The microsurgical characteristics of leaves, stems and heartwood powder of
the Dalbergia tonkinensis Prain have been described in detail, contributing to
standardize this precious wood species.
- Sequenced 3 chloroplast gene rbcL, rpoB and rpoC sequences of 02
Dalbergia species samples and compared the genetic distance of each gene regions
with 5 other Dalbergia species that have been sequenced on GenBank.
- 03 chloroplast gene rbcL, rpoB and rpoC are capable of distinguishing the
species of the Sua (Dalbergia) with rate 99%, 98% and 97% respectively.
- The sequence of three regions rbcL, rpoB and rpoC of (Dalbergia tonkinensis
Prain) in Vietnam has been registered on gene banks in the numbers of KY283103,
KY287755 and KY287750 respectively.
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3
CHAPTER 2: PRACTICAL TEST AND STUDYING METHODS
This section describes in detail the process of material samples, microsurgical
studying methods and gene sequences, making of extraction parts, chromatographic
separation, compounds isolation and bioactive testing methods.
2.1. Subject
Dalbergia tonkinensis Prain includes branches, leaves and brack that are collected
in Buon Ma Thuot city, Dak Lak province. The scientific name of the tree was
determined by Dr. Nguyen Quoc Binh, Vietnam Natural Museum, Vietnam Academy
of Science and Technology. The specimen sample of plants are stored in the plant's
specimen room, Institute of Ecology and Biological Resources and at the Bioactive
Department, Institute of Natural Products Chemistry, sample symbols are C-561 and
C-612.
After being collected, Sua samples are washed, removed skin, only get red cores
and dried to isolate the substance. Then the sample is minced and soaked several times
with MeOH at temperature room. After removing solvent, residue is fractionated with
solvents with increasing polarity such as: n-hexane, chloroform or dichloromethane,
ethyl acetate and water.
2.1. Subject
The plant and heartwood of Dalbergia tonkinensis Prain (over ten-year old) was
collected in Buon Ma Thuot city, Daklak province, Vietnam. The plant was identified
by botanist Dr. Nguyen Quoc Binh, Vietnam National Museum of Nature, VAST,
Hanoi, Vietnam. A voucher specimen (C-561) and (C-612) was deposited in
Department of Bioactive Products, Institute of Natural Products Chemistry, VAST,
Hanoi, Vietnam
Dried powdered heartwoods (3.7 kg) of Dalbergia tonkinensis Prain were
extracted with hot methanol (5 x 7.0 L) under reflux, filtered, and then concentrated
under decreased pressure giving a black crude methanol residue (120 g). The
suspension of the methanol residue in hot methanol-water (1:1, v/v) was successively
4
partitioned with n-hexane, dichloromethane and ethyl acetate to give n-hexane (29.0 g,
MH), dichloromethane (45.1 g, MD), ethyl acetate (11.1 g, ME) and water (12.0 g,
MW) fractions, respectively. And then we describe the isolation of the compounds
from the fractions of the methanol extract of Dalbergia tonkinensis Prain.
Figure 2.1. Processing sample and extracting from heartwood of
Dalbergia tonkinensis Prain
2.2. Methodology
2.2.1. Microsurgical study: Performed at the Department of Plant - Hanoi University
of Pharmacy.
2.2.2. Genetic sequence study: Performed at the Institute of Ecology and Biological
Resources - Vietnam Academy of Science and Technology.
2.2.3. Method of separating compounds obtained from the extraction
The methods have been used such as: Column chromatography was carried out on
silica gel (Si 60 F254, 40–63 mesh, Merck, St. Louis, MO, USA), or reverse phase
(ODS, YMC (30-50 μm)), column chromatography Dianion HP-20, Sephadex LH-20.
All solvents were redistilled before use. Besides, using crystallization method to collect
clean substances. Pre-coated thin layer chromatography (TLC) plates (Si 60 F254) were
used for analytical purposes. Compounds were visualized under UV radiation (254, 365
nm) and by spraying plates with 10% H2SO4 followed by heating with a heat gun.
