1STUDIES ON BIOLOGICALLY ACTIVESECONDARY METABOLITES FROM SOMEMEDICINAL PLANTS IN VIET NAM

NINH THE SON

0

Contents
Abstract

4

Chapter 1 An overview of studies on Dalbergia genus

8

1.1. Botany

8

1.2. Traditional uses

8

1.3. Phytochemical studies

9

1.4. Biological investigations

12

2. Studies on Dalbergia species in Vietnam

16

Chapter 2 Chemical constituents from Dalbergia tonkinensis Prain
2.1. Introduction

18
18

2.2. Phytochemical investigation of heartwood extract and structural 19
elucidation
2.2.1. Isolation procedure

19

2.2.2. Structural elucidation of new compounds 21, 22 and 25

21

2.3. Phytochemical investigation of leaf extract and structural elucidation29

2.3.1. Isolation procedure29

2.3.2. Structural elucidation of new compounds 31 and 33

30

2.4. Phytochemical investigation of root extract and structural elucidation

38

2.4.1. Isolation procedure38

2.4.2. Structural elucidation of new compound 47

39

2.5. Chemotaxonomic significances from the isolated constituents
Chapter 3 Chemical constituents from Cratoxylum formosum

44
50

3.1. Introduction

50

3.2. Phytochemical investigations of leaf extract and structural elucidation

51

3.2.1. Isolation procedure

51

Chapter 4 Biological activities

53

4.1. Introduction

53

4.2. Antimicrobacterial results

54

4.2.1. The first model

54

4.2.2. The second model

61

1

4.3. DPPH radical scavenging result

62

4.4. Enzyme active screening results

64

Conclusion

79

Experimental section

81

References

127

Acknowledgements

136

Publications based on this dissertation1382

Abbreviations
TLC

Thin layer chromatography

CC

Column chromatography

RP-18

Reversed phase C18 column

GC

Gas chromatography

HPLC

High performance liquid chromatography

MPLC

Medium performance liquid chromatography

CD

Circular dichroism

MS/HR-MS

Mass spectrum/High resolution-mass spectroscopy

NMR

Nuclear magnetic resonance

1D/2D

One dimension/two dimension

1

H/13C-NMR

Proton/Carbon-NMR

DEPTDistortionless enhancement by polarization transfer

COSY

Correlated spectroscopy

HMBC

Heteronuclear multiple bond correlation

HSQC

Heteronuclear single quantum coherence

NOESY

Nuclear Overhauser enhancement spectroscopy

δ/JChemical shift/coupling constantfr.sFractions

MIC

Minimum inhibitory concentration

mg/µg/mL

Milligram/microgam/millilitreMHz

Megahertz

m/zMass to charge ratio

3

Abstract
Historical records have evidently accumulated in showing that the use of traditional
antibiotics, which are derived from synthetic substances, is always accompanied by a long
duration of treatment, high costs, and drug resistances. Therefore, calls for new antibiotic drugs
from natural sources are warranted. For thousands of years, Vietnamese traditional herbal
medicine, also known as Southern Medicine, is considered as a useful tool to quickly drive
various diseases. From generation to generation, the recording medical documents exposured
that either of Vietnamese people or other parts of the world treated diseases with available
resources, utilizing orally herbs and plants which were gathered or cultivated. The search forbioactive molecules and novel drugs have always drawn much more attentions to scientists,
especially chemists. In the recent days, modern chromatography techniques have been applied toisolate numerous number of secondary metabolites. It, therefore, would lay a good platform toget further extensive steps in discovery of new drugs and preparations.
As my present work for Ph.D thesis, I would like to point out that my career would betaken good advances for the dissertation entitled “Studies on Biologically Active Secondary
Metabolites from Some Medicinal Plants in Viet Nam”, which emphasizes on phytochemical,
spectroscopic, and biological studies to enhance my responsibilities and skillful development of
future professionals. This study deals with two Vietnamese medicinal plants Dalbergia
tonkinensis Prain and Cratoxylum formosum (Jack) Dyer in Hook. The timber tree D. tonkinensis
is endemic to Viet Nam, which is valuable for wood craft, workship and ornamentation. D.
tonkinensis is on The IUCN Red List of Threatened Species and Vietnam Government DecreeNo, 32/2016/ND-CP, prohibiting exploitation, shipping, and storage. Furniture made from D.
tonkinensis heartwood is particularly prized in Vietnam, while commercial sales of this plant are4quietly banned. Of pharmacological aspect, D. tonkinensis species is recognized to be potential
agent in treating diabetes. C. formosum species grows up as shrubs or small to medium-sizedtrees. It has pink flowers and can be up to 20 meters tall. The leaf of this herbal plant has
traditionally been employed for tea, heat detoxification, digestive benefits, and anti-aging. As thechemical components of this plant are rich in tannins and flavonoids, the pharmacologicalreports of C. formosum species are associated with the significant antioxidant capacity. Despite
the fact that these two plants have been playing an important role in Vietnam, but to date,specific phytochemical investigations on these are quietly limited. In order to explore further
scientific aspects on these plants, my thesis has aimed at discovering the plant extracts/fractions
and natural compounds with new kinds of biological activities from several parts of these twoplants. The results are as follows:
Chapter 1 An overview of studies on Dalbergia genus:
It is necessary to demonstrate a board spectrum of the previous studies relating to theplants from Dalbergia species. As a consesquence, this chapter deals with an overview of the
botanical

description,

traditionaluses,

especially

phytochemical

investigations,andpharmacological results. Most of Dalbergia species have claimed for protection due to overexploitation. Adding that, the perennial plants from Dalbergia genus are well-known to be richin flavonoids and their glycosides. Thus, pharmacological outcomes arising from in vitro and invivo examimnations give a general insight in employing Dalbegria plants as good candidates
against micro-bacterial, free radicals, diabetes, cancer, cardiac heart failure, and so on.
Chapter 2 Chemical constituents from Dalbergia tonkinensis Prain:
In this chapter, the experimental performance mainly focused on the phytochemical
investigations on heartwood, leaf, and root of Vietnamese D. tonkinensis plant. Briefly, the firststage involved in the process to produce the crude extracts obtained by using methanol or ethanol
5solvents. These crude extracts were gone on to partition with an increase in solvents using suchas n-hexane, chloroform, dichloromethane, and ethyl acetate. Utilizing silica gel, Sephadex, RP18 and HPLC columns in multi-chromatographical actions, total 47 compounds were isolated.
Among them, two new rare flavanone derivatives (2S)-6,8-dicarboxyethylpinocembrin (20), and(2S)-8-carboxyethylnaringenin

(21)

and

one

new

sesquiterpenerel-(3R,6S,7S)-6α-hydroxycyclonerolidol (25) were isolated from heartwood, while one new unique alkaloid 3(1H-indol-3-yl)-2-methoxypropanamide (31) and one new isoflavone glycoside isocaviunin 7-Oβ-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (33) were identified presenting in the leaf. In
addition, other new sesquiterpene 2,5'-dimethyl-5'-vinylhexahydro-2,2'-bifuran-5(2H)-one (47)
was found in the root. The result also suggested that flavonoids and flavonoid glycosides
predominated over in D. tonkinensis plant. Of particular interests, among several secondary
metabolites, sesquisterpenes nerolidol derivatives were only found in Vietnamese D. tonkinensis
and Chinese D. odorifera, thereby making a close affinity between them.
Chapter 3 Chemical constituents from Cratoxylum formosum:
Phytochemical study on C. formosum led to the isolation of 9 compounds from themethanol extract of leaf. Isolated compounds were categorized as phytosterols, xanthone
glycosides, flavonoids, and flavonoid glycosides.
Chapter 4 Biological activities:
With regards to biological assays, antimicrobacterial, enzyme active and inhibitory, andantioxidant assays were carried out. In the present study, several kinds of the extracts, fractions,and isolated compounds from the plants of Dalbergia genus were submitted to bioassays. D.
tonkinensis methanol extracts of leaf, stem bark and root, choroform fraction of heartwood
methanol extract exhibited inhibitory percentage of more than 60% against Bacillus cereus,

6Escherichia coli and Streptococcus pneumoniae. Among the isolated compounds, flavanone
pinocembrin and isoflavone biochanin A suppressed 80% of the growth of the two bacterial
strains B cereus and S. pneumonia compared with the positive control. Likewise, the strongest
percentage of 80% against bacterium S. pneumonia was assigned to the isoflavanone sativanone
and chalcone isoliquiritigenin.
The products from D. tonkinensis (extracts, fractions, and isolated compounds) wereaccessed by serine protease active and inhibitive assays. The results suggest that atconcentrations of 25-250 ng/mL, the methanol extracts of leaf and root, the root ethanol extractand its dichloromethane fraction, and heartwood water decoction extract can serve as usefulsources to stimulate trypsin enzyme activity. In addition, water decoction extracts of leaf andstem bark may explain unknown ethno-pharmacology due to the high inhibitory percentages intrypsin-chymotrypsin-elastase experiments. Among isolated compounds, quercetin inhibited theactivities of trypsin and chymotrypsin with the IC50 9.7 µM.
Finally, for antioxidant evaluation, DPPH radical scavenging assay was used and theresult suggested that isoflavan 3R-vestitol (from D. tonkinensis) and isoquercetin (from C.
formosum) possessed the inhibitory capacity as comparable as standard compound catechin (IC5042.98 µg/mL).
In this thesis, the aforemenioned data and results are discussed in detail and settled in theexperimental section.

7

Chapter 1 An overview of studies on Dalbergia genus
1.1. Botany
Dalbergia, belonging to the pea family Fabaceae, has a wide distribution, native to all
over the world, especially in the tropical and subtropical regions [1]. According to database of
The Plant List (www.theplantlist.org, 2019), the following acceptable names of 274 of 647
Dalbergia species were listed at a level of high confidence [2]. The members of the genus arevarious considerably in shape but generally displayed as small to medium size trees, shrubs andlianas with leathery, alternate, and compound leaves. Leaflets are alternate, rarely, somesubopposite and glabrous. Corolla small, rarely fragrant, white to cream, sometimes flushed
purplish. Fruits are oblong-lanceolate, indehiscent, usually flattened, and seeded. Seeds arekidney shaped, thin and flat, light brown in color. Root nodulation is very common [1].
1.2. Traditional uses
Numerous species of Dalbegia genus have a long history of traditional medicinal use inmany countries. The heartwood of medicinal plant D. odorifera was found in China with brandname “Jiangxiang”, “Kangjinhyang” in Korean, and “Koshinko” in Japanese drugs for
cardiovascular diseases, cancer, diabetes, blood disorder, ischemia, swelling, necrosis, rheumatic
pains, and so forth [3-5]. In the employments of D. sissoo in Jharkhand-India, the decoction ofits leaf possessed acute stage of gonorrhea, removing pus in urine, as alleviates profuse
menstruation [6]. Generally, Dalbegia species have been employing either as single drugs or in
combination with other materials. Representative Table 1 reveals the most striking features in
ethnomedical uses of some Dalbergia species.

8

Table 1. Traditional and medicinal uses of some Dalbergia species
Name
D. cochinchinensis
D. lanceolaria

Parts
HeartwoodBark

D. latifolia

Whole plant

Pharmacological uses
Anti-tumor and blood stasis [7]A decoction of the bark is used in dyspepsia [1]Whole plant is used in diarrhea, dyspepsia, leprosy
and obesity [1]
In Senegal, stem and root bark are used in thetreatment of diarrhea with baobab or tamarind fruits[1]
Chewed leaf is applied to treat snakebites andleaves are rubbed on abscesses [1]A root infusion is used in the treatment of stomachache and toothache
Cardiovascular diseases, cancer, blood disorder,
ischemia, swelling, necrosis, rheumatic pains [3-5]The heartwood is extremely resistant to attack bymarine boring organisms [8]Decoction of leaf possesses acute stage ofgonorrhea, removing pus in urine, and alleviates
profuse menstruation [6].
Decoction of leaf is used as anti-inflammatory andantiarthritic, applied in aphthae, and also used insore-throat as gargle [1]Stem
D. melanoxylon
Root barkD. nitidula

Leaf

D. obovata

Root

D. odorifera

Heartwood

D. retusa

Heartwood

D. sissoo

Leaf

D. volubilis

Leaf

Beside the important role in traditional ethnopharmacology and pharmaceuticalapplications, many species of Dalbergia are important timber trees, valued for their decoratives
and often fragrant woods, rich in aromatic oils. For instances, D. sissoo wood is suitable for
quality furniture, plywoods, bridge piles, sport goods, railway sleepers due to strong and toughproperties,

and

D.

melanoxylon

isanintensely

black

wood

in

demandformaking woodwind musical instruments [9]. It should be noted that many wild plant Dalbergia
species have been threatening by habit loss and over exploitation for timber usage. It is not forcommercial sales, therefore, the protection and growth of this genus is an urgent task.
1.3. Phytochemical studies
Nowadays, the procedures of isolation, purification, and structural elucidation of theinteresting secondary metabolites are facilitated by continual development of chromatographic
9techniques such as thin layer chromatography (TLC), column chromatography (CC), gas
chromatography (GC), and high performance liquid chromatography (HPLC), ultra performanceliquid chromatography (UPLC), and spectroscopic analyses, for instance, nuclear magnetic
resonance (NMR), and mass spectrum (MS). Components of Dalbergia species are classified
into a wide range of compounds, comprising of the predominant amounts of flavonoids andphenolic compounds, as well as sesquiterpenes, aryl benzofurans, quinones, fatty acids, ketones,
and ester derivatives. As shown in Fig. 1, flavonoids in Dalbergia species have been found inseveral categories, such as flavones, isoflavones, flavanones, isoflavanones, isoflavans,
isoflavens, neoflavones, pterocarpans, chalcones, and bisflavonoids. While glucopyranosyl,
apiosyl-glucopyranosyl, and glucopyranosyl-glucopyranosyl units are the most common sugar of
the flavonoid glycosides. Particularly, isoflavone 7-O-β-apiofuranosyl-(1→6)-β-glucopyranosideis to be a crucial chemotaxonomic marker for this genus.

10Figure 1. Main skeletons found in isolated compounds of Dalbergia species
111.4. Biological investigations
The Dalbergia genus is shown to consist of main classes of flavonoid, phenol, andsesquiterpene derivatives, as well as several arylbenzofurans, quinones, and fatty acids.
On the other hand, biological studies on extracts, and isolated compounds from species ofDalbergia genus involve in cytotoxic, antibacterial, antioxidative, antiinflammatory,
antithrombotic,

antiplatelet,

antiosteosarcoma,

antiosteoporosis,

antiangiogenesis,

vasorelaxant activities, α-glucosidase inhibitory activities, and many other valuableeffects. Interestingly, as the valuable resources for natural new drug developments,
Dalbergia species is widely used as medicinal drugs in many countries for treatments,
such as cardiovascular diseases, cancer, blood disorder, ischemia, swelling, necrosis, or
rheumatic pains.
Regarding to the cytotoxic activity of chemical constituents of Dalbergia species,
Choi et al. reported the inhibitory effects of pterocarpan medicarpin and a phenolic
compound hydroxyobtustyrene on the proliferation of four human tumor cell lines,
human uterine carcinoma cell line (MES-SA), multidrug resistant subline of MES-SA(MES-SA/DX5), human colorectal adenocarcinoma cell line (HCT-15), and multidrug
resistant subline of HCT15/CL02 [10], in which medicarpin and hydroxyobtustyrene
established the significant ED50 values of 5.7-7.3 and 5.1-6.8 µM, respectively.
A poorly aqueous soluble chalcone, namely butein, was precipitated out of
methanol extract of D. odorifera species heartwood [11], which chelated with metal ions
Fe2+ (UV λmax: 286 and 422 nm) and Cu2+ (286 and 454 nm), and showed as potential
antioxidant agent with iron-induced lipid peroxidation inhibition in rat brain homogenate
12

in a concentration dependent manner with the IC50 value of 3.3 ± 0.4 µM, the IC50 valueof 9.2 ± 1.8 µM in DPPH reducing experiment (which was more potential than referencecompounds α-tocopherol 11.9 ± 0.2 µM, and BHT 14.5 ± 2.5 µM), the IC50 value of 5.9 ±0.3 µM in xanthine oxidase-induced uric acid formation inhibitory activity [12].
Notes on several antiinflammatory studies. Isoliquiritigenin did not show
cytotoxicity for RAW 264.7 macrophages event at the concentration of 20 µM, but, at theconcentration of 10 µM, displayed the results in inhibitory percentage of 86 and 79% for
NO and IL-1β production when LPS (200 ng/mL) stimulated RAW 264.7 macrophages
respectively, and absolutely inhibited iNOS mRNA and protein, TNF-α mRNA
expression [13]. A neoflavone 9-hydroxy-6,7-dimethoxydalbergiquinol and two
arylbenzofuran derivatives, (2R,3R)-obtusafuran and isoparvifuran were isolated from theheartwood of D. parviflora, D. odorifera and D. louvelii species, respectively. The
viability

of

cells

incubated

with

the

variousconcentrationsof

2,4,5-

trimethoxydalbergiquinol (5-50 µM), two arylbenzofurans (1-20 µM) did not affectsignificantly to BV2 microglia, whereas both 9-hydroxy-6,7-dimethoxydalbergiquinoland (2R,3R)-obtusafuran inhibited the levels of proinflammatory mediators NO, PGE2,
TNF-α, IL-1β, and decreased iNOS and COX-2 appearance when BV2 microglia wasstimulated by LPS at the doses of 500 ng/mL and 1 µg/mL, respectively. But
isoparvifuran did not show any decrease levels of above proinflammatory agents and
iNOS and COX-2 expressions event at the concentration of 20 µM [14-15].
The screening results for the motility and the viability of phytopathogenic
Aphanomyces cochlioides zoospores showed that three flavonoid derivatives daidzein,

