from rhizomes
have been in continuous use in food preparations [3].
Current users of traditional medicine claim the application
of its rhizome powders has antioxidant, antibacterial,
anticancer, anti-inflammatory, antimutagenic, antidiabetic,
and hepatoprotective activities [4-6].
The extracts of the
C. longa
species react with alkalis
to create red-brown salts.
C. longa
extracts are soluble
in alkalis, ethanol, ketone, acetic acid, and chloroform
[1].
This phenomenon is mainly due to the presence of
curcumin and its derivatives [1]. Chromatographic HPLC
is a powerful and robust technique for both qualitative and
quantitative analysis of curcumin and its derivatives [7-10].
In the current paper, we report the phytochemical and HPLC
quantitative procedures for identifying curcuminoids from
the Vietnamese
C. longa
rhizomes together with their DPPH
radical scavenging, α-glucosidase, acetylcholinesterase
(AChE), and cytotoxic
assays.
Materials and methods
General procedures
The Bruker Avance 500 MHz was used to measure 1D
and 2D-NMR with TMS as an internal standard. A Thermo
Scientific LTQ Orbitrap XL instrument was used to collect
ESI-MS data. Silica gel (40-63 μm mesh, Sigma) and
Sephadex LH-20 (75-150 μm, Bio-Science, Sweden) were
used for column chromatography (CC). TLC examination
was performed on plates that had been precoated with
silica gel 60 F
254
(Merck, Germany). Compounds were seen
using a UV lamp with wavelengths of 254 and 365 nm, as
well as spraying with indicators (5% H
2
SO
4
and vanillin).
Solvents for the HPLC analysis were purchased from
Merck, Germany. HPLC-DAD data were obtained using
a ZORBAX Eclipse XDB C18 column (150×4.6 nm, 5 m)
coupled with a ZORBAX Eclipse XDB guard C18 column
(12.5×4.6 mm, 5 m) on an Agilent Series 1260 (Agilent
Technologies, USA) system, which included a vacuum
degasser, a quaternary mixing pump, an auto-sampler, a
column oven, and a diode-array detector (DAD) (Agilent
Technologies, USA).
Curcuminoids from the Vietnamese
Curcuma longa
:
Phytochemical analysis and biological activities
Thi Thu Ha Nguyen
1, 2
, Thi Tu Anh Le
1
, Thanh Tra Nguyen
1, 2
, The Son Ninh
1*
1
Institute of Chemistry, Vietnam Academy of Science and Technology
2
Graduate University of Science and Technology, Vietnam Academy of Science and Technology
Received 16 September 2021; accepted 11 November 2021
*
Corresponding author: Email: [email protected]
Abstract:
A phytochemical investigation on the ethanolic (EtOH) extract of the Vietnamese
Curcuma longa
(
C. longa
) rhizomes has
led to the isolation of four curcuminoids including curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), and
cyclocurcumin (4). The chemical structure of compounds 1-4 was elucidated by Nuclear magnetic resonance (NMR) and
Mass spectrometry (MS) spectral data. Based on High-performance liquid chromatography (HPLC) quantitative analysis,
the amounts of three major compounds 1-3 in the
C. longa
extract were calculated and reached 7.215±0.101, 3.927±0.031,
and 2.255±0.049 mg/g, respectively. Curcuminoids 1-4 have induced IC
50
values of 9.23-14.6 μg/ml in a 2,2-diphenyl-1-
picrylhydrazyl (DPPH) radical scavenging assay as compared with that of the positive control resveratrol (IC
50
11.5 μg/
ml). Compounds 1-4 with IC
50
values ranging between 8.7-15.54 μg/ml were better than the positive control acarbose
(IC
50
169.14 μg/ml) in α-glucosidase inhibitory examination. In addition, the EtOH extract and compounds 1-4 were also
responsible for inhibitions against enzyme acetylcholinesterase and four cancer cell lines including including epidermoid
carcinoma (KB), hepatocellular carcinoma (HepG2), lung cancer (SK-LU-1), and breast cancer (MCF7).
Keywords:
Curcuma longa
, curcuminoids, cytotoxicity, DPPH radical scavenging, HPLC analysis, α-glucosidase inhibition.