Soaked MeOH (5 x 7,0L)
n-hexane
Allocated attraction
water ethyl acetate dichloromethane
Boiling water (4 x
3,0L)
Heartwood
(3,7 kg)
Residue after
extracting
ME
(11,1 g)
MH
(29,0 g)
W
(4,2 g)
MW
(12,0 g)
MD
(45,1 g)
Methanol attraction
(120 g, M)
5
2.2.4. Methods of determining chemical structure of the compounds
The general method for determining the chemical structure of substances is the
combination of physical parameters with modern spectral methods including: 1H-NMR
(500 MHz) and 13C-NMR (125 MHz) were measured on a Bruker Avance 500 MHz
spectrometer. HR-ESI-MS was obtained from a Varian FT-MS spectrometer and
MicroQ-TOF III (Bruker Daltonics, Ettlingen, Germany). IR spectroscopy was
fulfilled on Nicolet Impact 410 spectrometer. UV was performed in spectroscopic V-
630 UV-VIS instrument. The CD data was obtained from a Chirascan spectrometer.
2.2.5. Methods of testing biological activity
2.2.5.1. Method of testing anti microorganism activity
In vitro antimicrobial on some species (with a significant minimum inhibitory
concentration [MIC] value: Bacillus subtillis, Staphylococcus aureus, Aspergillus
niger, Fusarium oxyporum, Saccharomyces cerevisiae, Candida albicans. They
performed at the Institute of Natural Products Chemistry - Vietnam Academy of
Science and Technology
2.2.5.2. Method of testing enzyme -glucosidase inhibitory activity
The inhibitory activity against α-glucosidases was done according to the assay
described by Nguyen, et al. 2018.
Rice α-glucosidase (Type 4) was purchased from Sigma Aldrich (St. Louis City,
MO, USA). Enzyme α-glucosidases from Saccharomyces cerevisiae, Bacillus
stearothermophilus and from rice were provided by Sigma Aldrich, Singapore.
Acarbose and p-nitrophenyl glucopyranoside (pNPG) were purchased from Sigma
Aldrich, 3050 Spruce Street, St. Louis, MO, USA.
All these tests were performed at the Pharmacy Department, Tam Kang
University, Taiwan.
2.2.6. Physical and chemical parameters and spectral data of isolated compounds
This section shows details of the spectral data as well as the physical constants
of 18 compounds were isolated from heartwood of Dalbergia tonkinenis Prain.
6
CHAPTER 3. RESULTS AND DISCUSSIONS
This chapter shows the study results of microsurgery, DNA, the isolation result
and determination of chemical structure of compounds, antibacterial activity and
inhibition of a-glucosidase enzyme.
3.1. Research results on microsurgery and gene sequences of Dalbergia tonkinenis
Prain
This section describes in detail the microscopic morphology results of leaves,
stems and heartwood of Dalbergia tonkinensis Prain.
3.1.1. Results of microsurgery research
We had conducted research on microscopic leaves, stems and core powder of
Dalbergia tonkinensis Prain collected in Buon Ma Thuot. Its microcharacters were
described to support morphological classification as well as supplement genetic
database for this species. Some results are shown in the following figure:
Figure 3.1. Microscopy of leaves Figure 3.2. Microscopy of core
powder
3.1.2. Research results on gene sequences
In this study, we have identified the sequence of 3 chloroplast genome rbcL,
rpoB and rpoC of 2 collected samples in Buon Ma Thuot City (C-561-L) and Hanoi
(C-564-L) and compared the genetic distance of each genetic region with other 5
Dalbergia species that have announced their sequences on GenBank.
The results showed that (Figure 3.3), the three regions of rbcL, rpoB and rpoC
are suitable for the taxonomy study of species of the Sua genus (Dalbergia) with 99%,
7
98% and 97%. The sequence of three chloroplast genome rbcL, rpoB and rpoC of
Dalbergia tonkinenis Prain in Vietnam has been registered on gene banks with the
numbers of KY283103, KY287755 and KY287750 respectively.
Figure 3.3. Comparison of the ability to distinguish between Dalbergia of three
studied gene regions
3.2. Isolation results and determination of chemical structure of compounds from
heartwood of Dalbergia tonkinensis Prain
This section details the isolation work, spectrum analysis and determination of
chemical structure of Dalbergia tonkinensis Prain, including two new compounds and
the rest are known compounds.