13

dalbergin, and (6aR, 11aR)-variabiin from heartwood of D. stevensonii, D. odorifera andbark of D. frutescans, and medicarpin showed repellent activity at 150 µg/ml, whereas
claussequinone and formononetin showed stimulating and attracting activity at 100 and
50 µg/ml, respectively. A mixture of daidzein, dalbergin, and (6aR, 11aR)-variabiin(1:1:1, w/w/w) had advantages in repellent activity at 50 µg/ml [16].
As part of ongoing search for natural products with anti-cancers effects, 4methoxydalbergione was found to suppress growth and induce apoptosis in humanosteosarcoma cells in vitro and in vivo xenograft model through down-regulation of the
JAK2/STAT3 pathway [17].
The latent vasorelaxant relaxation of the butein on rat aortic rings precontractedwith various vasoconstrictors phenylephrine (3 µM), KCl (60 mM), U-46619 (1 µM),
endothelin-1 (1 nM), and angiotensin II (3 µM) had the EC50 values of 7.4 ± 1.6, 10.5 ±
2.3, 14.3 ± 3.3, 11.8 ± 2.0, 13.6 ± 3.7 µM, respectively [18]. Meantime, isoliquiritigeninfrom the heartwood of D. cochinchinensis, D. louvelii, and D. odorifera species wasfound to relax rat aorta contracted with phenylephrine, KCl, U-46619, endothelin and 5hydroxytryptamine, with EC50s of 7.4 ± 1.6, 10.5 ± 2.3, 14.3 ± 3.3, 11.8 ± 2.0 and 13.6 ±3.7 µM, respectively [19].
Taking a look at pharmacological screening of the extracts from genus Dalbergia,
the methanol extract (60%) of the Chinese medicinal plant D. odorifera heartwoodshowed potent repellent activity against A. cochlioides zoospores at the dose 200 µg/mL[16]. Taking the analgesic activtity into consideration, the methanol extract of D. spinosa
species was found to showe potential analgesic activity in the acetic acid-induced

14

writhing model with an inhibition of 40% at a dose of 500 mg/kg body weight ascompared with the control [1].
2. Studies on Dalbergia species in Vietnam
According to “An illustrated Flora of Vietnam” (2002), 48 plant species ofDalbergia genus were recorded in Vietnam, and distributed in different areas of thecountry, particularly in the North [15]. However, up to now, the phytochemical andbiological studies are quite limited. To the best of our knowledge, the phytochemical
investigation of methanol extracts of D. oliveri, D. vietnamensis, and D. tonkinensis led
to the isolation of total twenty two compounds, including daidzein, formononetin,pinocembrin,2′,4′,5′-trimethoxy-7-hydroxyisoflavanone,

liquiritigenin,

isoliquiritigenin, (3R)-5'-methoxyvestitol, sativanone, isoduratin,hydroxy-2,4,5-trimethoxydalbergiquinol,

butein,(6aR,11aR)-3,8-dihydroxy-9-methoxypterocarpan,

caviunin,violanone

naringenin, 3′-pratensein,

maackiain,

3-hydroxy-9-methoxypterocarpan,

lanceolarin, genistein-6-C-glucoside, daltonkins A and B, dalspinosin 7-O-β-Dapiofuranosyl-(1→6)-β-D-glucopyranoside, caviunin 7-O-β-D-apiofuranosyl-(1→6)-βD-glucopyranoside, caviunin 7-O-(5-O-trans-p-coumaroyl)-β-D-apiofuranosyl-(1→6)-βD-glucopyranoside,

and9,10-threo-3-[7-(3,10-dihydroxy-9-hydroxymethyl-2,5-

dimethoxy)-9,10-dihydrophenanthrenyl]-propenal [20-22].

15

Figure 2. Chemical constituents from the Vietnamese Dalbergia speciesInthecytotoxic

experiment,

twopterocarpans(6aR,11aR)-3,8-dihydroxy-9-

methoxypterocarpan and 3-hydroxy-9 methoxyterocarpan from D. oliveri heartwood werereported to inhibit four cell lines KB, LU-1, Hep G2, and MCF7 with the strong IC50 values of
3.76-7.09 µg/ml, whereas with regards to the antimicrobial assays, these compounds as well as
liquiritigenin, genistein 6-C-glucoside, maackiain, formononetin, pratensein, violanoneisoliquiritigenin, and (3R)-5'-methoxyvestiol were inactive against all tested bacteria
Staphylococcus aureus, Bacillus subtilis, Lactobacillus fermentum, Salmonella enterica,
Escherichia coli, Pseudomonas aeruginosa, and Candida albican [21].
Among Dalbergia species, the precious tree D. tonkinensis has established in thevulnerable level in the International Union for Conservation of Nature (IUCN) Red List of16

Threatened Species by excessive exploitation for commercial purpose. International publications
involved in D. tonkinensis which mostly focused on taxonomic differents between it and its otherrelated species principally based on using DNA identifications and chemical comparison with
GC-MS techniques [23,24]. Recently, a survey conducted by Nguyen et al suggested that themethanol extract of the trunk-bark, the heartwood, and the leaf of this plant had demonstrated
potential α-glucosidase inhibition [25].

17Chapter 2 Chemical constituents from Dalbergia tonkinensis
2.1. Introduction
Scientific name: Dalbergia tonkinensis Prain and local name: Sua do, Trac thoi;
Family: Fabaceae; genus: Dalbergia;
Morphology: The perennial trees, 5-13 m tall, leaves 9-20 cm, rachis glabrous, stipules
caducous, small, tawny, puberulent, leaflets (7) 9-11, petiolule glabrous, blades ovate, 4-9×(1) 35 cm, subleathery, sparsely puberulent when young, soon glabrous, base rounded, apex shortly
acuminate. Panicles corymbose, axillary, ca. 5×3.8 cm. Flowers white, fragrant. Legumedistinctly stipitate, ovoid or oblong, 5–7.5×ca. 2 cm, reticulate opposite seeds, base cuneate, apex
subacute. Seeds reniform, compressed, ca. 9×5 mm [26].
Distribution: Ha Noi, Vinh Phu, Ha Nam Ninh, Ha Son Binh, Dong Nai [15].
Conservation status: Vulnerable level.

Figure 3. Heartwood, root, and leaf of D. tonkinensis

18

2.2. Phytochemical investigation of heartwood extract and structural elucidation
2.2.1. Isolated proceduren-Hexane: H

Heartwood of D. tonkinensis

Choloroform: C(1 kg powder)

Dichloromethane: D
MeOH/5 x 1.5 L/5 days

Ethyl acetate: E
Acetone: A
Methanol: M

MeOH extract (46.53 g)

Powder

MeOH:H2O (1:1, v/v)

Water decoction, 4 x 2.0 L/4 h

Water: W

Water: W
Partioned with n-Hexane, CHCl3, and EtOAc

Fr. W (15.0 g)
Diaion HP-20, W:M (0:1→0:1)

Fr. E (1.3 g)

Fr. C (39.43 g)
Fr. W2 (0.9 g)

Fr. W4 (1.44 g)

Silica gel

Silica gel

Silica gel

n-H:A 2:1

C:M:W 3:1:0.1

D:M:W 1:1:0.1

27 (2.5 mg),
28 (7.4 mg)

20 (2 mg),
21 (4 mg)

Silica gel, C:A 3:1

Fr. E2 (0.8 g)

Fr. E3 (0.1 g)

Fr. C2 (6.1 g)

Fr. C3 (15.3 g)

Silica gel

Silica gel

Silica gel

Silica gel

C:E 3:1

D:M 3:1

C:A 70:1

C:A 30:1

9 (5.1 mg),
11 (4.0 mg)

10 (3.4 mg)

1 (70 mg), 2 (0.2 g), 3 (54mg), 4 (4 mg), 5 (45 mg), 6(6.1 mg), 7 (18 mg), 8 (3mg), 22 (4.2 mg), 23 (2 mg),24 (1.5 mg), and 25 (2 mg).

12 (5 mg), 13 (5 mg),
14 (3 mg), 15 (4 mg),
16 (6 mg), 17 (2 mg),
19 (3 mg), 18 (25 mg),and 26 (1.5 mg).

From the chloroform (CHCl3), ethyl acetate (EtOAc) fr.s of the methanol, and aqueous
decoction extracts of D. tonkinensis heartwood, three new compounds 20, 21 and 25, along withtwenty five known compounds 1-19, 22-24, and 26-28 were isolated. By comparison of their 1H,
and 13C-NMR spectroscopic data with the literature data, the chemical structures of the known
compounds were identiﬁed as formononetin (1), sativanone (2), naringenin (3), liquiritigenin (4),pinocembrin (5), medicarpin (6), 3'-hyrdoxy-2,4,5-trimethoxydalbergiquinol (7), buteaspermanol

19

(8), butin (9), 3'-hyrdoxymelanettin (10), biochanin A (11), calycosin (12), eriodictoyl (13),isoliquiritigenin (14), 3'-O-methylviolanone (15), luteolin (16), quercetin (17), apigenin (18),sulfuretin (19), dalbergin (22), homoferreirin (23), neroplomacrol (24), kaemfperol (26), formyl
phenol (27), and naringin (28) [9, 27-33].

Figure 4. Compounds isolated from D. tonkinensis heartwood20

2.2.2. Structural elucidation of new compounds 20, 21 and 25
Compound 20: (2S)-6,8-Dicarboxyethylpinocembrin

Figure 5. Structure, and COSY and HMBC key correlations of 20
Table 2. 1H- and 13C-NMR data of compounds 20 and 21 in CD3OD
Position

2021δH (J in Hz) a)

δC b)

δH (J in Hz) a)

δC b)

2

5.50 (dd, 13.0, 3.0)

80.2

5.34 (dd, 13.0, 2.5)

80.5

3

2.84 (dd, 17.0, 3.0)

44.2

2.72 (dd, 17.0, 2.5)

44.1

3.11 (dd, 17.0, 13.0)

3.14 (dd, 17.0, 13.0)

4

198.1

198.0

4a

103.5

103.2

5

159.9

162.9

6

109.1

7

163.9

166.3

8

108.3

108.6

8a

161.1

162.7

5.97 (s)95.7

92.88 (m)19.5

2.84 (t, 8.0)

18.8

10

2.54 (m)34.7

2.48 (t, 8.0)

34.6

11

178.7

9'

2.88 (m)

18.8

10'

2.54 (m)

34.3

11'

178.5

1'

140.6

21

177.9

131.2

a)

2',6'

7.54 (d, 7.5)

127.2

4'

7.39 (t, 7.5)

129.5

3',5'

7.44 (d, 7.5)

129.7

7.33 (d, 8.5)

129.0
159.0

6.84 (d, 8.5)

116.3

500 MHz, b) 125 MHz.
Compound 20 was isolated as a white amorphous solid. The molecular formula of 20 was

consistent with C21H20O8 deduced from the quasi-molecular ion peak at m/z 399.1080 [M-H]- inthe negative HR-ESI-MS. Its IR spectrum displayed the absorption bands at 3370 and 1721 cm-1,
due to hydroxyl and carbonyl groups, respectively. The 1H and 13C-NMR spectroscopic data of
20 were similar to those of (2S)-pinocembrin [34], except for the absence of two aromatic
protons H-6 and H-8 in (2S)- pinocembrin which were replaced by two carboxyethyl groups [H9' (δH 2.88)/C-9' (δC 18.8), H-10' (δH 2.54)/C-10' (δC 34.3), and C-11' (δC 178.5)] and [δH 2.88 (H9)/δC 19.5 (C-9), δH 2.54 (H-10)/δC 34.7 (C-10), and δC 178.7 (C-11)] in 20 (Table 2). Analysis
of 1H-1H correlation spectroscopy (COSY) and HMBC spectra enabled the linkage of one
carboxyethyl side chain to be on the carbon C-6 by COSY correlation of H-9' and H-10' and
HMBC correlations of H-9' and H-10'/C-11', and H-9'/C-5, C-6 and C-7 (Fig. 5). Similarly,
COSY cross peak between H-9 and H-10, as well as HMBC correlations between H-9 and H10/C-11, and H-9/C-7 and C-8a confirmed the connectivity of the other carboxyethyl side chain
on the carbon C-8. The CD spectrum of 20 revealed positive Cotton effect at ∆ε339 (nm) +0.83(n−π* transition) and negative Cotton effects at ∆ε290 (nm) −8.21 (π−π* transition), thereby
indicating the 2S-configuration [35]. Consequently, the chemical structure of 20 wascharacterized as (2S)-6,8-dicarboxyethylpinocembrin.

22

H-9,9'
H-10,10'
H-3

Ring B
H-2

Pinocembrin skeleton

CarboxyethylgroupsFigure 6. 1H-NMR/13C-NMR spectra of compound 20 in CD3OD
23

Compound 21: (2S)-8-Carboxyethylnaringenin

Figure 7. Structure, COSY and HMBC key correlations of 21
Compound 21 was obtained as a white amorphous solid, and its molecular formulaC18H16O7 was deduced from the quasi-molecular ion peak at m/z 343.0815 [M-H]- in thenegative high resolution-electrospray ionization mass spectroscopy (HR-ESI-MS). The IR
spectrum of 21 showed absorption bands at 3356 and 1737 cm-1, attributing to hydroxyl andcarbonyl groups, respectively. The 1H and 13C-NMR spectroscopic data of 21 were similar to
those of (2S)-naringenin [34], except for the absence of the aromatic proton H-8 in (2S)naringenin which was replaced by a carboxyethyl group [δH 2.84 (H-9)/δC 18.8 (C-9), δH 2.48 (H10)/δC 34.6 (C-10), and δC 177.9 (C-11)] existing in 21 (Table 2). This was further substantiatedby heteronuclear HMBC correlations between H-6 and C-5/C-7/C-8 (Fig. 7). In addition, the
HMBC correlations of H-9 to C-7/C-8/C-8a suggested that the carboxyethyl group was located at
C-8 (Fig. 7). The CD spectrum of 21 showed positive Cotton effect at ∆ε337 (nm) +0.19 (n−π*transition) and negative Cotton effect at ∆ε293 (nm) −0.71 (π−π* transition), which was consistent
with the 2S-configuration [35]. According to the analysis of spectroscopic data, the chemical
structure of compound 21 was elucidated as (2S)-8-carboxyethylnaringenin. Two rare isolated
compounds 20 and 21 indicated the same manner with the previous natural product torosaflavone
D and its derivative ananthoflavone [36].

24

H-9

H-6

H-2',6'H-3b

H-3',5'

H-3a

H-10

H-2

Carboxyethyl
group

Naringenin skeleton

Figure 8. 1H-NMR/13C-NMR spectra of compound 21 in CD3OD
25

Compound 25: Rel-(3R,6S,7S)-6α-hydroxycyclonerolidol

Figure 9. Structure, and COSY, HMBC and NOESY key correlations of 25
Table 3. 1H- and 13C-NMR data of compound 25 in CD3OD
Position δH (J in Hz) a)1
5.22 (dd, 17.5, 1.5)
5.04 (dd, 10.5, 1.5)
2
5.93 (dd, 17.5, 10.5)
34
1.52 (m)1.87 (m)51.40 (m)1.73 (m)6
3.27 (br d, 10.5)
78
1.46 (m)1.55 (m)a)δC b) Position
112.0
9δH (J in Hz) a)
2.08 (m)

δC b)
23.0

146.6
73.9
40.9

10
11
12

5.14 (br t, 7.5)
1.69 (s)

126.1
131.2
25.9

26.6

13

1.64 (s)

17.7

79.3
75.5
39.4

14
15

1.12 (s)1.28 (s)

22.1
27.9

500 MHz, b) 125 MHz.

Compound 25 was obtained as a white amorphous solid. The molecular formula of 25 wasdetermined to be C15H26O2 based on the quasi-molecular ion peak observed at m/z 239.2011[M+H]+ in the positive HR-ESI-MS spectrum. The IR spectrum showed absorption bands at
3362 and 1642 cm–1, which were attributed to hydroxyl and double bond groups, respectively.
The 1H NMR spectrum of 25 showed signals similar to those of 6α-hydroxycyclonerolidol andrel-(3R,6R,7S)-3,7,11-trimethyl-3,7-epoxy-1,10-dodecadien-6-ol [37, 38], as follows: olefinic
proton signals of H2-1 exo-olefinic methylene at δH 5.22 (dd, J = 17.5, 1.5 Hz, H-1a) and 5.04
(dd, J = 10.5, 1.5 Hz, H-1b), H-2 at δH 5.93 (dd, J = 17.5, 10.5 Hz) and H-10 at δH 5.14 (br t, J =7.5 Hz), oxygenated methine proton H-6 at δH 3.27 (br d, J = 10.5 Hz), two pairs of vicinalmultiplet methylene protons H2-4 at δH 1.52 (m, H-4a) and 1.87 (m, H-4b), H2-5 at δH 1.40 (m,26

H-5a) and 1.73 (m, H-5b), H2-8 at δH 1.46 (H-8a) and 1.55 (H-8b), H2-9 at δH 2.08, and four
methyl groups [H3-12 (δH 1.69), H3-13 (δH 1.64), H3-14 (δH 1.12), H3-15 (δH 1.28), each singlet].
The 13C-NMR/DEPT spectra of 25 contained 15 carbon signals, comprising four CH3, five CH2,
three CH, and three C4 groups, which were assigned to a sesquiterpene. Its COSY correlations
provided three partial units of C1–C2, C4–C6, and C8–C10, as indicated by bold lines in Fig. 9.
The structure of 25 was further confirmed by HMBC analysis. The HMBC spectrum supported
the C8–C13 carbon sequence due to both H3-12 and H3-13 signals showing correlations with two
olefinic carbons C-10 (δC 126.1) and C-11 (δC 131.2), while the H3-14 signal showed HMBC
cross peaks with C-6 (δC 79.3), C-7 (δC 75.5), and C-8 (δC 39.4) (Fig. 9). Furthermore, HMBC
correlations of H3-15 with quaternary carbon C-3 (δC 73.9) and C-4 (δC 40.9), and of H-2 withquaternary carbon C-3 confirmed that methyl group H3-15 and vinyl moiety C1–C2 wereattached to the C-3 position. The stereochemistry of the chiral centers was elucidated using
nuclear Overhauser effect spectroscopy (NOESY) correlations and by comparing the 13C NMR
spectrum with literature data [37, 38]. The chemical shift of the chiral center C-3 was similar to
those of reference compounds 6α-hydroxycyclonerolidol and rel-(3R,6R,7S)-3,7,11-trimethyl3,7-epoxy-1,10-dodecadien-6-ol, indicating that C-3 in 25 had the R*-configuration [37,38].
Furthermore, the NOESY spectrum showed selective correlations between CH3-14 and oxygenbearing H-6, and between H3-15 and H-8 signals, but had neither correlations between the H-6
and H2-8 or H3-15 signals, nor between the H3-14 to H3-15 signals. These NOESY data
supported that two methyl groups, H3-14 and H3-15, had a trans-configuration and adopted an αorientation with respect to the 6-OH group [37,38]. Compound 25 showed a negative opticalrotation value of [α]25D –15.0° (c 0.02; CHCl3), which was similar to that of glandulaurencianol
C [39]. From these results, the structure of compound 25 was assigned to be rel-(3R,6S,7S)-6αhydroxycyclonerolidol.
27