Classification number:
2.2
DOI : 10.31276/VJSTE.64(3).19-23
Physical s
ciences
|
Chemistry
20
SEPTEMBER 2022
Volume 64 Number 3
Plant material
The rhizomes of
C. longa
were collected in Bac Giang,
Vietnam in December 2020 and were identified by the taxonomist
Nguyen The Cuong of the Institute of Ecology and Biological
Resources. A voucher specimen labelled CL-2020 was
deposited in the Department of Applied Biochemistry, Institute
of Chemistry, Vietnam Academy of Science and Technology.
Extraction and isolation
C. longa
dried rhizomes (0.5 kg) were extracted with EtOH
(4 l x 3 times) under reflux for 3.5 h. The crude EtOH extract
(120 g) was obtained by evaporating the mixed extract under
reduced pressure.
The EtOH extract was subjected to silica gel CC and
eluted with
n
-hexane (1 l), CH
2
Cl
2
(1.5 l), and EtOAc (1.2 l)
to afford 7 fractions (CL1-CL7). Fraction CL2 (15.1 g) was
chromatographed on silica gel CC [
n
-hexane-acetone (3:1,
v/v)] to yield 7 fractions (CL21-CL27). The fraction CL22 (2.2
g) was subjected to sephadex LH-20 CC [MeOH/CH
2
Cl
2
(9:1,
v/v)] to give compound
2
(8.0 mg). The fraction CL3 (9.7 g)
was separated on sephadex LH-20 column [MeOH (100%)]
to yield 6 fractions (CL31-CL36). The fraction CL33 (1.2
g) was separated by sephadex LH-20 CC eluted with MeOH
(100%) to provide 3 fractions (CL331-CL333). Compound
4
(1.5 mg) was isolated from the fraction CL332 (0.2 g) by
using preparative TLC [CH
2
Cl
2
/EtOAc (6:1, v/v)]. Silica gel
CC [CH
2
Cl
2
-EtOAC, 9:1 to 1:1, v/v)] was utilized for fraction
CL24 (8.9 g) to produce 8 fractions (CL241-CL248). Both
compound
1
(9.1 mg) and compound
3
(12.1 mg) were derived
from the fraction CL242 (1.5 g) by using silica gel CC [CH
2
Cl
2
/
CH
3
COCH
3
(3:1, v/v)].
Curcumin
(
1
):
y
ellow powder; ESI-MS (+):
m/z
369.1
[M+H]
+
(calcd for C
21
H
21
O
6
, 369.0);
1
H-NMR (500 MHz,
CD
3
OD,
δ
H
ppm): 7.60 (2H, d, 16.0 Hz, H-4), 7.23 (2H, d, 1.5
Hz, H-6), 7.13 (2H, dd, 1.5, 8.0 Hz, H-10), 6.85 (2H, d, 8.0 Hz,
H-9), 6.62 (2H, d, 16.0 Hz, H-3), 5.99 (1H, s, H-1), 3.93 (6H, s,
7-OCH
3
);
13
C-NMR (125 MHz, CD
3
OD,
δ
C
ppm): 184.8 (C-2),
150.5 (C-8), 149.5 (C-7), 142.1 (C-4), 128.6 (C-5), 124.1 (C-
3), 122.3 (C-10), 116.6 (C-9), 111.8 (C-6), 101.4 (C-1), 56.5
(7-OCH
3
).
Demethoxycurcumin
(
2
):
y
ellow powder; ESI-MS (+):
m/z
339.2 [M+H]
+
(calcd for C
20
H
19
O
5
, 339.0);
1
H-NMR (500
MHz, DMSO-
d
6
,
δ
H
ppm): 10.10 (1H, s, 8’-OH), 9.72 (1H, s,
8-OH), 7.61 (3H, m, H-4’, H-6’, H-10’), 7.57 (1H, d, 15.0 Hz,
H-4), 7.20 (1H, d, 10.0 Hz, H-10), 7.32 (1H, s, H-6), 6.87 (3H,
d, 10.0 Hz, H-9, H-7’, H-9’), 6.79 (1H, d, 15.0 Hz, H-3), 6.74
(1H, d, 15.0 Hz, H-3’), 3.90 (3H, s, 7-OCH
3
);
13
C-NMR (125
MHz, DMSO-
d
6
,
δ
C
ppm): 184.1 (C-2’), 183.8 (C-2), 160.1
(C-8’), 148.7 (C-7), 148.5 (C-8), 141.5 (C-4), 141.0 (C-4’),
131.0 (C-7’, C-9’), 126.8 (C-5), 126.1 (C-5’), 123.9 (C-3),
121.5 (C-3’), 115.2 (C-6’, C-10’), 111.9 (C-6), 101.4 (C-1),
56.4 (7-OCH
3
).