18 compounds from heartwood of Dalbergia tonkinensis Prain were isolated and
determined their chemical structure. They include: 08 compounds from
dichloromethane extraction, 07 compounds from ethyl acetate extraction, and 03
compounds from water extraction. The structure of 18 isolated compounds are in Table
3.1 below.
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
rpoB rpoC rbcL
8
Table 3.1. Chemical structure of isolated compounds from the heartwood of
Dalbergia tonkinensis Prain
.
Pinocembrin
Naringenin
3'-hydoxy-2,4,5-trimethoxydalbergiquinol
Medicarpin
Buteaspermanol
Daltonkin A
(2S)-8-carboxyethylpinocemprin
(New)
Daltonkin B
(2S)-2,6-dicarboxyethylnaringenin
(New)
Dalbergin
9
Liquiritigenin
7,3',5'-trihydroxyflavanone
Sativanone
3'-O-methylviolanone
Vestitone
Calycosin
7,3',4'-trihydroxyaurone (Sulfuretin)
Formononetin
Isoliquiritigenin
4',7-dihydroxy-3-methoxyflavone
3.2.1. New compound daltonkin A
Daltonkin A was isolated as a white amorphous solid. In negative high-
resolution electrospray ionisation mass spectrometry (HR-ESI-MS), the quasi-
molecular ion peak at m/z = 327.0868 [M – H]– suggested that the molecular formula
10
of compound daltonkin A is C18H16O6. Its infrared (IR) spectrum showed absorption
bands at 3354 and 1701 cm-1, which can be attributed to hydroxyl and carbonyl groups,
respectively. In addition to the phenyl rings A and B, a methylene carbon at δC 44.3
(C-3), an oxymethine carbon at δC 80.5 (C-2), and a carbonyl carbon at δC 197.5 (C-4)
of ring C in the 13C-NMR spectrum indicated that compound daltonkin A is a
flavanone. The 1H and 13C-NMR spectra of compound daltonkin A are similar to that
of the flavanone pinocembrin isolated from Dalbergia odorifera, except for the
replacement of the aromatic proton H-8 in pinocembrin by a carboxyethyl group
(CH2CH2COOH) (H-9 [δH 2.86, t, 8.0 Hz]/C-9 [δC 18.7], H-10 [δH 2.49, t, 8.0
Hz]/C-10 [δC 34.1], and C-11 [δC 177.7]) (Figure 3.43.6 and Table 3.2).
Figure 3.4. The 1H-NMR of compound daltonkin A
Figure 3.5. 13C-NMR of compound daltonkin A
11
Figure 3.6. DEPT of compound daltonkin A
This was confirmed by cross-peaks between the protons H-9 and H-10 in
correlation spectroscopy (COSY), as well as between protons H-9 and H-10, and the
carbonyl carbon C-11 in the heteronuclear multiple-bond correlation (HMBC)
spectrum (Figure 3.7-3.8).
Figure 3.7. COSY spectrum of
compound daltonkin A
Figure 3.8. HMBC spectrum of compound
daltonkin A
Moreover, correlations between carboxyethyl proton H-9 and carbons C-7, C-8,
and C-8a in the HMBC spectrum indicated that the position of the carboxyethyl group
was at C-8 of ring A.
12
Table 3.2. 1H -NMR and 13C- NMR spectroscopic data for daltonkin A
Position
daltonkin A (*)
δH (ppm) (J in Hz) δC (ppm)
2 5.47, dd, 13.0; 3.0 80.5, d
3
2.80, dd, 17.0; 3.0, H-3eq
3.11, dd, 17.0; 13.0, H-3ax
44.3, t
4 197.5, s
4a 103.3, s
5 162.8, s
6 6.02, s 95.6, d
7 166.1, s
8 108.5, s
8a 162.8, s
9 2.86, t, 8.0 18.7, t
10 2.49, t, 8.0 34.1, t
11 177.7, s
9'
10'
11'
1' 140.5, s
2',6' 7.52, d, 7,5 127.3, d
4' 7.39, t, 7,5 129.6, d
3',5' 7.44, d, 7,5 129.7, d
(*)1H-NMR (500 MHz, methanol-d4);
13C-NMR (125 MHz, methanol-d4)
Flavanones with a 2S-configuration exhibit a positive Cotton effect at ~330 nm
(n–* transition), and a negative Cotton effect at 270–290 nm (–* transition) in the
CD spectrum. The CD (c 0,4, MeOH) curve of compound daltonkin A showed a
positive Cotton effect at 330 + 2.10 (n–* transition), and a negative Cotton effect at
288 – 10.43 (–* transition); this suggested a 2S-configuration for compound
daltonkin A.