15-CH3
13-CH314-CH312-CH3

H-10
H-1
H-6
H-2

Figure 10. 1H-NMR/13C-NMR spectra of compound 25 in CD3OD
28

2.3. Phytochemical investigation of leaf extract and structural elucidation
2.3.1. Isolation procedure
Leaf of D. tonkinensis(2 kg, Powder)
MeOH/10 L/4 h
MeOH extract, 300 g
MeOH:H2O (1:1, v/v)
Partitoned with n-Hexan, CHCl3, and EtOAc

Fr. C (60.7 g)

Fr. E (15.3 g)

C:E, 99:1

Fr.W (95.5 g)

Sephadex, M:W 1:1

Silica gel

Fr. E4 (2.3 g)

Diaion HP-20, W:M (0:1→0:1)
Fr.W2 (4.57 g)

RP 18, M:W 1:1

Silica gel C:M:W 3:1:0.1

31 (4.4 mg)
Fr. C2 (5.1 g)

Fr. C3 (4.3 g)

Fr. C6 (5.7 g)

Silica gel

Silica gel

Silica gel

C:A 30:1

n-H:A 6:1

C:M 3:1

11 (45.7 mg)

29 (34.6 mg)

30 (15.6 mg)

32(20.6 mg)

Fr. W27(0.9 g)Fr. W29(0.6 g)Fr. W28(1.9 g)

HPLC,

HPLC,

HPLC,

M:W 7:3,

M:W 7:3,

M:W 3:1,

1 mL/min

1 mL/min

0.1% HCOOH

33 (1.2 mg)

34 (4.6 mg)

2 mL/min35 (1.6 mg)

From the fr. CHCl3 and aqueous residue of the methanol extract of D. tonkinensis leaf,
two new compounds 31 and 33, and six known compounds 11, 29, 30, 32, 34 and 35 wereisolated. The chemical structures of the known compounds were identiﬁed as biochanin A(11), genistein (29), orobol (30), dalsissooside (32), ambocin (34), lanceolarin (biochanin A
7-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside) (35) by comparison of their NMR
spectroscopic data with the literature data, respectively [11, 40-43]. Among them, biochanin29A (11) had also been found in the heartwood, and ambocin (34), an isoflavone glycoside, wasisolated for the first time from genus Dalbergia.

Figure 11. Compounds isolated from leaf of D. tonkinensis
2.3.2. Structural elucidation of new compounds 31 and 33
Compound (31): 3-(1H-indol-3-yl)-2-methoxypropanamide

Figure 12. Structure, and COSY, HMBC and NOESY key correlations of 33

30

Table 4. 1H-, and 13C-NMR data of compound 31 in CD3OD
3-(1H-indol-3-yl)-2-methoxypropanamide (31)
δH (J in Hz)a)δCb)
PositionδH (J in Hz)
170.3
4'
7.59 (d, 8.0 Hz)
3.85 (dd, 9.0, 5.5 Hz)
79.2
5'6.98 (td, 8.0, 1.0 Hz)
3.39 (dd, 9.0, 5.5 Hz)
23.3
6'7.01 (td, 8.0, 1.0 Hz)
7.27 (s)124.0
7'7.28 (d, 8.0 Hz)
107.6
7'a
126.9
2-OCH33.25 (s)a)b)
500 MHz, 125 MHz

Position
1
2
3
2'
3'
3'a

δC
117.6
118.6
121.2
111.0
136.7
51.3

Compound 31 was obtained as a white powder and its molecular formula C12H14N2O2
was deduced from its quasi-molecular ion peak at m/z 241.0953 [M+Na]+ in the its positive
HR-ESI-MS data. Prominent fragment ions at m/z 188, 144 and 118 from APCI mass were
due to the loss of a methoxy group and an amide group, thereby supporting the presence of an
indolyl unit. The IR spectrum of 31 showed absorption bands at 3416 and 1629 cm-1, which
could be attributed to amine and amide groups, respectively [44]. The UV spectrum showedabsorption maxima at λmax 281 and 288 nm, which are characteristic of a tryptophan
derivative [44]. The 1H-NMR spectrum of 31 displayed signals similar to those of 2-methoxy3-(3-indolyl)-propionic acid [45] with resonances from an indolyl ring [H-4' (δH 7.59, 1H, d,
8.0 Hz), H-5' (δH 6.98, 1H, td, 8.0, 1.0 Hz), H-6' (δH 7.01, 1H, td, 8.0, 1.0 Hz), H-7' (δH 7.28,
1H, d, 8.0 Hz) and H-2' (δH 7.27, s)] and a 2-methoxypropanoyl moiety [H-2 (δH 3.85, 1H, dd,
9.0, 5.5 Hz), H2-3 (δH 3.39, 2H, dd, 9.0, 5.5 Hz), and 2-OCH3 (δH 3.25, s)]. The 13C-NMR and
HSQC spectra of 31 exhibited 12 carbon signals, comprising of 8 aromatic carbons from δC
107.6–136.7, a carboxyl carbon at δC 170.3 (C-1), an O-bearing methine carbon at δC 79.2 (C2), a methylene carbon at δC 23.3 (C-3) and a methoxy carbon at δC 51.3 (2-OCH3) (Fig. 12).
The structures of 31 was also conﬁrmed by analysis of its COSY, HMBC and NOESY spectra(Fig. 12). The 1H-1H COSY spectrum showed cross peaks for H-4'/H-5'/H-6'/H-7' and H2/H2-3 and the HMBC spectrum established the indolyl ring by the J2 and J3 correlations from31

H-2' to C-3', C-3'a and C-7'a. Meanwhile, the HMBC cross peaks from OCH3 to H-2, from H2 and H2-3 to C-1, and from H2-3 to C-2' and C-3' confirmed the presene of a 2methoxypropanoyl unit and its connection to carbon C-3'. In accordance with 2-methoxy-3(3-indolyl)-propionic acid, the 1H-1H NOESY correlations between H-2 and H-4' and between
2-OCH3 and H-2' were observed [45]. Based on these spectroscopic data, compound 31 wasdetermined to be 3-(1H-indol-3-yl)-2-methoxypropanamide.

2- OCH3
Overlap

Indolyl ring
H-3
H-2

32

2- OCH3
C-2
C-1

Figure 13. 1H-NMR/13C-NMR spectra of compound 31 in CD3OD
Compound 33: Isocaviunin 7-O-β-apiofuranosyl-(1→6)-β-glucopyranoside

Figure 14. Structure, and COSY and HMBC key correlations of 33

33

C-3

Table 5. 1H-, and 13C-NMR data of compound 33 in C5D5N
Isocaviunin 7-O-β-apiofuranosyl-(1→6)-β-glucopyranoside (33)
Position δH (J in Hz)a)δCb)
PositionδH (J in Hz)
δC2
8.24 (s)155.7
2''4.34 (m)
74.5
3
120.6
3''
4.78, (m)
77.7
4
181.2
4''
4.11 (m)
71.2
4a
106.9
5''
4.27 (m)
77.3
5
157.8
6''
4.71 (d, 11.0 Hz)
68.6
4.14 (m)67.30 (s)100.1
1'''5.70d) (d, 2.5 Hz)
111.0
7
157.2
2'''
4.32 (m)
78.4
8
130.0
3'''
80.2
8a
150.9c)4'''4.63 (d, 9.0 Hz)
75.0
4.36 (m)
1'
111.3
5'''
4.20 (t, 11.5 Hz)
65.6
2'
152.6
5-OH
13.20 (s)3'
6.78 (s)98.9
8-OCH33.99 (s)
61.5
c)
4'
150.9
2'-OCH3
3.73 (s)
55.9
5'
143.6
4'-OCH3
3.80 (s)56.3
6'
7.21 (s)117.05'-OCH3
3.83 (s)56.7
1''
5.68d) (d, 7.5 Hz) 102.2a)
500 MHz, b)125 MHz, c)may be interchangeable, d)the assignments were made by HSQC
and HMBC correlations and comparison with 32
Compound 33 was obtained as white amorphous powder. Its molecular formula
C30H36O17 was deduced from the quasi-molecular ion peak at m/z 707.1577 [M+K]+ in thepositive HR-FAB-MS data and from the m/z 707 [M+K]+, 669 [M+H]+, and 667 [M–H]–
peaks in the positive (negative) FAB-MS data. The IR spectrum of 33 showed absorption
bands at 3510, 3378, 1654, and 1581 cm-1, which could be attributed to hydroxyl, chelatedhydroxyl, α,β-unsaturated carbonyl, and carbon-carbon double bond conjugated carbonylgroups, respectively. The UV spectrum of 33 showed absorption maxima at λmax values of 205,
263 and 295 nm, which are characteristic of an isoflavone derivative [40]. The 1H-NMR
spectrum of 33 displayed a spectral pattern similar to that of isocaviunin 7-gentibioside(compound isolated from D. sissoo) with resonances from two components [40], which34

include an isocaviunin nucleus [5-OH (δH 13.20, 1H, s), H-2 (δH 8.24, 1H, s), H-6 (δH 7.30,
1H, s), H-6' (δH 7.21, 1H, s), H-3' (δH 6.78, 1H, s), 8-OCH3 (δH 3.99, 3H, s), 5'-OCH3 (δH 3.83,
3H, s), 4'-OCH3 (δH 3.80, 3H, s), and 2'-OCH3 (δH 3.73, 3H, s)] and an β-apiofuranosyl(1→6)-β-glucopyranosyl moiety [H-1''' (δH 5.70, d, 2.5 Hz), H-1'' (δH 5.68, d, 7.5 Hz), and
11H (δH 3.99-4.78)] (Table 5 and Fig. 14). The 13C-NMR and HSQC spectra of 33 (C5D5N)
contained 30 carbon signals (12×C, 11×CH, 3×CH2, 4×CH3), which were assignable to an
isoflavone aglycone, a β-apiofuranosyl residue [H-1''' (J = 2.5 Hz)], and a β-glucopyranosyl
bridge [H-1'' (J = 7.5 Hz)] [40]. Regarding the 2D-NMR analysis, the HMBC spectrum
established the isocaviunin ring by the J2 and J3 correlations H-2/C-3, C-4 and C-8a; H-6/C4a, C-5, C-7, and C-8; 5-OH/C-4a and C-5; H-3'/C-1' and C-5'; H-6'/C-5' and C-3; 8-OCH3/C8; 2'-OCH3/C-2; 4'-OCH3/C-4; and 5'-OCH3/C-5, and a glycone unit was confirmed by thecross peaks H-2''/C-3''; H-3''/C-4''; H-4'''/C-1''' and C-2'''; and H-5'''/C-4''' in the HMBC
spectrum as well as H-1''/H-2''; H-4''/H-5''; H-5''/H-6''; and H-1'''/H-2''' in the COSY spectrum(Fig. 14). Furthermore, the key 1H-13C HMBC correlation H-1'''/C-6'' evidently generated a
1→6 linkage between two sugar units, and the cross peak H-1''/C-7 indicated the connection
between glycone and aglycone at C-7. In addition, the acid hydrolysis of 33 yielded theaglycone, the NMR and MS data of which were identical to those of isocaviunin [46]. Basedon these findings, compound 33 was elucidated to be isocaviunin-7-O-β-apiofuranosyl(1→6)-β-glucopyranoside, trivially named daltonkinensiside (33).

35

Anomeric protons
H-1'', H-1'''

H-6'
H-6
H-2

H-3'

5-OH

8-OCH

Sugar unit

36

35'-OCH

4'-OCH
3

32'-OCH3

Anomeric carbons
C-1'''
GlyconeIsocaviunin sekeleton
C-1''

Figure 15. 1H-NMR/13C-NMR spectra of compound 33 in C5D5N

378, 2', 4', 5'-OCH3

2.4. Phytochemical investigation of root extract and structural elucidation
2.4.1. Isolation procedure
Root of D. tonkinensis(EtOH extract, 200.0 g)
Partioned with n-hexane, CH2Cl2, and EtOAc
Fr. CH2Cl2 (45.5 g)
Silica gel, D:A 3:1
D1 (2.1 g)

D3 (2.7 g)

D2 (3.2 g)

Total 6 Fr.s

H:A 3:1

Sephadex

Silica gel

39 (15.0 mg), 40(5.5 mg)

LH-20

C:M 3:1

M:W 1:1

45 (2.0 mg),46 (2.5 mg)

D31 (20 mg)

36 (4.2 mg), 41 (3.1 mg),

HPLC, M:W 3:1,

43 (2.1 mg), 44 (3.99 mg)

0.1% HCOOH
2 mL/min
UVmg)
210 nm47 (1.1

Fr. EtOAC (30.5 g)
Silica gel, C:M:W 3:1:0.1
E2 (3.2 g)

HPLC, M:W
7:3, 2 ml/min
UV 210 nm
2 (1.3 mg), 6 (0.7mg), 13 (1.2 mg),
14 (2.3 mg), 22(1.5 mg), 26 (0.5mg), 42 (1.3 mg)

E4 (1.2 g)

E3 (1.2 g)
Sephadex
LH-20
M:W 1:1
37 (5.1 mg)

Silica gel

Sephadex

C:M 3:1

LH-20

Total 7 Fr.s

M:W 1:1

4 (2.4 mg)
8 (1.8 mg)

1 (7.2 mg)

15 (4.0 mg)

9 (2.1 mg)

38 (25.2 mg)

From fr.s CH2Cl2 and EtOAc of the ethanol extract of D. tonkinensis root, one newcompound 47 and twenty-two other known compounds were isolated. Among them,compounds 1-2, 4, 6, 8, 9, 13-15, 22 and 26 have already been isolated from the heartwood.

38

The chemical structures of the other known compounds were identified to be tectorigenin (36),tectoridin (37), tectorigenin 7-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (38), 1monolaurin (39), and 1-monomyristin (40), (3R)-vestitol (41), melilotolcarpan D (42),daidzein (43), 3'-hydroxyldaidzein (44), formononetin-7-O-β-D-glucopyranoside (45), andmethyl 4-hydroxybenzoate (46) [1, 47-51]. Two isoflavone glycosides 37 and 38 wereisolated for the first time from the root, whereas mono-acylglycerides 39 and 40 and monophenol 46 have never been identified in the family Fabaceae before.

Figure 16. Compounds isolated from D. tonkinensis root
2.4.2. Structural elucidation of new compound 47
2,5'-Dimethyl-5'-vinylhexahydro-2,2'-bifuran-5(2H)-one (47)Rel-(2S,2'S,5'S)-2,5'-Dimethyl-5'-vinylhexahydro-2,2'-bifuran-5(2H)-one (47a)Rel-(2S,2'S,5'R)-2,5'-Dimethyl-5'-vinylhexahydro-2,2'-bifuran-5(2H)-one (47b)39

Figure 17. Structure, and COSY and HMBC key correlations of isolated compound 47
Table 6. 1H-, and 13C-NMR data of compound 47 in CD3OD
Position2

δH (J in Hz)a)

δCb)
88.3

Position
3'2-CH3

1.35 (s)

22.4

4'

3

1.91 (m)2.31 (m)2.62 (m)

28.1

5'

29.1
178.4

5'-CH36'

4
5
2'

4.10 (dd, 3.0, 83.6
7'
10.0)
a)
500 MHz, b)125 MHz, *47a, **47b

δH (J in Hz) a)
1.70 (m)1.99 (m)1.76 (m)1.95 (m)
-

δCb)
26.7

1.28 (s) and 1.30 (s)5.97 (dd, 10.5, 17.5)*
5.84 (dd, 10.5, 17.5)**
5.18 (d, 17.5)*
4.99 (d, 10.5)**

25.7
143.7

36.9
83.2

110.6

Compound 47 was obtained as yellowish amorphous powder. The molecular formulaof 47 corresponded to C12H18O3, as deduced from the molecular ion peak at m/z 210.1253[M]+ in its HR-EI-MS data. The absorptions at 1771 and 1607 cm-1 in the IR spectrum showed
the presence of a γ-lactone and a double bond, respectively. The 1H, and 13C-NMR data of 47
included two methine groups, five methylene groups, two methyl groups, two oxygenatedquaternary carbons, and one lactone carbon (Table 6 and Fig. 17). Based on the HSQC,
HMBC, COSY, and NOESY extensive analysis and comparison with data recorded in theliterature [37], compound 47 was assigned as a stereoisomer of rel-(2R,2'R,5'S)-2,5'-dimethyl5'-vinylhexahydro-2,2'-bifuran-5(2H)-one (a component was isolated from D. odorifera T.