Bisdemethoxycurcumin
(
3
):
y
ellow powder; ESI-MS
(+):
m/z
308.1 [M+H]
+
(calcd for C
19
H
17
O
4
, 308.0);
1
H-NMR
(500 MHz, DMSO-
d
6
,
δ
H
ppm): 10.03 (2H, s, 8-OH), 7.55
(6H, m, H-4, H-6, H-10), 6.82 (4H, d, 8.5 Hz, H-7, H-9), 6.70
(2H, d, 15.5 Hz, H-3), 6.04 (1H, s, H-1);
13
C-NMR (125 MHz
DMSO-
d
6
,
δ
C
ppm): 184.8 (C-2), 161.1 (C-8), 141.8 (C-4),
131.1 (C-7, C-9), 126.0 (C-5), 122.0 (C-3), 116.9 (C-6, C-10),
101.5 (C-1).
Cyclocurcumin
(
4
):
y
ellow powder; ESI-MS (+):
m/z
369.2 [M+H]
+
(calcd for C
21
H
21
O
6
, 369.0);
1
H-NMR (500
MHz, CDCl
3
,
δ
H
ppm): 7.07 (1H, d, 10.0 Hz, H-9), 7.03 (4H,
m, H-6, H-15, H-18, H-19), 6.95 (1H, d, 10.0 Hz, H-10), 6.50
(1H, d, 14.5 Hz, H-12), 7.33 (1H, d, 1.4.5 Hz, H-13), 5.60 (1H,
s, H-3), 5.40 (1H, dd, 1.5, 14.5 Hz, H-4), 3.99 (3H, s, 7-OCH
3
),
3.94 (3H, s, 16-OCH
3
), 3.04 (1H, dd, 15.0, 20.0 Hz, H-2a),
2.90 (1H, d, 1.5, 14.5 Hz, H-2b);
13
C-NMR (125 MHz, CDCl
3
,
δ
C
ppm): 194.1 (C-1), 170.0 (C-11), 147.7 (C-17), 147.0 (C-
16), 146.8 (C-7), 146.1 (C-8), 146.5 (C-7), 137.9 (C-13), 130.4
(C-5), 127.7 (C-14), 123.0 (C-19), 120.1 (C-10), 118.1 (C-12),
114.1 (C-18), 113.9 (C-9), 109.2 (C-6), 108.9 (C-15), 105.7 (C-
3), 80.9 (C-4), 43.5 (C-2), 56.5 (16-OCH
3
), 56.1 (16-OCH
3
).
HPLC quantitative analysis
Quantitative analysis by HPLC has been applied to
identify the amounts of curcuminoids in the EtOH extract of
the Vietnamese
C. longa
rhizomes. Due to a material shortage
of compound
4,
the analysis was focused on compounds
1-3
.
The HPLC method has been carefully described in a previous
publication [11]. Briefly, three isolated compounds were used
as standard compounds in which their calibration solutions
were prepared in a concentration range of 10-400 μg/ml by
MeOH dilution. Linearity was evaluated in this range three
times. The extract of
C. longa
was also diluted by MeOH and
filtered before use. The mobile phase was made up of 0.1%
acetic acid in water (mobile phase A) and ACN (mobile phase
B). The HPLC running conditions were the same for standard
compounds and extracts, which include sample injection (2
μl), flow rate (1.6 ml/min), column temperature (40°C), UV
detector (λ=425 nm) for 0 min (75% A, 25% B), 0-20.0 min
(30% A, 70% B), and 20-25 min (100% B).
The limit of detection (LOD) is the lowest concentration
of the standard substance that can be detected. Similarly, the
limit of quantitation (LOQ) shows the lowest concentration
of analyte that can be quantified. Both LOD and LOQ were
calculated using a calibration curve based on the standard
deviation (SD or σ) of the data response and the slope of the
calibration curve (
a
) using the following equations: LOD=3.3
σ/a and LOQ=10 σ/a.
Biological assays
Antioxidative assay:
O
ur earlier reports have detailed
descriptions of the DPPH antioxidant assay [12-14]. In brief,
DPPH (0.1 mM) was diluted in MeOH. DPPH (200 μl) was

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