13
Figure 3.9. CD spectrum of compound daltonkin A
Based on these spectroscopic results, compound named daltonkin A was
determined to be (2S)-8-carboxyethylpinocembrin, a new monocarboxyethylflavanone
isolated from the heartwood of Dalbergia species.
Figure 3.10. Chemical structure of compound daltonkin A
Figure 3.11. Selected HMBC (H→C) and COSY (H→H) correlations of compound
daltonkin A
3.2.2. New compound daltonkin B
Daltonkin B was isolated as a white amorphous solid, with a suggested molecular
formula of C21H20O9 according to a quasi-molecular ion peak at an m/z of 415.1014 [M
– H]– (calculated for C21H19O9 415.1024) in negative HR-ESI-MS. The IR spectrum
indicated bands at 3369 cm-1 (hydroxyl groups) and 1751 cm-1 (carbonyl groups).
Similar to daltonkin A, daltonkin B has characteristic signals for flavanone compounds;
14
specifically, a methylene carbon at δC 44.0 (C-3), an oxymethine carbon at δC 80.3 (C-
2), and a carbonyl carbon at δC 198.6 (C-4) in the 13C-NMR spectrum.
The 1H and 13C-NMR spectra of daltonkin B was similar to that of the flavanone
naringenin isolated from Dalbergia odorifera, except for the replacement of two
aromatic protons (H-8 and H-6) in compound naringenin by two carboxyethyl groups
(H-9 [δH 2.85, m]/C-9 [δC 18.8], H-10 [δH 2.55, m]/C-10 [δC 34.4], and C-11 [δC 179.1])
and (H-9' [δH 2.81, m]/C-9' [δC 19.4], H-10' [δH 2.51, m]/C-10' [δC 34.8], and C-11' [δC
179.2]) in daltonkin B (Figure 3.11-3.14 and Table 3.3)
Figure 3.12. HR-ESI-MS high resolution spetrum of compound daltonkin B
Figure 3.13. 1H-NMR spectrum of compound daltonkin B
15
Figure 3.14. 13C-NMR spectrum of compound daltonkin B
Cross-peaks between H-9/H-10 and H-9'/H-10' in the COSY spectrum, as well
as correlations between protons H-9 and H-10 to C-11, and H-9' and H-10' to C-11',
further confirmed the existence of two carboxyethyl groups. Moreover, the correlations
between proton H-9' to C-5/C-6 and C-7, as well as proton H-9 to C-7/C-8 and C-8a,
were observed in the HMBC spectrum. This suggests that two carboxyethyl groups
were located.
Moreover, the correlations between proton H-9' to C-5/C-6 and C-7, as well as
proton H-9 to C-7/C-8 and C-8a, were observed in the HMBC spectrum. This suggests
that two carboxyethyl groups were located at C-6 and C-8 of ring A (Figure 3.15-3.16)
Figure 3.15. COSY spectrum of
compound daltonkin B
Figure 3.16. HMBC spectrum of
compound daltonkin B
16
Table 3.3. 1H NMR and 13C NMR spectroscopic data for daltonkin B
Position
daltonkin B (*)
δH (ppm) (J in Hz) δC (ppm)
2 5.39, dd, 13.0, 3,0 80.3, d
3
2.79, dd, 17.0, 3.0, H-3eq
3.13, dd, 17.0, 13.0, H-3ax
44.0, t
4 198.6, s
4a 103.4, s
5 159.8, s
6 109.0, s
7 163,9, s
8 108.3, s
8a 161.1, s
9 2.85, m 18.8, t
10 2.55, m 34.4, t
11 179.1, s
9' 2.81, m 19.4, t
10' 2.51, m 34.8, t
11' 179.2, s
1' 131.4, s
2',6' 7.36, d, 8.5 128.8, d
4' 158.9, s
3',5' 6.85, d, 8.5 116.4, d
(*)1H-NMR (500 MHz, methanol-d4); 13C-NMR (125 MHz, methanol-d4)
Similar to daltonkin A, daltonkin B was determined to be (2S). Thus, compound
daltonkin B was determined to be (2S)-6,8-dicarboxyethylnaringenin, a new
dicarboxyethylflavanone.