40

Chen) [37]. As shown in Fig. 17, the appearance of tetrahydrofuran (THP) and γ-lactone rings
was confirmed by the COSY correlations H-3/H-4, H-2'/H-3', and H-3'/H-4' as well as the
HMBC cross peaks H-3 and H-4/C-5 and H-4'/C-5'. Furthermore, the COSY correlation H6'/H-7' and the HMBC interactions H-6'/C-5', 5'-CH3/C-5' and C-4', and 2-CH3/C-2 and C-3
indicated that the vinyl group occurred at C-5', and two methyl groups were attached at the C2 and C-5' positions. The connection between the THP ring and the γ lactone ring can be
explained by the key HMBC correlation 2-CH3/C-2'.
Compound 47 was found to have diffential δH at proton 5'-CH3 and H-6' and a small opticalrotation of –1.2 (c 1.1, MeOH). As a consequence, we supposed that compound 47 was adiastereomeric mixture. Thus, compound 47 was further analyzed by a chiral HPLC column(OD-RH, 150 x 4.6 mm) with a mobile phase of n-hexane-2-propanol (80:20, v/v), thereby
showing two peaks at tR = 11.2 (35%) and tR = 15.81 (65%). Therefore, compound 47 was
identified as two diastereomers rel-(2S,2'S,5'S)-2,5'-dimethyl-5'-vinylhexahydro-2,2'-bifuran5(2H)-one (47a) and rel-(2S,2'S,5'R)-2,5'-dimethyl-5'-vinylhexahydro-2,2'-bifuran-5(2H)-one(47b).

41

Figure 18 Chiral HPLC chromatogram of diastereomers 47a and 47b2-CH35'-CH3H-3', H-4'
H-7'
H-6'

H-3
H-2'

H-4

42

C-3, C-4, C-3'
5'-CH
3
2-CH

C-2 C-2', C-5'
C-5

C-7'

C-6'

C-4'

3

Figure 19. 1H-NMR/13C-NMR spectra of compound 47 in CD3OD

Figure 20. NOESY spectrum of compound 47 in CD3OD
43

2.5. Chemotaxonomic significances from the isolated constituentsA vast data evidently showed that flavonoids and their glycosides possessed the highamounts in the species of the genus Dalbergia and the family Fabaceae [1]. The present
phytochemical studies on D. tonkinensis species led to the isolation and identification of 47
constituents from its several parts for the first time.
Two new unusual flavanones (2S)-6,8-dicarboxyethylpinocembrin (20) and (2S)-8carboxyethylnaringenin (21) could be seen as the first products of mono-and/or di-substitutionof carboxyethyl group at the positions C-6 and/or C-8 of pinocembrin (5) and naringenin (3).
The occurrence of 20 and 21 reveals not only a key feature of chemical structure but also thesignificant findings regarding the species D. tonkinensis and the pea family for the first time.
Hence, two new carboxyethylflavanones 20 and 21 may be valuable chemotaxonomic
markers for the family Fabaceae.
This study also provides new information concerning isolated flavanone metabolites,such as buteaspermanol (8), which is characteristic of heartwood and root of D. tonkinensis
species, since this compound had only been found in bean family [this compound was onlyisolated from two species of the subfamily Faboideae, Butea monosperma (from stem bark),
and Spatholobus suberectus (from stem)] [52,53]. Therefore, compound 8 was isolated fromthe genus Dalbergia for the first time, and its occurrence supports the arguments to define theclose releationship among the species, the genera of the subfamily Faboideae. Likewise,
another flavanone glycoside naringin (28), which is common in plants and mostly distributedin the Citrus genus [54], and in Securigera securidaca flower (Fabaceae) [55], but it isisolated for the first time from the genus Dalbergia.

44

Of the remaining flavonoids, formononetin (1), liquiritigenin (4), medicarpin (6), andisoliquiritigenin (14) are also constituents of the bark of the Vietnamese plant D. oliveri [56],and sativanone (2), naringenin (3), pinocembrin (5), biochanin A (11), calycosin (12), 3'-Omethylviolanone (15), and genistein (29) are constituents of D. parwiﬂora heartwood [48]. 3'Hydroxy-2,4,5-trimethoxydalbergiquinol (7), butin (9), 3'-hydroxymelanettin (10), eriodictoyl(13), sulfuretin (19), dalbergin (22), homoferreirin (23), orobol (30), tectorigenin (36), (3R)vestitol (41), melilotocarpan D (42), hydroxyldaidzein (43), and 3'-hydroxyldaidzein (44) are
isolated from D. odorifera T. Chen [1], while luteolin (16), and apigenin (18) are derived
from D. paniculata flower [57]. Quercetin (17) is a constituent of D. sericea leaf [58] andkaemfperol (26) is precipitated out of tender leaf of D. sissoo [59].
In accordance with results of the Vietnamese plant D. vietnamensis, isoflavone
glycosides 32-35, 37, and 38 were found to have 7-glycosyl moieties of O-β-Dglucopyranosyl, O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranosyl, and/or O-(5-O-trans-pcoumaroyl)-β-D-apiofuranosyl-(1→6)-β-D-glucopyranosyl [60]. Among them, isoflavone
glycoside (33) is a new isomer of compound 32. Ambocin (34), an isoflavone glycoside isalso presented in the D. tonkinensis leaf but it is found for the first time in genus Dalbergia.
In the mean time, tectoridin (37) has been isolated from D. volubilis flower [61], but
dalsissooside (32), and tectorigenin 7-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (38)are new compounds to date, and they have only been found in D. sissoo leaf [40,49].
Formononetin-7-O-β-D-glucopyranoside (45) has been identified presenting in stem bark of D.
paniculata [62]. However, both 32, 37 and 38 were first observed in D. tonkinensis.
Based on these results, a wide variety of flavonoids isolated from D. tonkinensis has
proven the close relationship among the species of the genus Dalbergia, and the related
genera of the family Fabaceae.
45

HPLC-MS and GC-MS techniques were used to detect indole-3-acetic acid derivatives in theseeds of D. dolichopetala [63,64], but to date, no alkaloids had been isolated from the genus
Dalbergia. Thus, 3-(1H-indol-3-yl)-2-methoxypropanamide (31), a unique alkaloid, is likely
to be an important chemotaxonomic marker for this genus. Significantly, 1H-indole-3propanamide, the other indole alkaloid, showed a similar substitution pattern to 31, but had
only ever been isolated from two species of the subfamily Faboideae Erythrina brucei and E.
addisoniae [65,66]. These results add to the evidence demonstrating the close relationshipamong the species in both subfamily.
Sesquiterpenes are rarely reported from the genus Dalbergia. To date, several isolated
sesquiterpenes, such as (3S,6S,7R)-3,7,11-trimethyl-3,6-epoxy1,10-dodecadien-7-ol and rel(3S,6S,7R)-3,7,11-trimethyl-3,6-epoxy-1,10-dodecadien-7-ol, or 3,7,11-trimethyldodeca-1,10diene-3,6,7-triol, which have been identified as derivatives of nerolidol, have only been foundin D. odorifera growing in China [37,67]. However, an isolated heterocyclic metabolite, 3,10epoxy-3,7,11-trimethyldodeca-1,6-dien-11-ol, trivially named neroplomacrol (24), is one of
the new compounds isolated from Oplopanax horridus (Sm.) Miq. (family Araliaceae), andthis is the second time to report the isolation of 24 from a natural source. Compound 24 was
first isolated from a plant in the legume family and the type of nerolidol derivatives present
can also be seen as a phylogenetic differentiator between the Vietnamese D. tonkinensis
species and D. odorifera species [68]. Last of this, rel-(3R,6S,7S)-6α-hydroxycyclonerolidol(25), and 2,5'-dimethyl-5'-vinylhexahydro-2,2'-bifuran-5(2H)-one (47), which are newsesquiterpenes in nature, are diastereoisomers of rel-(3R,6R,7S)-3,7,11-trimethyl-3,7-epoxy1,10-dodecadien-6-ol

andrel-(2R,2'R,5'S)-2,5'-dimethyl-5'-vinylhexahydro-2,2'-bifuran-5(2H)-one (components were isolated from D. odorifera heartwood), respectively [37]. It

46therefore suggests that the environments such as geographic factors might be playing a role inaffecting the native plants.
Despite the fact that phenolic compounds were found to be the second most common
type of component isolated from Dalbergia species [1], aldehydic phenols were not available.
To date, only two compounds, voludal and 2-hydroxy-3,4-dimethoxybenzaldehyde have been
isolated from the non-green branches of D. volubilis and D. odorifera heartwood, respectively[69,70]. Hence, the small molecule 4-formyl phenol (27) is a useful agent to recognize either
D. tonkinensis or genus Dalbergia. A similar compound, named killitol (46), was one ofcytotoxic potential agents of chloroform fraction of marine plant Thalassia testudinum but it
was found in the family Fabaceae for the first time [51].
Last but one least, mono-acylglycerides named 1-monolaurin (39) and tridecanoic αmonoglyceride (40) widely distributed in plant kindom. For instance, compound 39 presents
in coconut oils, or is one of glycerols of the powder of berries of Serenoa repens [50].
Nevertheless, from chemotaxonomic point of view, we recognize that these compounds andrelated glycerides have never been found in the family Fabaceae.
In conclusion, the present studies imply that D. tonkinensis Prain contains interesting
secondary metabolites, consisting of various typical flavonoids, along with alkaloids,
sesquiterpenes, phenols, and glycerides, which provide a basic foundation for further
researches.

47

Table 7. Chemotaxonomic summaries of phytochemicals of D. tonkinensis
Compounds
Formononetin (1)Sativanone (2)Naringenin (3)Liquiritigenin (4)Pinocembrin (5)Medicarpin (6)3'-Hydroxy-2,4,5-trimethoxydalbergiquinol (7)Buteaspermanol (8)Butin (9)3'-Hyrdoxymelanettin (10)Biochanin A (11)Calycosin (12)Eriodictoyl (13)Isoliquiritigenin (14)3'-O-Methylviolanone (15)Luteolin (16)
Quercetin (17)Apigenin (18)
Sulfuretin (19)(2S)-6,8-Dicarboxyethylpinocembrin (20)(2S)-8-Carboxyethylnaringenin (21)Dalbergin (22)
Homoferreirin (23)Neroplomacrol (24)Rel-(3R,6S,7S)-6α-hydroxycyclonerolidol (25)
Kaemfperol (26)Formyl phenol (27)Naringin(28)Genistein (29)
Orobol (30)
3-(1H-indol-3-yl)-2-methoxypropanamide (31)Dalsissooside (32)
Daltonkinensiside (33)
Ambocin (34)
Lanceolanrin (35)Tectorigenin (36)
Tectoridin (37)
Tectorigenin 7-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (38)1-Monolaurin (39)1-Monomyristin (40)(3R)-Vestitol (41)Melilotolcarpan D (42)Daidzein (43)3'-Hydroxyldaidzein (44)Remarks
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
A, B, E
A, B, E
B
B
B, D, F
A, B, F
B
B, D
B, C
B
B
A, B, D
B
A, B
B, C
B
B
B
B
B, D
B, D
B
B
B
B

48

Formononetin-7-O-β-D-glucopyranoside (45)B
Killitol (46)B, D
2,5'-Dimethyl-5'-vinylhexahydro-2,2'-bifuran-5(2H)-one (47)
A, B, F
A: new compound; B: It is the first time to isolate from D. tonkinensis; C-D: it is reported
for the first time from genus Dalbergia, and the family Fabaceae, respectively; E: its rare innature, F: The class of compound only found in D. tonkinensis and D. odorifera

49

Chapter 3 Chemical constituents from Cratoxylum formosum
3.1. Introduction
Scientific name: Cratoxylum formosum (Jack)
Dyer in Hook;
Other name: Cratoxylum formosum subsp.
formosum, Cratoxylum formosum ssp. formosum,
Cratoxylum formosum Dyer, and Mampat;

Figure 21. Leaf of C. formosum

Subspecies: C. formosum subsp. pruniflorum (Kurz) Gogelein;
Family: Clusiaceae (Guttiferae);
Distribution: southeast Asia countries Brunei, Burma, Cambodia, China, Indonesia,
Laos, Malaysia, Philippines, Singapore, Thailand, and Vietnam [71].
Morphology: The tropical plant C. formosum, all parts of which are glabrous. Leaves
elliptic to oblong (to lanceolate) 31/2-14 by 2-7 cm, petiole 6-15 mm. Connective notglandulose. Hypogynous scales linguiform often attenuate, sometimes blunt or truncate.
Ecology: Granitic sand, dry, red soil, clay, rocky hill slopes, or fresh-water swamps, along
rivers or near mangrove swamps, in open or dense primary forests or old secondary forests,
thickets. Altitude mostly low, sometimes up to 600 m, or seldom more 1200 m. Flowering:
Noted about March-July. Field characters: Trees maximum 35 m tall, young specimens andsaplings spiny, base of the trunk spiny. Flowers pink or white, once noted purplish.
Hypogynous scales orange-red [71].
Phytochemical investigations and biological evaluations: To the best of our knowledge,
there was only one account reporting the isolation and antioxidant activities of 4 flavonol
50

derivatives quercetin, isoquercitrin, hyperin, and quercitrin from C. formosum [72]. At
concentration of 1 µM, these compound could be claimed responsible for in vitro anti-oxidant
capacity through peroxy radical scavenging experiments, and reducing powers of convert
metal ion Cu2+ into Cu+. Further, in the dose dependent manners from 1 to 10 µM, thesecompounds also demonstrated the cellular antioxidant behaviour when AAPH, Cu2+ or H2O2
stimulated intracellular oxidative stress on hepatoma HepG2 cell line. Its worth noting thatmonosaccharide such as glucose, galactose, or rhamnose in these glycoside derivatives arelikely to be related to the primary reason for anti-oxidant enhancement [72].
3.2. Phytochemical investigation of leaf extract and structural elucidation
3.2.1. Isolation procedure
Leaf of Cratoxylum formosum(MeOH extract, 220 g)
Silica gel

D:E
8:2

CH2Cl2n-hexane
Fr1

Fr3

Fr2

D:E
1:1
Fr4

EtOAc
Fr5

E:M
9:1

E:M
8:2
Fr7

Fr6

Silica gel

Silica gel

Sephadex

H:A 3:1

C:M 8:1

M:W 2:1

48 (45 mg)

Fr42
Fr44 4 Fractions*
Sephadex
Silica gel

49 (15 mg)

M:W 1:1
50 (37.5 mg)

D:M:W 3:1:0.1

Washing by MeOH
54 (240 mg)

52 (15 mg) Fr61
HPLC, M:W 7:3
UV: 210 nm
1.0 mL/min

51 (200 mg)

53 (5.4 mg), 55 (4.2 mg), 56 (3.2 mg)

From methanol extract of C. formosum leaf, nine known compounds were isolated.
The chemical structures of the known compounds were to be β-sitosterol (48), sitgmasterol(49), (+)-catechin-3-O-(3,4-dihydroxybenzoyl) (50), quercetin (51), quercitrin (52), hyperin(53), magniferin (54), afzelin (55), and isoquercetin (56) [72-76]. Despite the fact that flavans
51widely distributed in C. formosum leaf, it is the first time to isolate (+)-catechin-3-O-(3,4dihydroxybenzoyl) (50) from genus Cratoxylum.

Figure 22. Chemical constituents from C. formosum leaf

52

Chapter 4 Biological activities
4.1. Introduction
Currently, infectious diseases have been emerging as the complicated public healthproblem around the worldwide, especially in terms of developing countries [76].
Consequently, microbacterial organisms are harmful to healthy invidiuals, leading to a further
increase in infections. Considering several microbacteria, the microbacterial pathogenic strain
S. pneumoniae is a major cause of superficial skin infection in children, infections of the ear,sinus, meningitis, and pneumonia disease, while the gram positive aerobic bacterium B.
cereus is always considered a contaminant organism, widely existing in soil, dust, water or
hospital environments, mainly causing food poisoning and necrotizing skin infection indiabetic patients [78,79]. Additionally, regarding the antimicrobial chemotherapy, clinicalapplications are seeking drugs and methods to treat bacterial infections. Historical records
have accumulated evidence showing that the use of traditional antibiotics, which are derived
from synthetic substances, is always accompanied by a long duration of treatment, high costs,
and drug resistances [80]. Therefore, calls for new antibiotic drugs from natural sources in thefight against multidrug-resistant bacteria are warranted.
Serine proteases are a family of enzymes containing serine as a nucleophilic site when
the peptide bonds of the proteins are cleaved. Based on substrate specificity, they are divided
into three categories, including trypsin-like, chymotrypsin-like, and elastase-like proteases[81]. Serine protease enzymes can be both beneficial and harmful. They display critical roles
as activators in foods and medicines, especially in milk, but are also related to numerous
diseases, such as chronic inflammatory disease [82]. Much evidence shows that natural
products from terrestrial plants have acted as useful serine protease inhibitors [83,84].
53

Among recently explored sources, Santhos and Suriyanarayanan pointed out thatnaturally occurring flavonoids should be the suitable priority with anti-microbacterial
experimental model manipulation since they are available in quantity from plants and have thesimple and small properties in molecular structure [85]. In other case, flavonoids could be a
new class of candidates of serin protease inhibitors. Non-competitive inhibitory flavonoids,
like agathisflavone and myricetin, were reported to be the best short-term solution for dengue
virus serotype 2 NS2B-NS3 protease [86]. In the current thesis, we carry out both animicrobacterial and serine protease assays for extracts and isolated flavonoids from D.
tonkinensis species in anticipation of developing them into potent anti-microbacterial
antibiotics as well as serine protease activators and inhibitors.
4.2. Anti-microbacterial results
4.2.1. The first model
Table 8. Plant extracts and isolated compounds for antimicrobacterial assays-Leaf methanol extract (DB1)-Stem bark methanol extract (DB3)-Root ethanol extract (DB5)-Dichloromethane fraction of root ethanol
extract (DB7)-Root water decoction extract (DB9).
-Heartwood chloroform fraction (DB11)-Formononetin (1)-Naringenin (3)-Pinocembrin (5)-3'-Hydroxy-2,4,5-trimethoxydalbergiquinol(7)-Butin (9)-Biochanin A (11)-Eriodictoyl (13)-3'-O-Methylviolanone (15)-Quercetin (17)-Sulfuretin (19)-(2S)-8-Carboxyethylnaringenin (21)

-Leaf water decoction extract (DB2)-Stem bark water decoction extract (DB4)-Root methanol extract (DB6)-Ethyl acetate fraction of root ethanol extract(DB8)-Heartwood water decoction extract (DB10)-Sativanone (2)-Liquiritigenin (4)-Medicarpin (6)-Buteaspermanol (8)-3'-Hyrdoxymelanettin (10)-Calycosin (12)-Isoliquiritigenin (14)-Luteolin (16)-Apigenin (18)-(2S)-6,8-Dicarboxyethylpinocembrin (20)-Mixture of 2 and 15