Figure 3.17. Chemical structure of compound daltonkin B
17
Figure 3.18. Selected HMBC and COSY correlations of daltonkin B
3.2. The result of testing biological activity
3.2.1. Antimicrobial activity of isolated compounds
In vitro antimicrobial results are displayed in Table 2. Among the tested
compounds, pinocembrin (3) exhibited inhibitory effects on filamentous fungi (with a
significant minimum inhibitory concentration [MIC] value of 50 µg/mL), while
compounds pinocembrin (3) and naringenin (4) showed moderate MIC values of 100
µg/mL against yeast and the Gram-positive bacterium Staphylococcus aureus.
Compound 3'-hydroxy-2,4,5-trimethoxydalbergiquinol (5) failed to show any activity.
Table 3.4. Antimicrobial activity of isolated compounds from
(Dalbergia tonkinensis Prain)
(-): IC50 ≥ 200 µg/mL
3.2.2. The α-glucosidase inhibitory activity
3.2.2.1. New records of Dalbergia tonkinensis Prain extracts
To determine which part used is the most active, the heartwood, trunk bark, and
leaves of Dalbergia tonkinensis were collected and extracted with methanol; their
activity was then tested. As shown in Figure 1A, all the parts used of Dalbergia
tonkinensis show stronger activity (98–100%) than that of positive control (62%).
These results highlighted the promising in vitro antidiabetic property of the extracts
from the heartwood, trunk bark and leaves of Dalbergia tonkinensis. To clarify the
N
o
Antimicrobial activity (MIC: µg/mL)
Gram-positive bacteria Filamentous fungi Yeast
Bacillus
subtillis
Staphylococcus
aureus
Aspergillus
niger
Fusarium
oxyporum
Saccharomyces
cerevisiae
Candida
albicans
3 (-) 100 50 50 100 100
4 (-) 100 100 (-) (-) (-)
5 (-) (-) (-) (-) (-) (-)
18
results, the activity was also expressed as IC50 value. The heartwood extract
demonstrated the strongest activity among the Dalbergia tonkinensis parts used due to
its smallest IC50 value of 0.17 mg/mL; therefore, it was used in conducting subsequent
experiments, including stability tests and purification.
Figure 3.19. The inhibition α-glucosidase of different of Dalbergia tonkinensis Prain;
aGIs: α-glucosidase inhibitors
3.2.2.2. Primary partitioned separation of methanol extract from heartwood of
Dalbergia tonkinensis Prain
The methanol extract of heartwood of Dalbergia tonkinensis (HDT) was
successively partitioned with n-hexane, dichloromethane, ethyl acetate and water to
obtain 04 fractions. The yield and activity of HDT and its fractions are recorded in
Table 3.5. HDT-3 partitioned by ethyl acetate demonstrated the strongest activity due
to its greatest inhibition (%) and lowest EC50 value of 95% and 0.069 mg/mL,
respectively. Thus, this fraction was chosen for the isolation of active compounds. The
water fraction (HDT-4) showed acceptable activity. Since it had the good profile of
TLC separation, it was also considered for subsequent experiments of purification.
Table 3.5. α-glucosidase inhibition of HDT and its fractions after partition
Samples
Yield
(g)
α-Glucosidase Inhibition
IC50 (mg/mL) Inhibition (%) *
HDT (crude MeOH extract) 60.3 0.172 ± 0.011 98 ± 3.2
HDT-1 (Hexane Fr.) 1.6 1.712 ± 0.210 73 ± 4.1
HDT-2 (Dichloromethane Fr.) 27.2 0.124 ± 0.003 90 ± 2.5
HDT-3 (Ethyl acetate Fr.) 11.1 0.069 ± 0.001 95 ± 3.7
HDT-4 (Water Fr.) 12.0 0.513 ± 0.051 82 ± 2.3
Acarbose (positive control) 1.357 ± 0.03 62 ± 1.8
*: the inhibition of acarbose and fractions were detected at their concentration range of 0.1–5 mg/mL;
results are means ± SD of multi tests (n = 3).
3.2.2.3. Sub-fractionation of the extract, and identification of active compounds
A
Concentration (mg/mL)
0 1 2 3 4 5
a
G
I
( %
)
0
20
40
60
80
100
Heartwood
Trunk bark
Leaves
Acarbose
B
Part used
Heartwood Bark Leaves Acarbose
E
C
5
0
(
m
g
/m
L
)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
0.17
0.57
0.78
1.25
IC50
(mg/
mL)
19
The most active fraction (HDT-3) was separated by a silica open column to
obtain 12 sub-fractions. The activity of these sub-fractions was tested and expressed as
%. As shown in Figure 3.20, HDT-3.1 showed the best activity (98%) while the others
demonstrated weak inhibition (2–36%) at the same tested concentration of 0.1 mg/mL.
Further separation of HDT-3.1 was applied to the most active sub-fraction (HDT-
3.1.2), showing inhibition of 99% (Figure 3.20).
Figure 3.20. Inhibitory activity (%) of sub-fractions and purified compounds;
aGIs: α-glucosidase inhibitors
3.2.2.4. Specific inhibitory activity of the purified compounds
Specific inhibition of the purified compounds was detected to characterize them
as active antidiabetic drugs; a total of 04 enzymatic sources, including α-glucosidases
from rat, yeast, bacterium, and rice were conducted to test the inhibition. The activity
was expressed as IC50 value and presented in Table 3.6
Table 3.6. Specific inhibitory activity of sativanone, formononetin, and acarbose
No. Enzyme Source
Inhibition Expressed as IC50 (mg/mL)
Sativanone Formononetin Acarbose
1 Yeast α-glucosidase 0.23 ± 0.012 0.06 ± 0.002 1.321 ± 0.048
2 Rat α-glucosidase 0.37 ± 0.022 0.23 ± 0.037 0.121 ± 0.001
3 Bacterial α-glucosidase 0.07 ± 0.001 0.03 ± 0.002 0.001 ± 0.000
4 Rice α-glucosidase 0.81 ± 0.023 0.98 ± 0.029 0.031 ± 0.005
All tests were performed in triplicate; results are means ± SD of multi tests (n = 3)
In this study, both sativanone and formononetin demonstrated much stronger
inhibition against yeast α-glucosidase than did acarbose, and showed their effects on
A
3
.1
3
.2
3
.3
3
.4
3
.5
3
.6
3
.7
3
.8
3
.9
3
.1
0
3
.1
1
3
.1
2
a
G
I
(%
)
0
20
40
60
80
100
Sub-fractions of HDT-3
B
3
.1
.1
3
.1
.2
3
.1
.3
3
.1
.4
3
.1
.5
3
.1
.6
3
.1
.7
3
.1
.8
a
G
I
(%
)
0
20
40
60
80
100
Components separated from HDT-3.1
C
4
.1
4
.2
4
.3
4
.4
4
.5
4
.6
4
.7
4
.8
a
G
I
(%
)
0
20
40
60
80
100
Sub-fractions of HDT-4
D
4
.3
.1
4
.3
.2
4
.3
.3
4
.3
.4
4
.3
.5
4
.3
.6
a
G
I
(%
)
0
20
40
60
80
100
Components separated from HDT-4.3
E
Concentration (mg/mL)
0 1 2 3 4 5
a
G
I
(%
)
0
20
40
60
80
100
Compound 1 (purified HDT-3.1.2)
Compound 2 (purified HDT-4.3.3)
Acarbose (positive control)
20
rat α-glucosidase comparable to that of acarbose. The inhibitory activity of sativanone
and formononetin against rat α-glucosidase was newly investigated in the current
study, based on our current references review. Therefore, they were determined as
new mammalian.