54

Figure 23. Chemicals for assay
The antimicrobial activities of the extracts (1.0 mg/mL) and isolated compounds (0.1mg/mL) are presented by absorbance at λ 620 nm in Fig.s 24a-24k. The interest resuts were
described as the percent observations of relative bacterial growth (or relative bacterial growth
inhibition) when compared with those of the positive and negative controls, and medium only
(the standard compound ampicillin sodium salt evidently generated the inhibitory percentage
of greater than or equal to 80% and 50% for skin and oral microbacterial, respectively; data
not shown). Among the extracts, at the concentration of 1.0 mg/mL, the crude methanol
extracts of leaf (DB1), stem bark (DB3), root (DB6), and heartwood chloroform fraction(DB11) showed the significant values of 80, 70, 80, and 70% in inhibiting the microbacterium
S. pneumoniae, respectively, whereas the extract remainders showed no activity (Fig. 24e).
Additionally, the stem bark methanol extract (1.0 mg/mL) also possessed the rate of 60% for55

preventing the growth of two types of oral bacteria E. coli and B. Cereus (Fig.s 24a-24b).
Based on these findings, the methanol solvent and Soxhlet extraction method may be
responsible for the antimicrobacterial mode, along with the latent chloroform fraction DB11
would be further developments in phytochemical investigations. Similarly, the chloroform
extract of D. coromandeliana stem, and the dichloromethane extract of D. melunoxylon bark
were found to show notable activity against bacteria, and fungi, respectively [87,88].
Structural differences, stereochemistry, molecular size, the lengths of atomic bonds,
extra or intramolecular hydrogen bonding, especially in terms of functional groups and theirlocal substituents were greatly related to the outcomes in bioassays. Regarding to the isolated
flavonoids, among the tested compounds (0.1 mg/mL), the flavanone pinocembrin (5), andisoflavone biochanin A (11) suppressed 80% of the growth of the two bacterial strains B.
cereus and S. pneumonia compared with the positive control (Fig.s 24a and 24e). Likewise,
the strongest percentage of 80% against bacterium S. pneumonia also accounted for theisoflavanone sativanone (2), chalcone isoliquiritigenin (14), two other flavanones naringenin(3), and liquiritigenin (4), and the mixture of two isoflavanones 2+15 (Figure 6). Hence, thesetypical flavonoids of flavanone, isoflavanone, isoflavone, or chalcone could be the best choice
to argue for the mode of antimicrobacterial activity.
More recently, Cuong et al. found that compound 5 indicated an inhibitory effect on
the filamentous fungus A. niger with a significant MIC value at 50 µg/mL [89]. Furthermore,in contrast to the results of the isoflavanone 15, the mixture of 2+15 revealed positive signals
against 70 and 50% growth inhibition of the two pathogens B. cereus and S. epidermidis,
respectively (Fig.s 24a and 24d). Hence, the 2'- and 4'-methoxy groups of isoflavanones 2 and15 could serve as the most striking feature in these efficacious aspects. Meanwhile,
considering three flavones (16-18) and one aurone (19), only the flavanone luteolin (16)56established an inhibitory effect on the oral bacterium S. epidermidis with a relative growth
inhibition of 50%. Presumably, regarding the flavanones, an additional hydroxyl group at C-3'
produced a significant effect, whereas hydroxylation at C-2 failed to do so.
Sotres et al. indicated that the n-hexane extract of D. congestiflora heartwood, whichcontained a major flavonoid derivative of skeletal pterocarpan named medicarpin (6), caused
100% growth inhibition of the Trametes versicolor fungus at the dose of 250 mg/mL [90]. In
the present study, as shown in numerous Figures 24a, 24c, 24g, 24h, and 24k, compound 6
showed remarkably antimicrobial activities against B. cereus and V. parahaemolyticus (70%growth inhibition), and S. aureus (60%), as well as the oral pathogens S. mutans and P.
gingivalis (50%), whereas the inhibitory numbers of 70% growth were also calculated for aneoflavanoid 7, and a flavanone 3 (the growth inhibition of B. cereus), and two otherflavanones 5 (V. parahaemolyticus), and 9 (S. pneumoniae). Significantly, in the comparison,
the other neoflavanoid 3'-hyrdoxymelanettin (10) was formed by plausible hypothesis oflactone intramolecular cyclization between the exo-methylene group at the carbon C-3'' and 2methoxy group, and hydroxylation at the carbon C-4' in compound 7 showed the inactive
results in any antimicrobacterial experiments. In addition, the flavanone naringenin (3) was
considerably estimated to show 60% growth suppression of two types of bacteria S.
epidermidis, and V. parahaemolyticus. In general, the most striking feature was that four
isolated flavanones 3-5, and 9 indicated the mode regarding the substitution of hydroxyl
groups at C-5, C-7, and C-3', or at C-4', except for compound 13, while replacement ofprotons H-3, and H-5, or H-8 by hydroxyl, or carboxyethyl groups in flavanones 8, 20, or 21
significantly resulted in reducing the inhibitory values. On the other hand, among three
isoflavones 1, 11, and 12, with an opposite property of chemical structure, compounds 1 and
12 showed no significant inhibitory activity, while isoflavone 11 was not only revealed as a57

latent agent that repressed the pathogenic bacteria B. cereus and S. pneumoniae but also
showed 50% growth inhibition of two other types of S. epidermidis, and P. gingivalis,presumably due to the substitution at the carbon C-5 rather than C-3'.

Figure 24a. The relative growth of microbacterium B. cereus

Figure 24b. The relative growth of microbacterium E. coli

Figure 24c. The relative growth of microbacterium S. aureus

58Figure 24d. The relative growth of microbacterium S. epidermidis

Figure 24e. The relative growth of microbacterium S. pneumoniae

Figure 24f. The relative growth of microbacterium S. pyogenes

Figure 24g. The relative growth of microbacterium V. parahaemolyticus
59Figure 24h. The relative growth of microbacterium S. mutans

Figure 24i. The relative growth of microbacterium S. mitis

Figure 24j. The relative growth of microbacterium S. sobrinus

Figure 24k. The relative growth of microbacterium P. gingivalis

60

4.2.2. The second model
Table 9. Anti-microbacterial results of heartwood extracts and its isolated compounds
Anti-microbial acitivities (MIC: µg/mL)
Gram bacteria
Filamentous fungi
No
P.
B.
S.
A.
F.
E. coliaeruginosa subtillis aureus
niger
oxyporum
CH3OH extract*
(-)
(-)
(-)
(-)
200
(-)
CHCl3 fraction*
(-)
(-)
(-)
400
200
(-)
Compound 4
(-)
(-)
(-)
100
(-)
(-)
Compound 8
(-)
(-)
(-)
50
100
(-)
Compound 11
(-)
(-)
(-)
50
50
(-)
Compound 21
(-)
(-)
(-)
(-)
100
(-)
*
Extract and fraction of heartwood

Yeast
S.
C.
cerevisiae albicans
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)

Antimicrobial experiments were carried out and the results were shown in Table 9. Of
the heartwood extract, and fraction, the CH3OH extract had the weak MIC value of 200µg/mL against only Aspergillus niger (439), whereas the CHCl3 fraction showed theinhibition with the MIC value at 200, and 400 µg/mL against A. niger (439), and Gram
positive bacterium S. aureus subsp. aureus (ATCC 11632), respectively. Among the isolated
compounds, the known flavanone buteaspermanol (8), and the new flavanone (2S)-8carboxyethylnaringenin (21) showed the inhibitory effects on pathogenic fungus fungus A.
niger (439) at the weak MIC value of 100 µg/mL, and the MIC value of 50 µg/mL was
assigned for biochanin A (11). Furthermore, the flavanone derivatives 8, and 11 are hopefulagents against the filamentous fungus A. niger, revealing the effective MIC value of 50µg/mL, whereas the remaining subtance liquiritigenin (4) possessed the weak MIC value of
100 µg/mL. However, none of extracts, fractions, and isolated compounds exhibited
inhibitory effects on Gr negative bacteria E. coli and P. aeruginosa, Gr positive bacterium B.
subtillis (ATCC 11774), yeast fungi, and filamentous fungus F. oxysporum (M42).

61

4.3. DPPH Radical scavenging capacity result
Table 10. Compounds for DPPH radical scavenging assay
D. tonkinensis
Medicarpin (6)Orobol (30)(3R)-Vestitol (41)
Melitocarpan D (42)

C. formosum
Catechin-3-O-(3,4-dihydroxylbenzoyl) (50)
Quercetin (51)Hyperin (53)Magniferin (54)Afzelin (55)Isoquercetin (56)Standard compound: Catechin

Figure 25. Chemicals for assay
The DPPH radical scavenging assay was carried out on several isolated compounds
according to the protocol described by Tram and co-workers [91], and the results are given in
Table 11. With regards to the several tested compounds from D. tonkinensis species, (3R)vestitol (41) possessed the strongest anti-oxidative IC50 value of 42.20 (µg/mL), which wascomparable to that of positive control catechin (IC50 42.98 µg/mL). Pterocarpan 42 revealed a62strong IC50 value of 54.14 (µg/mL) when compared with the moderate IC50 value of 69.45(µg/mL) of pterocarpan 6 and the weak one of 91.18 (µg/mL) of isoflavone 30. It therefore
can be concluded that isoflavans from D. tonkinensis species should be the best choice foranti-oxidant model as compared with pterocarpans or isoflavones. In addition, comparing thetwo pterocarpans medicarpin (6) and melilotolcarpan D (42), hydroxyl groups occurred at the
carbons C-1 and C-8, and methoxyl group attached at the carbon C-2 can be claimed to be
responsible for anti-oxidant increase.
Regarding to the isolated compounds from C. formosum leaf, the IC50 values
established a consistent arrangement as follows: catechin (IC50 42.98 µg/mL) > isoquercetin(56) (IC50 45.63 µg/mL) > magniferin (54) (IC50 64.03 µg/mL) > hyperin (53) (IC50 64.22µg/mL) > quercetin (51) (IC50 64.54 µg/mL) > catechin-3-O-(3,4-dihydroxylbenzoyl) (50)
(IC50 75.37 µg/mL) > afzelin (55) (IC50 > 100 µg/mL, inactive). As a consequence, flavonols,
flavonol glycosides, and xanthone glycosides may be regarded as promising anti-oxidant
agents, but flavans cannot be considered as such. Taking flavonol derivatives into
consideration, glycosylation and hydroxylation occured at the respective carbons C-3 and C-3'
enhanced an increase in anti-oxidant capacity. Similarly, 3-glucopyranosylated flavonol(compound 56) showed a better anti-oxidant poteny than 3-galactopyranosylated flavonol(compound 53).

63

Table 11. DPPH Radical scavenging activity
Compounds
Medicarpin (6)Orobol (30)(3R)-Vestitol (41)
Melitocarpan D (42)
Catechin-3-O-(3,4-dihydroxylbenzoyl) (50)
Quercetin (51)Hyperin (53)Magniferin (54)Afzelin (55)Isoquercetin (56)
Catechin

IC50 (µg/mL)
69.45
91.18
42.20
54.14
75.37
66.54
66.22
64.03
> 100
45.63
42.98

4.4. Enzyme active screenings
Table 12. Plant extracts and isolated compounds for enzyme serine protease assays-Leaf methanol extract (DB1)-Stem bark methanol extract (DB3)-Root ethanol extract (DB5)-Dichloromethane fraction of root ethanol
extract (DB7)-Root water decoction extract (DB9).
-Heartwood chloroform fraction (DB11)-Formononetin (1)-Naringenin (3)-Pinocembrin (5)-3'-Hydroxy-2,4,5-trimethoxydalbergiquinol(7)-Butin (9)-Biochanin A (11)-Eriodictoyl (13)-3'-O-Methylviolanone (15)-Quercetin (17)-4-Formyl phenol (19)-Naringin (21)-(2S)-8-Carboxyethylnarigenin (23)Datosativanone (2b)-Leaf water decoction extract (DB2)-Stem bark water decoction extract (DB4)-Root methanol extract (DB6)-Ethyl acetate fraction of root ethanol extract (DB8)-Heartwood water decoction extract (DB10)-Sativanone (2)-Liquiritigenin (4)-Medicarpin (6)-Buteaspermanol (8)-3'-Hyrdoxymelanettin (10)-Calycosin (12)-Isoliquiritigenin (14)-Luteolin (16)-Apigenin (18)
-Sulfuretin (20)-(2S)-6,8-Dicarboxyethylpinocembrin (22)7-Dehydroxy-7-(prop-2-yn-1-yl)sativanone (2a)Mixture of 2 and 15

64

Figure 26. Chemicals for assay
The results for the serine protease assays are summarized in Table 13 and also Fig. 27.
Regarding the conceptual activation, root ethanol and methanol extracts increased the activityof trypsin in a concentration-dependent manner ranging from 25 ng/mL to 250 ng/mL. In themeantime, both 3'-O-methylviolanone (15) and (2S)-6,8-dicarboxyethylpinocembrin (20)65showed the same tend at the concentration of 100 µM, in particular, medicarpin (6) can beseen as a good candidate for trypsin activator at concentration of 10 µM. Besides trypsin
active capacity, root dichloromethane fraction of ethanol extract (DB7) also enhanced theactivity of chymotrypsin at a concentration of 250 ng/mL, meanwhile heartwood waterdecoction extract (DB6) increased the activity of both trypsin and chymotrypsin at 25 ng/mL.
Of inhibitors, at the concentration of 100 µM, isoflavanone 2 and neoflavanoid 7
specifically prohibited the activity of chymotrypsin, and also formononetin (1) and butin (9)indicated weaker inhibition than compounds 2 and 7. Both root water decoction extract (DB9)(250 ng/mL), and flavanone 5 (100 µM) showed inhibitory activities of trypsin-chymotrypsin.
Similarly, root methanol extract (DB6) (250 ng/mL) and a flavonoid type aurone 19 (100 µM)
suppressed the activity of either chymotrypsin or elastase delete. Stem bark methanol extract(250 ng/mL) exhibited inhibition for three tested enzymes when compared to methanol, andwater decoction extracts of root (DB9). Heartwood chloroform fraction (DB11), whichcontained the enriched flavonoids, showed a trypsin-chymotrypsin-elastase inhibitory activity
in a concentration-dependent manner ranging from 25 ng/mL to 100 ng/mL. Among the tested
flavonoids, quercetin (17) was ascertained to possess the most potent activity in suppressing
trypsin and chymotrypsin delete, and significant capability for elastase delete [92]. Numerous
flavonoids 2b, 3, 4, and 18 at the concentration of 100 µM, showed weak inhibition of threeexperimental serine proteases enzymes. It was noted that the leaf methanol extract merged asagonist, and antagonist, which significantly activated trypsin at 25 ng/mL, in contrast to
reduce the activity of chymotrypsin at the high concentration of 250 ng/mL. Similar activity
pattern is also assigned for isoflavone biochanin A (11). Root methanol extract (DB6) (25ng/mL) can possibly be interpreted as a trypsin activator, along with the inhibitory role forchymotrypsin and elastase at the concentration of 250 ng/mL. Ethyl acetate fraction of root66

ethanol extract and secondary metabolites 2a, 2+15, 8, 10, 12-14, 16, 21, and 27-28 showedno effects on the tested three proteases.
In the model of casein cleavage activity, at the dose of 25 ng/mL, leaf water decoction(DB2) extract inhibited casein cleavage by trypsin, ethanol (DB5) and methanol (DB6)extracts of root activated the activity (Fig. 28). The result was same as the protease activity
analysis using fluorogenic synthetic peptide substrates. However, leaf methanol extract (DB1)and heartwood water decoction extract (DB10) (25 ng/mL), and medicarpin (6) and quercetin(17) had no effect.
Fig. 27 showed that at the lowest dose of 2.5 ng/mL and 1.0 µM, no crude extracts andisolated compounds achieved the remarkable effects, but the changes corresponded to theconcentration adjustment. Taking extracts into consideration, the leaf, root, and heartwood of
D. tonkinensis may be suitable for trypsin activators in food. Furthermore, leaf, stem bark,and heartwood have significantly emerged as promising trypsin-chymotrypsin-elastase
inhibitors (Table 13). Leaf water decoction extract (DB2) (250 ng/mL) completely controlled
the activity of elastase delete to explain the great value of unique Vietnamese plant withunknown ethno-pharmacological aspect, especially in terms of water decoction traditional
method.
Structural differences, stereochemistry, functional groups, and their local substituentsmight be related to the outcome in protease bioassays. Taking the isolates from this plant into
consideration, flavonoids categorized as flavanone, isoflavanone, flavone, isoflavone,pretocarpan, aurone, and neoflavanone could be the best choice to argue for this issue.
Calycosin (12) failed to show activity due to 3'-OH comparing to 1. Taking a close look at
flavones, quercetin (17) can be seen as a comparative standard one because of absolutely

67attenuating actions of trypsin and chymotryspin to 100% at 100 µM, whereas apigenin (18), a
second flavone, is not to do so, and luteolin (16) has no activity. The presence of hydroxyl
group at C-3 and C-3' turns out to be the differential result among three flavones 16-18, andflavononols seem generally stronger than flavones. As the third case, in accordance with ourprevious findings in antimicrobacterial assay, 3'-hyrdoxy-2,4,5-trimethoxydalbergiquinol (7),an opened ring lactone type neoflavanone, dramatically decreased chymotrypsin activity to
95% in contrast to compound 10 at the concentration of 100 µM.
Buteaspermanol (8), a unique flavanonol of Dalbergia genus showed no effect onprotease activities. This means that hydroxylation at C-3 in flavanone does not show
significant contribution to protease delete assay. Compound 20 having carboxylethyl groups
at C-6 and C-8 was quite the reverse with compound 5. Comparing protease inhibitory
activity for naringenin (3), (2S)-8-carboxyethylnarigenin (21), and naringin (28), 7glycosylation and 8-carboxyethylation may led to inactivation. Eriodictoyl (13) with anadditional 5-OH resulted in reducing protease inhibitory activity, in comparison with butin (9).
Comparing structure and activity between 4 and 9, hydroxylation at C-3' is attributable to theattenuation of protease activity.
Naturally occurring isoflavanones 2 and 15, their mixture 2+15, and semi-synthetic
products 2a and 2b showed similar relation in proteases delete assay. Among them, thenegative and/or positive signals in outcome basically depended on the presence of
methoxylation at C-3' or not. In addition, a semi-synthetic compound, datosativanone (2b)was prepared from 2a and chalcone azide has been emerged as a trypsin-chymotrypsinelastase inhibitor.