3.2.2.5. The rat α-glucosidase inhibitory activity of crude extracts, fractions, sub-
fractions and isolated compounds from Dalbergia tonkinensis Prain
All the parts used extracts (the heartwood, trunk bark, and leaves) of Dalbergia
tonkinensis Prain, the fractions, sub-fractions, and purified compounds separated from
HDT, as well as acarbose, were tested their inhibition against α-glucosidase from rat.
The results were expressed as IC50 and max inhibition then summarized in Table 3.7.
Table 3.7. Rat α-glucosidase inhibitory activity of crude extracts, fractions, sub-
fractions, and isolated compounds from Dalbergia tonkinensis Prain extract
Components
Rat α-glucosidase inhibitory activity
IC50 (mg/mL) Max Inhibition (%)
Heartwood Extract (HDT) 1.72 ± 0.116b 61 ± 3.46e
Trunk bark extract 2.91 ± 0.289a 51 ± 4.62f
Leaves extract 2.78 ± 0.173a 54 ± 4.60f
HDT-3 1.31 ± 0.057c 68 ± 5.77d
HDT-3.1 1.13 ± 0.058cd 75 ± 5.20c
HDT-3.1.2 0.92 ± 0.023d 77 ± 5.18c
Sativanone 0.357 ± 0.006fg 91 ± 4.61a
HDT-4 1.43 ± 0.115bc 67 ± 2.89d
HDT-4.3 0.87 ± 0.035de 78 ± 4.61c
HDT-4.3.3 0.55 ± 0.012ef 84 ± 4.62b
Formononetin 0.251 ± 0.006fg 94 ± 5.11a
Acarbose 0.119 ± 0.005g 93 ± 2.5a
Coefficient of variation 12.50026 1.853018
The samples and acarbose were tested at their concentration range of 0.05–3.2 mg/mL;
results are means ± SD of multi tests (n = 3); the means of IC50 and max inhibition values
Among the 3 parts used extracts, HDT also demonstrated strongest inhibitory
activity due to its smallest IC50 and greatest inhibition values of 1.72 µg/mL, and 61%,
respectively. The activity was gradually increased via steps of partial purification. The
02 purified compounds showed their much stronger activity than that of the crude
sample (HDT) and its fractions (HDT-3, HDT-4), and sub-fractions (HDT-3.1, HDT-
3.1.2, HDT-4.3, and HDT-4.3.3).
21
CONCLUSION AND SUGGESTION
The results of plant research
-The microsurgical characteristics of leaves, stems and heartwood powder of
the Dalbergia tonkinensis Prain have been described in detail, contributing to
standardize this precious wood species.
- Sequenced 3 chloroplast gene rbcL, rpoB and rpoC sequences of 02
Dalbergia species samples and compared the genetic distance of each gene regions
with 5 other Dalbergia species that have been sequenced on GenBank.
- 03 chloroplast gene rbcL, rpoB and rpoC are capable of distinguishing the
species of the Sua (Dalbergia) with rate 99%, 98% and 97% respectively.
- The sequence of three regions rbcL, rpoB and rpoC of (Dalbergia tonkinensis
Prain) in Vietnam has been registered on gene banks in the numbers of KY283103,
KY287755 and KY287750 respectively.
The chemical composition
- The first time from heartwood of Dalbergia tonkinensis Prain has isolated and
determined the chemical structure of 18 flavonoid compounds: pinocembrin, naringenin,
3'-hydroxy-2,4,5-trimethoxydalbergiquinol, medicarpin, buteaspermanol, daltonkin A [(2S)-
8-carboxyethylpinocembrin], daltonkin B [(2S)-2,6-dicarboxyethylnaringenin], dalbergin,
isoliquiritigenin, 7,3',5'-trihydroxyflavanone, vestitone, calycosin, 4',7-dihydroxy-3-
methoxyflavone, liquiritigenin, sativanone, 3'-O-methylviolanone, 7,3',4'-trihydroxyaurone
and formononetin.
- 02 compounds daltonkin A and daltonkin B are new mono- and di-
carboxyethylflavanones.
3. Biological effects of extracts and compounds were isolated
3.1. In vitro antimicrobial: pinocembrin compound exhibits inhibitory ac
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