68

Table 13. Summary results of serine proteases analysis
No

Activation
Trypsin
Chymotrypsin(Increased rate/concentration)

Inhibition
Trypsin
Chymotrypsin
Elastase(Decreased rate/concentration)

Extracts and fractions
Leaf methanol extract (DB1)

1.5 times/A
1.4 times/B

66%/B
82%/A
94%/B
24%/B
63%/A
88%/B

Leaf water decoction extract (DB2)
Stem bark methanol extract (DB3)
Stem bark water decoction extract (DB4)
Root ethanol extract (DB5)
Root methanol extract (DB6)
Dichloromethane fraction (DB7)
Root water decoction extract (DB9)Heartwood water decoction extract (DB10)

1.4 times/A
1.9 times/B
1.9 times/A
1.4 times/B

1.8 times/B

1.5 times/A

1.6 times/A

Heartwood chloroform fraction (DB11)
Isolated compounds
Formononetin (1)Sativanone (2)Naringenin (3)Liquiritigenin (4)Pinocembrin (5)Medicarpin (6)3'-Hyrdoxy-2,4,5-trimethoxydalbergiquinol (7)Butin (9)3'-O-Methylviolanone (15)
Quercetin (17)86%/B100%/B

80%/B

39%/B

63%/B

38%/B

44%/B

38%/B

74%/B

30%/B

30%/A
50%/B

54%/A
89%/B

55%/D
27%/D
54%/D

45%/D
77%/D
57%/D
31%/D
60%/D

35%/A
67%/B

39%/D
52%/D

1.6 times/C
95%/D
45%/D
1.5 times/D
90%/C
69

86%/C

65%/D

100%/D
100%/D
Apigenin (18)
79%/D
41%/D
57%/D
Sulfuretin (20)
74%/D
38%/D
(2S)-6,8-Dicarboxyethylpinocembrin (22)
1.6 times/D
Datosativanone (2b)
39%/D
74%/D
53%/D
No samples increased the growth of elastase enzyme. Ethyl acetate fraction (DB8) of root ethanol extract, buteaspermanol (8), 3'hyrdoxymelanettin (10), biochanin A (11), calycosin (12), eriodictoyl (13), isoliquiritigenin (14), luteolin (16), 4-formyl phenol (19),naringin (21), (2S)-8-carboxyethylnarigenin (23), 7-dehydroxy-7-(prop-2-yn-1-yl)sativanone (2a), and 2+15 show insignificant effects
at any concentrations.
A: 25 ng/mL, B: 250 ng/mL, C: 10 µM, D: 100 µM; Increased and decreased rates compared with non-treatment group.

70

Leaf water decoction extract (DB2)

200

150
Activity(%)

Activity(%)

Leaf methanol extract (DB1)

Elastase

100

Chymotrypsin

0
0

2.5

25

250

Trypsin

Activity(%)

Activity(%)

Elastase
Chymotrypsin
0

2.5

25

250

0

Elastase

50

Chymotrypsin0

Trypsin

Trypsin

0

2.5
25
Concentration (ng/mL)

0

2.5

25

250

Elastase
Chymotrypsin
Trypsin

Activity(%)

Activity(%)

300

0

250

Root methanol extract (DB6)

Root ethanol extract (DB5)100

Trypsin250

100

Concentration (ng/mL)

200

2.5
25
Concentration (ng/mL)

150

150

0

Chymotrypsin

0

Stem bark water decoction extract (DB4)

Stem bark methanol extract (DB3)50

Elastase

50-50

Concentration (ng/mL)

100

100

300
200

Elastase

100

Chymotrypsin

0
0

2.5

25

Concentration (ng/mL)

Concentration (ng/mL)

71

250

TrypsinRoot ethyl acetate fraction (DB8)

300
200

Activity(%)

Activity(%)

Root dichloromethane fraction (DB7)

Elastase

100

Chymotrypsin

0
0

2.5

25

250

150
100

Elastase

50

Chymotrypsin

00

TrypsinConcentration (ng/mL)

Activity(%)

100

Elastase

50

Chymotrypsin

0
2.5

25

250

TrypsinHeartwood water decoction extract (DB10)

150

25

250

Trypsin

200
150
100
50
0

Elastase
Chymotrypsin
0Concentration (ng/mL)

2.5

25

Concentration (ng/mL)

Heartwood chloroform fraction (DB11)
Activity(%)

Activity(%)

Root water decoction extract (DB9)0

2.5Concentration (ng/mL)

150
100

Elastase

50

Chymotrypsin

0
0

2.5

25

Concentration (ng/mL)

72

250

Trypsin250

TrypsinComp. 2

150
100

Activity(%)

Activity(%)

Comp. 1

Elastase

50

Chymotrypsin

0
0

1

10

100

Trypsin

150
100

Elastase

50

Chymotrypsin

00Concentration (µM)

1

Activity(%)

Activity(%)

100

Elastase

50

Chymotrypsin

0
10

100

150
100

Elastase

50

Chymotrypsin

00

TrypsinConcentration (µM)

1

Activity(%)

Activity(%)

100

Elastase

50

Chymotrypsin

0
10

100

Trypsin

Comp. 6

150

1

10

Concentration (µM)

Comp. 50Trypsin

Comp. 4

150

1

100

Concentration (µM)

Comp. 3

0

10

100

200
150
100

Elastase

50

Chymotrypsin0

Trypsin0

Concentration (µM)

110Concentration (µM)

73

100

TrypsinComp. 8

150
100

Activity(%)

Activity(%)

Comp. 7

Elastase

50

Chymotrypsin

0
0

1

10

100

Trypsin

150
100

Elastase

50

Chymotrypsin

00Concentration (µM)

1

Activity(%)

100

Elastase

50

Chymotrypsin

0
10

100

Trypsin

150
100

Elastase

50

Chymotrypsin

00Concentration (µM)

110Concentration (µM)

Comp. 12
Activity(%)

Activity(%)

Trypsin

Comp. 10

150

1

100

Concentration (µM)

Comp. 9

0

10

150
100

Elastase

50

Chymotrypsin

0
0

1

10

Concentration (µM)

74

100

Trypsin100

TrypsinComp. 14

150
100

Activity(%)

Activity(%)

Comp. 13

Elastase

50

Chymotrypsin

0
0

1

10

100

150
100

Elastase

50

Chymotrypsin

00

TrypsinConcentration (µM)

1

200
150
100
50
0

Elastase
Chymotrypsin
1

10

100

Trypsin100

Elastase

50

Chymotrypsin

001

Activity(%)

Activity(%)

100

Elastase

50

Chymotrypsin

0
10

100

Trypsin

Comp. 18

150

1

10

Concentration (µM)

Comp. 170

Trypsin150

Concentration (µM)

-50

100

Comp. 16
Activity(%)

Activity(%)

Comp. 150

10Concentration (µM)

100

150
100

Elastase

50

Chymotrypsin0

Trypsin0

Concentration (µM)

110Concentration (µM)

75

100

TrypsinComp. 20

150
100

Activity(%)

Activity(%)

Comp. 19

Elastase

50

Chymotrypsin

0
0

1

10

100

Trypsin

150
100

Elastase

50

Chymotrypsin

00Concentration (µM)

1

Activity(%)

Activity(%)

100

Elastase

50

Chymotrypsin

010

Trypsin

Comp. 2+15

150

1

100

Concentration (µM)

Comp. 23

0

10

100

Trypsin

150
100

Elastase

50

Chymotrypsin

00Concentration (µM)

110Concentration (µM)

76

100

TrypsinComp. 2aComp. 2b
Activity(%)

Activity(%)

150
100
Elastase
50

Chymotrypsin
Trypsin

0
0

1

10

100

120
100
80
60
40
20
0

Elastase
Chymotrypsin
Trypsin0

Concentration (µM)

1

10

100

Concentration (µM)

Figure 27. Effects of extracts DB1-DB11 ((2.5, 25, and 250 ng/mL) and isolated compounds 1-10, and 12-25 (1, 10, and 100 µM) on theamounts of serine proteases enzymes trypsin, chymotrypsin, and elastases

77Marker (kDa)
24
20
120

remained casein (% )

100

*

80

#

#

60
40
20
0

Figure 28. Effect of extracts and compounds on casein-cleavage-activity by trypsin. Data present mean ± SD values of 7 independent
determinations. Differences were determined using student t-test and considered to be significant when p-value was less 0.05. *p<0.01
vs. without additives; #p<0.01 vs. DMSO
78

Conclusion
The current thesis have carried out the phytochemical studies of two Vietnameseplants D. tonkinnensis and C. formosum demonstrating a variety of flavonoids in both twoplants. The results can remark as follows:
1) Forty seven secondary metabolites were isolated from heartwood, leaf and root of D.
tonkinnensis, consisting of thirty nine flavonoids and flavonoid glycosides, threesesquiterpenes, two monoacylglycerides, two mono-phenols, and one alkaloid.
2) Nine compounds were isolated from C. formosum, comprising of six flavonols, two
phytosterols, and one xanthone.
3) New compounds were found in D. tonkinnensis, including two flavanones (2S)-6,8dicarboxyethylpinocembrin (20), and (2S)-8-carboxyethylnaringenin (21), one isoflavone
glycoside

isocaviunin

7-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside(daltonkinensiside) (33), two sesquiterpenes rel-(3R,6S,7S)-6α-hydroxycyclonerolidol (25),
and 2,5'-dimethyl-5'-vinylhexahydro-2,2'-bifuran-5(2H)-one (47), and one indole alkaloid 3(1H-indol-3-yl)-2-methoxypropanamide (31).
4) Flavonoid from D. tonkinnensis species was assigned to flavones and isoflavones,
flavanones and isoflavanones, pterocarpans, neoflavonoids, chalcones, and isoflavone-7glycosides.
5) Two new compounds 20 and 21 are rare in nature and new indole 47 is a unique
alkaloid in genus Dalbergia. Monoacylglycerides were reported for the first time from thefamily Fabaceae.

796) D. tonkinesis extracts and fractions as well as isolated compounds were found to bepromising agents against the growth of bacterial strains. For instance, at the concentration of
1.0 mg/mL, the crude methanol extracts of leaf (DB1), stem bark (DB3), root (DB6), andheartwood chloroform fraction (DB11) showed the significant values of 80, 70, 80, and 70%
in inhibiting the microbacterium S. Pneumonia, or at the concentration of 0.1 mg/mL, theflavanone pinocembrin (5), and isoflavone biochanin A (11) suppressed 80% of the growth of
the two bacterial strains B. cereus and S. pneumonia.
7) In enzyme serine protease screening, at the concentrations of 25-250 ng/mL, suchas methanol extracts of leaf and root, root ethanol extract and its dichloromethane fraction,and heartwood water decoction extract of D. tonkinensis may be served as useful sources tostimulate trypsin enzyme activity (1.4-1.9 times increase). In addition, water decoction
extracts of leaf and stem bark may explain unknown ethno-pharmacology due to the highinhibitory effects in enzyme assays using trypsin, chymotrypsin, and elastase (86-100%
inhibition), or quercetin (17) inhibits the activities of trypsin and chymotrypsin with IC50 9.7µM.
8) Regarding DPPH radical scavenging assay, (3R)-vestitol (5) and isoquercetin (24)possessed the strongest antioxidative IC50 values of 42.20 µg/mL and 45.63 µg/mL,
respectively, and their values were comparable to that of the positive control catechin (IC5042.98 µg/mL).
9) The effects of substituted groups of flavonoids accounted for biological results.

80

Experimental section
General analytical instruments- Specific optical rotation ([α]D): JASCO P-1030 digital polarimeter.
- Infrared spectroscopy (IR): JASCO FT-IR 410 infrared spectrophotometer.
- Ultraviolet spectroscopy (UV): Shimadzu UV-1650 PC spectrophotometer.
-Circular dichroism (CD): JASCO-J-725 spectropolarimeter.
-HR-MS: MStation JMS-700 instrument.
-HPLC: JASCO PU-1580 equipped with a JASCO UV-1575 detector.
-COSMOSIL 5C18-AR-II: Nacalai tesque.
-500 NMR spectra: Bruker Avance 500 MHz spectrometer.
Reagents
The dehydrated reactions were carried out under argon atmosphere in anhydrous solvent. Allother reagents were used as supplied.
Silica gel column was performed on Kieselgel 60 (40-63 mesh, MERCK); Thin-layer
chromatography (TLC) was operated using Merck Silica gel 60 F254 pre-coated plates.
Chemical shifts are reported in δ (ppm) with reference to solvent signals [1H NMR: CDCl3(7.26), CD3OD (3.31); 13C NMR: CDCl3 (77.0), CD3OD (49.0)]. The signal patterns areindicated as: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet.
Plant materials

81The whole plant of D. tonkinensis was collected in Quangbinh Province, Vietnam in 2016.
The leaves of C. formosum were collected in Ninhbinh Province, Vietnam in 2010. This
plants were identified by Dr. Nguyen Quoc Binh, Vietnam National Museum of Nature,
Hanoi, Vietnam. The voucher specimens were deposited in Department of Bioactive Products,
Institute of Natural Products Chemistry, VAST, Hanoi, Vietnam.

82

Chapter 2 Chemical constituents from Dalbergia tonkinensis
2.2. Phytochemical investigation of heartwood and structural elucidation
2.2.1. Isolation procedure
Dried powdered heartwood of D. tonkinensis (1.0 kg) were extracted with methanol(5x1.5 L) over the period of 5 days at room temperature and concentrated under decreased
pressure to yield a black crude MeOH extract (46.53 g). The crude MeOH extract wassuspended in hot MeOH-H2O (1:1, v/v) and successively partitioned with n-hexane, CHCl3,
EtOAc, and H2O. The resulting fractions (fr.s) were concentrated under decreased pressure to
give the corresponding solvent-soluble fr.s n-hexane (5.3 g), CHCl3 (39.43 g), EtOAc (1.3 g)and H2O (0.5 g) residue, respectively.
The fr. CHCl3 (39.43 g) was separated by a silica gel column (40-63 mesh, Merck),
eluting with a gradient of n-hexane-CH3COCH3 (2:1, v/v), to give 6 fr.s (C1-C6). The fr. C2(6.1 g) was then chromatographed on a silica gel column (40-63 mesh, Merck) eluting with asolvent mixture of CHCl3-CH3COCH3 (70:1, v/v) to produce 8 fr.s (C21-C28). Using several
column chromatographies filled with silica gel or reversed RP-18, compounds 5 (45 mg), 6(6.1 mg), 7 (18 mg), 8 (3 mg), 22 (4.2 mg), and 23 (2 mg) were obtained from the fr. C22 (1.1g); compounds 1 (70 mg), 2 (0.2 g), and 4 (4 mg) were derived from the fr. C24 (1.5 g);compounds 24 (1.5 mg), and 25 (2 mg) were came from the fr. C27 (0.4 g); the remainder
compound 3 (54 mg) was saperated from the fr. C26 (1.0 g). In the same manner, the fr. C3(15.3 g) was subjected to a silica gel column with mobile phase of CHCl3-CH3COCH3 (30:1,v/v) to give 8 fr.s (C31-C38). Following the applications of normal silica gel, Sephadex LH20, and RP-18 comlumns, total 9 compounds had been isolated, including compounds 12 (583mg), 13 (5 mg), 14 (3 mg), 15 (4 mg), 16 (6 mg), 17 (2 mg), 18 (25 mg), 19 (3 mg), and 26(1.5 mg).
In other case, to give total 3 fr.s (E1-E3), the EtOAc fr. (1.3 g) was fractionated byusing the combination of a silica gel column and CHCl3-CH3COCH3 (3:1, v/v). After that, thefr. E2 (0.8 g) was completely chromatographed on a silica gel column eluting with a solvent
mixture of CHCl3-EtOAc (3:1, v/v) to afford compounds 9 (5.1 mg), and 1 1 (4 mg), whereas
chromatography on silica gel with CH2Cl2-MeOH (3:1, v/v), compound 10 (3.4 mg) had
successfully been isolated from the fr. E3 (0.1 g).
With regards to power after extracting methanol, it was decocted in hot water (4 x 2.0
L) over the period of 4 h and concentrated under decreased pressure to yield brownish black
crude (15.0 g), which was then subjected to a Diaion HP-20 column, eluting with gradientsolvent mixture of H2O-MeOH (1:0→0:1) to produce 5 fr.s (W1-W5). The fr. W2 (0.9 g) was
further chromatographed on a silica gel column, eluting with a gradient of CHCl3-MeOH-H2O(3:1:0.1, v/v/v) to afford compounds 27 (2.5 mg) and 28 (7.4 mg). Two newcarboxyethylflavanone 20 (2 mg) and 21 (4 mg) were derived from fr. W4 (1.44 g) by using asilica gel column with CHCl3-MeOH-H2O (1:1:0.1, v/v/v).
2.2.2. Chemical and physcical data of isolated compounds
Compound 1: Formononetin

84

White amorphous solid, EI-MS (+): m/z 268 [M]+, C16H12O4; 1H-NMR (500 MHz,
CD3OD, δ ppm): 8.17 (1H, s, H-2), 8.08 (1H, d, 9.0 Hz, H-5), 7.49 (2H, d, 8.5 Hz, H-2', H-6'),
7.01 (2H, d, 8.5 Hz, H-3', H-5'), 6.97 (1H, dd, 9.0, 2.0 Hz, H-6), 6.88 (1H, d, 2.0 Hz, H-8),
3.85 (3H, s, 4'-OCH3); 13C-NMR (125 MHz, CD3OD, δ ppm): 178.1 (s, C-4), 164.9 (s, C-7),
161.1 (s, C-4'), 159.8 (s, C-8a), 154.8 (d, C-2), 131.4 (d, C-2', C-6'), 128.5 (d, C-5), 125.7 (s,C-1'), 125.6 (s, C-3), 118.1 (s, C-4a), 116.6 (d, C-6), 114.9 (d, C-3', C-5'), 103.3 (d, C-8), 55.7
(q, 4'-OCH3).
Compound 2: Sativanone

White amorphous solid, EI-MS (+): m/z 300 [M]+, C17H16O5; 1H-NMR (500 MHz,
CD3OD, δ ppm): 7.78 (1H, d, 8.5 Hz, H-5), 7.00 (1H, d, 8.5 Hz, H-6'), 6.57 (1H, d, 2.0 Hz, H8), 6.52 (1H, dd, 8.5, 2.0 Hz, H-6), 6.48 (1H, dd, 8.5, 2.0 Hz, H-5'), 6.35 (1H, d, 2.0 Hz, H-3'),
4.56 (1H, t, 11.0 Hz, H-2a), 4.40 (1H, dd, 11.0, 5.5 Hz, H-2b), 4.16 (1H, dd, 11.0, 5.5 Hz, H3), 3.79 (3H, s, 2'-OCH3), 3.76 (3H, s, 4'-OCH3); 13C-NMR (125 MHz, CD3OD, δ ppm):
194.4 (s, C-4), 166.4 (s, C-7), 165.7 (s, C-8a), 162.2 (s, C-4'), 159.9 (s, C-2'), 132.0 (d, C-5),
130.4 (d, C-6'), 117.4 (s, C-1'), 115.7 (s, C-4a), 111.7 (d, C-3'), 106.0 (d, C-5'), 103.6 (d, C-6),
99.9 (d, C-8), 70.0 (t, C-2), 56.0 (q, 4'-OCH3), 55.8 (q, 2'-OCH3), 49.0 (d, C-3).
Compound 2a: 7-Dehydroxy-7-(prop-2-yn-1-yl)sativanone

85

White amorphous solid, HR-EI-MS: m/z 338.1151 [M+Na]+ calcd. for C20H18O5Na,
1

found: 338.1151; H-NMR (500 MHz, DMSO, δ ppm): 7.76 (1H, d, 8.5 Hz, H-5), 7.01 (1H, d,
8.0 Hz, H-6'), 6.71 (1H, dd, 8.0, 2.0 Hz, H-5'), 6.59 (1H, d, 2.5 Hz, H-8), 6.64 (1H, d, 2.0 Hz,H-3'), 6.49 (1H, dd, 8.5, 2.5 Hz, H-6), 4.90 (2H, d, 2.0 Hz, H-7'), 4.60 (1H, t, 11.0 Hz, H-2a),4.47 (1H, dd, 11.0, 5.5 Hz, H-2b), 4.20 (1H, dd, 11.0, 5.5 Hz, H-3), 3.75 (3H, s, 4'-OCH3),13

3.72 (3H, s, 2'-OCH3), 3.65 (1H, t, 2.5 Hz, H-9'); C-NMR (125 MHz, DMSO, δ ppm): 190.6(s, C-4), 163.1 (s, C-7), 163.0 (s, C-8a), 160.1 (s, C-4'), 158.1 (s, C-2'), 130.7 (d, C-5), 128.6(d, C-6'), 115.8 (s, C-1'), 115.4 (s, C-4a), 110.2 (d, C-3'), 105.0 (d, C-5'), 102.0 (d, C-6), 98.9
(d, C-8), 78.8 (s, C-8'), 78.5 (d, C-9'), 70.5 (t, C-2), 55.9 (t, C-7'), 55.6 (q, 4'-OCH3), 55.2 (q,2'-OCH3), 46.8 (d, C-3).
Compound 2b:

7-Dehydroxy-7-((1-(2-(3-hydroxy-4-(3-(3,4,5trimethoxyphenyl)acryloyl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)sativanone,

trivialname Datosativanone: Yellow amorphous solid; HR-MS-MALDI-TOF: m/z 760.2482
1[M+Na]+ calcd. for C40H39N3O11Na, found: 760.2484; H-NMR (500 MHz, DMSO, δ ppm):
8.35 (1H, s, H-2''), 8.27 (1H, d, 9.0 Hz, H-5'''), 7.91 (1H, d, 15.5 Hz, Hα), 7.78 (1H, d, 15.5
Hz, Hβ), 7.74 (1H, d, 8.5 Hz, H-5), 7.25 (2H, s, H-2'''', H-6''''), 6.99 (1H, d, 8.5 Hz, H-6'), 6.74
(2H, d, 2.5 Hz, H-8, H-2'''), 6.56 (1H, dd, 8.5, 2.5 Hz, H-5'), 6.54 (1H, d, 2.5 Hz, H-3'), 6.53

86

(1H, dd, 9.0, 2.5 Hz, H-6'''), 6.47 (1H, dd, 8.5, 2.5 Hz, H-6), 5.26 (2H, s, H-3''), 4.83 (2H, t,
6.0 Hz, H-7'''), 4.60 (1H, t, 11.0 Hz, H-2a), 4.54 (2H, t, 6.0 Hz, H-8'''), 4.46 (1H, dd, 11.0, 5.5
Hz, H-2b), 4.19 (1H, dd, 11.0, 5.5 Hz, H-3), 3.86 (6H, s, 3''''-OCH3, 5''''-OCH3), 3.74 (3H, s,
4'-OCH3), 3.72 (3H, s, 4''''-OCH3), 3.70 (3H, s, 2'-OCH3);

13

C-NMR (125 MHz, DMSO, δppm): 191.9 (s, CO), 190.5 (s, C-4), 164.4 (s, C-1'''), 166.9 (s, C-3'''), 163.9 (s, C-7), 163.2 (s,C-8a), 160.0 (d, C-4'), 130.6 (d, C-5), 158.1 (s, C-2'), 153.1 (s, C-3'''', C-5''''), 144.9 (d, Cβ),142.0 (s, C-1''), 140.0 (s, C-4''''), 132.7 (d, C-5'''), 129.9 (d, C-1''''), 125.4 (d, C-2''), 120.9 (d,Cα), 115.8 (s, C-4a), 115.1 (s, C-1'), 114.2 (s, C-4'''), 110.2 (d, C-3'), 107.5 (d, C-6'''), 106.9(d, C-2'''', C-6''''), 104.9 (d, C-5'), 101.8 (d, C-2'''), 101.6 (d, C-6), 98.8 (d, C-8), 72.8 (t, C-7'''),
70.4 (t, C-2), 60.1 (q, 4''''-OCH3), 56.1 (q, 3''''-OCH3, 5''''-OCH3), 55.6 (q, 2'-OCH3), 55.2 (q,4'-OCH3), 48.7 (t, C-8'''), 46.7 (d, C-3), 38.1 (t, C-3'').
Semi-synthesis of compounds 2a and 2b: To a solution of compound 2 (20 mg),
K2CO3 (18 mg), propargyl bromide (0.15 mL) in dimethylformamide (4.0 mL) was stirred atroom temperature for 5h. The mixture reaction was then diluted with water and extracted
three times with EtOAc (3 × 50 mL). The organic layer was washed with brine, dried overanhydrous Na2SO4 and concentrated under vacuum. This residue was purified by silica gelcolumn and mobile phase n-hexane/acetone 3:1 to give compound 2a (14.4 mg).
A mixture of 2a (1.0 mol) and the chalcone azide (1.0 mol) in DMSO (7 mL) was
stirred at room temperature for 10 min. A catalytic amount of CuI was then added and stirred
overnight at room temperature. The product was subsequently participated in distilled water,
and filtered by Büsner funnel. Compound 2b (8.25 mg) was obtained by silica gel column
using CH2Cl2/MeOH 9:1 as eluting system.

87

Figure 26. Semisynthetic reactions of 2a and 2b
Compound 3: Naringenin

Yellow amorphous solid, EI-MS (+): m/z 272 [M]+, C15H12O5; 1H-NMR (500 MHz,
CD3OD, δ ppm): 7.34 (2H, d, 8.5 Hz, H-2', H-6'), 6.84 (2H, d, 8.5 Hz, H-3', H-5'), 5.91 (2H, d,
2.0 Hz, H-6, H-8), 5.36 (1H, dd, 13.0, 3.0 Hz, H-2), 3.13 (1H, dd, 17.0, 13.0 Hz, Hb-3), 2.72
(1H, dd, 17.0, 3.0 Hz, Ha-3); 13C-NMR (125 MHz, CD3OD, δ ppm): 197.8 (s, C-4), 168.4 (s,C-7), 165.5 (s, C-5), 164.9 (s, C-8a), 159.1 (s, C-4'), 131.8 (s, C-1'), 129.1 (d, C-2', C-6'),
116.4 (d, C-3', C- 5'), 103.4 (s, C-4a), 97.1 (d, C-6), 96.2 (d, C-8), 80.5 (d, C-2), 44.1 (t, C-3).
Compound 4: Liquiritigenin

88White amorphous solid, EI-MS (+): m/z 256 [M]+, C15H12O4; 1H-NMR (500 MHz,
CD3OD, δ ppm): 7.74 (1H, d, 8.5 Hz, H-5), 7.33 (2H, d, 8.5 Hz, H-2', H-6'), 6.83 (2H, d, 8.5
Hz, H-3', H-5'), 6.51 (1H, dd, 8.5, 2.5 Hz, H-6), 6.37 (1H, d, 2.5 Hz, H-8), 5.39 (1H, dd, 13.0,
3.0 Hz, H-2), 3.06 (1H, dd, 17.0, 3.0 Hz, Ha-3), 2.71 (1H, dd, 17.0, 3.0 Hb-3); 13C-NMR (125MHz, CD3OD, δ ppm): 193.5 (s, C-4), 169.9 (s, C-8a), 165.6 (s, C-7), 158.9 (s, C-4'), 131.4(s, C-1'), 129.8 (s, C-5), 129.0 (d, C-2', C-6'), 116.3 (d, C-3', C- 5'), 114.9 (s, C-4a), 111.8(d, C-6), 103.8 (d, C-8), 81.0 (d, C-2), 44.9 (t, C-3).
Compound 5: Pinocembrin

White amorphous solid, EI-MS (+): m/z 256 [M]+, C15H12O4; 1H-NMR (500 MHz,
CD3OD, δ ppm): 7.52 (2H, d, 7.5 Hz, H-2', H-6'), 7.44 (2H, t, 8.0 Hz, H-3', H-5'), 7.39 (1H, t,
7.0 Hz, H-4'), 5.96 (1H, d, 2.0 Hz, H-6) 5.92 (1H, d, 2.0 Hz, H-8), 5.49 (1H, dd, 12.5, 3.0, H2), 3.12 (1H, dd, 17.5, 13.0, H-3a), 2.81 (1H, dd, 17.0, 3.0, H-3b); 13C-NMR (125 MHz,
CD3OD, δ ppm): 197.3 (s, C-4), 168.4 (s, C-7), 165.5 (s, C-5), 140.4 (s, C-1'), 129.7 (d, C-3',
C- 5'), 129.6 (d, C-4'), 103.4 (s, C-8a), 127.4 (d, C-2', C-6'), 103.4 (s, C-4a), 97.2 (d, C-6),
96.2 (d, C-8), 80.5 (d, C-2), 44.2 (t, C-3).

89

Compound 6: Medicarpin

White amorphous solid, EI-MS (+): m/z 284 [M]+, C17H16O4; 1H-NMR (500 MHz,
CD3OD, δ ppm): 7.18 (1H, d, 8.0 Hz, H-6), 7.30 (1H, d, 8.5 Hz, H-1), 6.46 (1H, dd, 8.0, 2.0
Hz, H-7), 6.40 (1H, d, 2.0 Hz, H-9), 6.32 (1H, d, 2.5 Hz, H-4), 5.48 (1H, t, 6.5 Hz, H-1b),4.32 (1H, dd, 12.0, 6.0 Hz, H-5a), 3.76 (3H, s, 8-OCH3), 3.54 (2H, m, H-5); 13C-NMR (125MHz, CD3OD, δ ppm): 162.6 (s, C-3), 162.1 (s, C-4a), 160.1 (s, C-8), 158.1 (s, C-9a), 133.2(d, C-1), 128.9 (s, C-6a), 125.9 (d, C-6), 112.9 (s, C-1a), 110.7 (d, C-2), 107.3 (d, C-7), 104.1(d, C-4), 97.6 (d, C-9), 80.1 (d, C-1b), 67.6 (t, C-5), 55.9 (8-OCH3), 40.9 (d, C-5a).
Compound 7: 3'-Hydroxy-2,4,5-trimethoxydalbergiquinol

White amorphous solid, EI-MS (+): m/z 300 [M]+, C18H20O4; 1H-NMR (500 MHz,
CD3OD, δ ppm): 7.08 (1H, dt, 8.0, 4.5 Hz, H-5'), 6.74 (1H, s, H-6), 6.66 (1H, s, H-3), 6.64
(1H, brd, 7.5 Hz, H-6'), 6.61 (1H, brd, 2.5 Hz, H-2'), 6.60 (1H, overlap, H-4'), 6.26 (1H, ddd,
17.0, 10.5, 7.0 Hz, H-2''), 5.16 (1H, dt, 10.0, 1.5 Hz, Ha-3''), 5.00 (1H, d, 6.5 Hz, H-1''), 4.90
(1H, dt, 17.0, 1.5 Hz, Hb-3''), 3.85 (3H, s, 4-OCH3), 3.74 (3H, s, 5-OCH3), 3.73 (3H, s, 290

OCH3); 13C-NMR (125 MHz, CD3OD): 158.2 (s, C-3'), 153.1 (s, C-2), 150.0 (s, C-4), 146.4 (s,C-1'), 144.3 (s, C-5), 142.0 (d, C-2''), 130.0 (d, C-5'), 125.1 (s, C-1), 120.9 (d, C-6'), 116.5 (d,C-4'), 116.0 (t, C-3''), 115.9 (d, C-6), 113.9 (d, C-2'), 99.9 (d, C-3), 57.6 (q, 2-OCH3), 57.0 (s,5-OCH3), 56.8 (q, 4-OCH3), 48.5 (d, C-1'').
Compound 8: ButeaspermanolWhite amorphous solid, EI-MS (+): m/z 286 [M]+, C16H14O5; 1H-NMR (500 MHz,
CD3OD, δ ppm): 7.57 (2H, t, 7.0 Hz, H-2′, 6′), 7.40-7.45 (3H, m, H-3′, 4′, 5′), 7.31 (1H, s, H5), 6.43 (1H, s, H-8), 5.09 (1H, d, 12.0 Hz, H-2), 4.53 (1H, d, 12.0 Hz, H-3), 3.88 (3H, s, 7OCH3); 13C-NMR (125 MHz, CD3OD, δ ppm): 194.28 (s, C-4), 159.7 (s, C-9), 156.8 (s, C-7),
145.5 (s, C-1′),138.8 (s, C-6), 129.8 (s, C-4′), 129.3 (d, C-3′, 5′), 128.9 (d, C-2′, 6′), 112.1 (s,C-10), 108.2 (d, C-5), 104.5 (d, C-8), 85.90 (d, C-2), 74.7 (d, C-3), 56.7 (q, 7-OCH3).
Compound 9: Butin

White amorphous solid, EI-MS (+): m/z 272 [M]+, C15H12O5; 1H-NMR (500 MHz,
CD3OD, δ ppm): 7.72 (1H, d, 9.0 Hz, H-5), 6.95 (1H, d, 1.5 Hz, H-8), 6.81 (2H, m, H-5', H6'), 6.48 (1H, dd, 9.0, 2.5 Hz, H-6), 6.32 (1H, d, 2.0 Hz, H-2'), 5.32 (1H, dd, 13.0, 3.0 Hz, H2), 3.01 (1H, dd, 17.0, 3.0 Hz, H-3a), 2.67 (1H, dd, 17.0, 3.0 Hz, H-3b). 13C-NMR (125 MHz,91

CD3OD, δ ppm): 193.5 (s, C-4), 165.7 (s, C-7), 165.3 (s, C-4a), 146.8 (s, C-3'), 146.5 (s, C4'), 132.2 (s, C-1'), 129.8 (s, C-5), 119.2 (d, C-6'), 116.3 (d, C- 5'), 116.2 (s, C-8a), 114.7 (d,C-2'), 111.5 (d, C-6), 104.1 (d, C-8), 81.0 (d, C-2), 45.0 (t, C-3).
Compound 10: 3'-Hyrdoxymelanettin

White amorphous powder, EI-MS (+): m/z 300 [M]+, C16H12O6; 1H-NMR (500 MHz,
CD3OD, δ ppm): 7.09 (1H, s, H-8), 7.05 (1H, s, H-5), 6.95 (1H, d, 8.0 Hz, H-5'), 6.94 (1H, s,H-2'), 6.88 (1H, dd, 8.0, 2.0 Hz, H-6'), 6.16 (1H, s, H-3), 3.99 (3H, s, 7-OCH3); 13C-NMR(125 MHz, CD3OD, δ ppm): 164.3 (s, C-2), 158.4 (s, C-4), 153.4 (s, C-7), 150.5 (s, C-8a),148.4 (s, C-4'), 146.8 (s, C-3'), 145.0 (s, C-6), 121.5 (d, C-5'), 116.7 (d, C-2'), 116.2 (d, C-6'),
113.3 (s, C-4a), 112.2 (d, C-5), 111.3 (s, C-3), 101.5 (d, C-8), 56.9 (q, 7-OCH3).
Compound 11: Biochanin A

Yellow amorphous solid, EI-MS (+): m/z 284 [M]+, C16H12O5; 1H-NMR (500 MHz,
CD3OD, δ ppm): 8.11 (1H, s, H-2), 7.50 (2H, d, 9.0 Hz, H-2', H-6'), 7.00 (2H, d, 8.5 Hz, H-3',
H-5'), 6.38 (1H, d, 2.0 Hz, H-8), 6.25 (2H, d, 2.0 Hz, H-6), 3.85 (3H, s, 4'-OCH3); 13C-NMR(125 MHz, CD3OD, δ ppm): 182.2 (s, C-4), 166.0 (s, C-7), 163.9 (s, C-5), 161.2 (s, C-4' ),92

159.7 (s, C-8a), 155.0 (d, C-2), 131.3 (d, C-2', C-6' ), 124.6 (s, C-1'), 124.5 (s, C-3), 114.9 (s,C-3', C-5'), 106.3 (s, C-4a), 100.2 (d,C-6), 94.8 (d, C-8), 55.8 (q, 4'-OCH3 ).
Compound 12: Calycosin

White amorphous solid, EI-MS (+): m/z 284 [M]+, C16H12O5; 1H-NMR (500 MHz,
CD3OD, δ ppm): 8.14 (1H, s, H-2), 8.07 (1H, d, 8.5 Hz, H-5), 7.06 (1H, overlap, H-2'), 6.99
(1H, brs, H-5'), 6.98 (1H, brs, H-6'), 6.96 (1H, dd, 8.5, 2.0 Hz, H-6), 6.87 (1H, d, 2.0 Hz, H-8),
3.85 (3H, s, 4'-OCH3); 13C-NMR (125 MHz, CD3OD, δ ppm): 178.0 (s, C-4), 164.7 (s, C-7),
159.8 (s, C-8a), 158.4 (d, C-2), 149.2 (s, C-4'), 147.4 (s, C-3'), 128.5 (d, C-5), 126.2 (s, C-1'),
125.8 (s, C-3), 121.6 (d, C-6'), 118.2 (s, C-4a), 117.4 (d, C-2'), 112.7 (d, C-5'), 116.4 (d, C-6),
103.3 (d, C-8), 56.5 (q, 4'-OCH3).
Compound 13: Eriodictoyl

White amorphous solid, EI-MS (+): m/z 288 [M]+, C15H12O6; 1H-NMR (500 MHz,
CD3OD, δ ppm): 6.94 ( 1H, s, H-2'), 6.81 (2H, s, H-5', H-6'), 5.92 (1H, d, 2.0 Hz, H-6), 5.90
(1H, d, 2.0 Hz, H-8), 5.31 (1H, dd, 13.0, 3.0 Hz, H-2), 3.09 (1H, dd, 17.0 Hz, 13.0, H-3a),2.72 (1H, dd, 15.0, 5.0 Hz, H-3b); 13C-NMR (125 MHz, CD3OD, δ ppm): 197.8 (s, C-4),
164.8 (s, C-5), 163.0 (s, C-7), 155.8 (s, C-8a), 146.9 (s, C-4'), 146.5 (s, C-3'), 131.8 (s, C-1'),93119.3 (d, C-6'), 116.3 (d, C-5'), 114.7 (d, C-2'), 103.4 (s, C-4a), 97.1 (d, C-8), 96.3 (d, C-6),
80.5 (d, C-2), 44.1 (t, C-3).
Compound 14: Isoliquiritigenin

Yellow amorphous solid, EI-MS (+): m/z 256 [M]+, C15H12O4; 1H-NMR (500 MHz,
CD3OD, δ ppm): 7.90 (1H, d, 9.0 Hz, H-6'), 7.81 (1H, d, 15.5 Hz, H-7), 7.64 (1H, d, 15.5 Hz,H-8), 7.65 (2H, dd, 15.5, 8.5 Hz, H-2, H-6), 6.87 (2H, d, 8.5 Hz, H-3, H-5), 6.31 (1H, dd, 9.0,
2.0 Hz, H-5'); 13C-NMR (125 MHz, CD3OD, δ ppm): 193.6 (s, CO), 167.5 (s, C-4'), 166.6 (s,C-2'), 161.6 (s, C-4), 145.6 (d, C-7), 133.4 (d, C-6'), 131.8 (d, C-2, C-6), 127.8 (s, C-1), 118.4(d, C-8), 116.7 (d, C-3, C-5), 114.7 (s, C-1'), 109.2 (d, C-5'), 103.9 (d, C-3').
Compound 15: 3'-O-Methylviolanone

White amorphous solid, EI-MS (+): m/z 330 [M]+, C18H18O6; 1H-NMR (500 MHz,
CD3OD, δ ppm): 7.79 (1H, d, 8.5 Hz, H-5), 6.86 (1H, d, 8.5 Hz, H-5'), 6.75 (1H, d, 8.5 Hz, H5'), 6.54 (1H, dd, 8.5, 2.0 Hz, H-6), 6.37 (1H, d, 2.0 Hz, H-8), 4.57 (1H, t, 11.5 Hz, H-2a),4.43 (1H, dd, 11.0, 5.5 Hz, H-3), 4.14 (1H, dd, 12.0, 5.5 Hz, H-2b), 3.83 (3H, s, 4'-OCH3),
3.83 (3H, s, 2'-OCH3), 3.82 (3H, s, 3'-OCH3); 13C-NMR (125 MHz, CD3OD, δ ppm): 194.2 (s,C-4), 166.5 (s, C-7), 165.8 (s, C-8a), 153.2 (s, C-2'), 155.1 (s, C-4'), 143.6 (s, C-3'), 130.4 (d,94C-5), 126.0 (s, C-6'), 123.1 (s, C-1'), 111.8 (d, C-6), 115.5 (s, C-4a), 108.8 (s, C-5'), 103.7 (d,C-8), 72.4 (t, C-2), 61.2 (q, 3'-OCH3), 61.0 (q, 2'-OCH3), 56.6 (q, 4'-OCH3), 49.5 (d, C-3).
Compound 16: Luteolin

Yellow amorphous powder, EI-MS (+): m/z 286 [M]+, C15H10O6; 1H-NMR (500 MHz,DMSO, δ ppm): 7.42 (1H, dd, 8.5, 2.0 Hz, H-6'), 7.39 (1H, d, 8.5 Hz, H-2'), 6.89 (1H, d, 8.5
Hz, H-5'), 6.66 (1H, s, H-3), 6.44 (1H, d, 2.0 Hz, H-8), 6.19 (1H, d, 2.0 Hz, H-6); 13C-NMR(125 MHz, DMSO, δ ppm): 181.6 (s, C-4), 164.1 (s, C-7), 163.9 (s, C-2), 161.5 (s, C-8a),
157.3 (s, C-5), 149.7 (s, C-4'), 145.7 (s, C-3'), 121.5 (d, C-6'), 119.0 (s, C-1'), 116.0 (d, C-6'),
113.4 (d, C-2'), 103.7 (s, C-4a), 98.8 (d, C-6), 93.8 (d, C-8).
Compound 17: Quercetin

Yellow amorphous powder, CI-MS (+): m/z 303 [M+1]+, C15H10O7; 1H-NMR (500
MHz, CD3OD, δ ppm): 7.75 (1H, d, 2.5 Hz, H-2'), 7.65 (1H, dd, 8.5, 2.5 Hz, H-6'), 6.91 (1H,d, 8.5 Hz, H-5'), 6.44 (1H, d, 2.0 Hz, H-8), 6.20 (1H, d, 2.0 Hz, H-6), 6.99 (1H, brs, H-5');
13

C-NMR (125 MHz, CD3OD, δ ppm): 177.3 (s, C-4), 165.6 (s, C-7), 162.1 (s, C-5), 158.3 (s,95C-8a), 148.7 (s, C-4'), 148.0 (s, C-2), 146.2 (s, C-3'), 137.2 (s, C-3), 124.2 (s, C-1'), 121.7 (d,C-6'), 116.2 (d, C-2'), 116.1 (d, C-5'), 104.4 (s, C-4a), 99.3 (d, C-6), 94.4 (d, C-8).
Compound 18: Apigenin

Yellow amorphous powder, EI-MS (+): m/z 270 [M]+, C15H10O5; 1H-NMR (500 MHz,
MeOD, δ ppm): 7.91 (2H, d, 9.0 Hz, H-2', H-6'), 6.92 (2H, d, 9.0 Hz, H-3', H-5'), 6.76 (1H, s,H-3), 6.47 (1H, d, 2.0 Hz, H-8), 6.19 (1H, d, 2.0 Hz, H-6); 13C-NMR (125 MHz, MeOD, δppm): 181.7 (s, C-4), 164.1 (s, C-2), 163.7 (s, C-7), 161.4 (s, C-4'), 161.1 (s, C-5), 157.3 (s,C-8a), 128.4 (d, C-2', C-6'), 121.2 (s, C-1'), 115.9 (d, C-3', C-5'), 103.7 (s, C-4a), 102.8 (d, C3), 98.8 (d, C-6), 93.9 (d, C-8).
Compound 19: Sulfuretin

White amorphous solid, EI-MS (+): m/z 270 [M]+, C15H10O5; 1H-NMR (500 MHz,
CD3OD, δ ppm): 7.61 (1H, d, 9.0 Hz, H-5'), 7.54 (1H, d, 2.0 Hz, H-2'), 7.25 (1H, dd, 8.5, 2.0
Hz, H-6), 6.86 (1H, d, 8.5 Hz, H-5), 6.69 (3H, overlap, H-6, H-8, H-6'); 13C-NMR (125 MHz,
MeOD, δ ppm): 184.3 (s, C-4), 170.0 (s, C-7), 168.7 (s, C-8a), 149.4 (s, C-4'), 147.8 (s, C-3),

96146.7 (s, C-3'), 126.8 (d, C-5), 126.3 (s, C-6'), 125.6 (s, C-1'), 118.9 (d, C-5'), 116.7 (d, C-6),
114.6 (d, C-2), 114.4 (d, C-2'), 114.2 (s, C-4a), 99.5 (d, C-8).
Compound 20: (2S)-6,8-Dicarboxyethylpinocembrin

White amorphous solid;
25[α] D +45.0 (c 0.22, MeOH);
IR (film): 3370.12, 2931.19, 1721.05, 1683.56, 1585.53, 1444.84, 1382.38, 1216.64,
1090.62, 899.53, 693.56 cm-1;
UV λmax (MeOH) nm (log ε): 296 (3.64);
CD (c 0.2, MeOH): ∆ε290 (nm) −8.21, ∆ε339 (nm) +0.83;
HR-ESI-MS: m/z [M−H]− 399.1080 calcd. for C21H19O8; found: 399.1079);1

H- and 13C-NMR Data of compound 20: See Table 2.

Compound 21: (2S)-8-Carboxyethylnaringenin

White amorphous solid;9725[α] D +37.0 (c 0.53, MeOH);
IR (film): 3356.96, 3089.96, 2924.09, 1737.86, 1685.78, 1576.65, 1477.47, 1377.17,
1215.15, 1035.77, 759.95, 675.09, 428.20 cm-1;
UV λmax (MeOH) nm (log ε):294 (3.73);
CD (c 0.1, MeOH): ∆ε293 (nm) −0.71; ∆ε337 (nm) +0.19;
HR-ESI-MS: m/z [M-H]− 343.0815 calcd. for C18H15O7; found: 343.0817);
1

H- and 13C-NMR Data of compound 21: See Table 2.

Compound 22: DalberginLight yellow solid, EI-MS (+): m/z 268 [M]+, C16H12O4; 1H-NMR (500 MHz, CD3OD,
δ ppm): 7.57 (3H, m, H-3', H-4', H-5'), 7.51 (2H, m, H-2', H-6'), 7.06 (1H, s, H-8), 6.88 (1H, s,H-5), 6.21 (1H, s, H-3), 3.99 (3H, s, 7-OCH3); 13C-NMR (125 MHz, CD3OD, δ ppm): 163.9(s, C-2), 158.2 (s, C-4), 153.6 (s, C-7), 113.1 (s, C-4a), 145.2 (s, C-6), 137.1 (s, C-1'), 130.8 (d,C-4'), 129.9 (d, C-3', C-5'), 129.5 (d, C-2', C-6'), 112.3 (d, C-3), 111.9 (d, C-5), 150.4 (s, C8a), 101.1 (d, C-8), 56.9 (q, 7-OCH3).

98

Compound 23: Homoferreirin

White amorphous solid, EI-MS (+): m/z 330 [M]+, C18H18O6; 1H-NMR (500 MHz,
CD3OD, δ ppm): 7.04 (1H, d, 8.5 Hz, H-6'), 6.59 (1H, s, H-3'), 6.52 (1H, d, 7.5 Hz, H-5'), 5.92
(1H, s, H-6), 5.90 (1H, s , H-8), 4.51 (1H, t, 11.0 Hz, Hb-2), 4.39 (1H, dd, 11.0, 5.5 Hz, H-3),
4.25 (1H, dd11.0, 5.0 Hz, Ha-2), 3.80 (3H, s, 2'-OCH3), 3.81 (3H, s, 4'-OCH3); 13C-NMR (125MHz, CD3OD, δ ppm): 199.0 (s, C-4), 168.2 (s, C-7), 165.8 (s, C-5), 165.2 (s, C-8a), 162.3 (s,C-4'), 159.9 (s, C-2'), 132.1 (d, C-6'), 116.8 (s, C-1'), 106.1 (d, C-5'), 103.3 (s, C-4a), 100.1 (s,C-3'), 97.1 (d, C-4), 96.0 (d, C-8), 71.5 (t, C-2), 56.1 (q, 2'-OCH3), 55.8 (q, 4'-OCH3), 48.5 (d,C-3).
Compound 24: Neroplomacrol

White amorphous solid, C15H26O2; 1H-NMR (500 MHz, CD3OD, δ ppm): 5.93 (1H,
dd,17.5, 11.0 Hz, H-2), 5.21 (2H, dd, 17.5, 1.5 Hz, H-1), 5.204 (1H, overlap, H-6), 3.25, (1H,
dd, 10.5, 1.5 Hz, H-10), 2.25 (1H, m, Ha-8), 2.04 (2H, m, H-5, Hb-8), 1.64 (3H, s, H-14), 1.73,(1H, m, Ha-9), 1.54 (1H, dtd, 2.5, 3.0, 3.0 Hz, H-4), 1.35 (1H, m, Hb-9), 1.27 (3H, s, H-15),
1.18 (3H, s, H-12), 1.14 (3H, s, H-13); 13C-NMR (125 MHz, CD3OD, δ ppm): 146.4 (d, C-2),
136.1 (s, C-7), 125.8 (d, C-6), 112.0 (t, C-1), 79.0 (s, C-10), 73.8 (s, C-11), 73.8 (s, C-3), 43.4

99

(t, C-4), 37.9 (t, C-8), 30.8 (t, C-9), 27.6 (q, C-15), 25.6 (q, C-12), 24.9 (q, C-13), 23.7 (C-5,t), 16.1 (q, C-14).
Compound 25: Rel-(3R,6S,7S)-6α-hydroxycyclonerolidol

White amorphous solid;
25[α] D −15.0 (c 0.02, CHCl3);
IR (film) νmax: 3362, 2923, 1642, 1434, 1092, 866 cm-1;
HR-ESI-MS: m/z 239.2011 [M+H]+ (calcd. for C15H27O2 239.2010), 243.1724[M+Na-H2O]+ (calcd. for C15H24ONa 243.1724);
1

H- and 13C-NMR Data of compound 25: See Table 3.

Compound 26: Kaemfperol

Yellow amorphous powder, EI-MS (+): m/z 286 [M]+, C15H10O6; 1H-NMR (500 MHz,
MeOD, δ ppm): 8.1 (2H, d, 8.5 Hz, H-2', H-6'), 6.91 (2H, d, 8.5 Hz, H-3', H-5'), 6.40 (1H, d, s,H-8), 6.19 (1H, s, H-6); 13C-NMR (125 MHz, MeOD, δ ppm): 177.3 (s, C-4), 165.5 (s, C-7),
162.4 (s, C-5), 160.4 (s, C-4'), 158.2 (s, C-8a), 148.0 (s, C-2), 137.1 (s, C-3), 130.6 (d, C-2',
C-6'), 123.7 (s, C-1'), 116.3 (d, C-3', C-5'), 104.6 (s, C-4a), 99.3 (d, C-6), 94.5 (d, C-8).

100

Compound 27: 4-Hydroxybenzaldehyde (Formyl phenol)

White amorphous solid, EI-MS (+): m/z 123 [M]+, C7H7O2; 1H-NMR (500 MHz,
CD3OD, δ ppm): 9.80 (1H, s, 1-CHO), 7.80 (2H, d, 8.5 Hz, H-2, H-6), 6.94 (2H, d, 8.5, H-3,
H-5); 13C-NMR (125 MHz, CD3OD, δ ppm): 191 (d, CHO), 164.1 (s, C-4), 132.4 (d, C-2, C6), 129.0 (s, C-1), 116.1 (d, C-3, C-5).
Compound 28: Naringin

White amorphous solid, EI-MS (+): m/z 580 [M]+, C27H32O14; 1H-NMR (500 MHz,
CD3OD +DMSO, δ ppm): 12.04 (1H, s, 5-OH), 9.62 (1H, s, 4'-OH), 7.33 (2H, m, H-2', H-6'),
6.80 (2H, d, 7.0 Hz, H-3', H-5'), 6.11 (1H, d, 2.0 Hz, H-6), 6.09 (1H, t, 3.0 Hz, H-8), 5.50 (1H,
ddd, 3.0, 2.5, 2.5 Hz, H-2), 5.31 (1H, d, 4.5 Hz, 3''-OH), 5.10-5.15 (3H, m, H-1'', H-1''', 5''OH), 4.72 (1H, d, 4.5 Hz, 4'''-OH), 4.66 (1H, d, 4 Hz, 2'''-OH), 4.58 (1H, s, 6''-OH), 4.47 (1H,d, 4.5 Hz, 3''-OH), 3.65-3.72 (3H, m, H-6''a, H-2''', H-5'''), 3.43 (3H, overlap, H-2'', H-3'', H5''), 3.29-3.39 (2H, m, H-3a, H-6''b), 3.18-3.23 (2H, m, H-4'', H-4'''), 2.73 (1H, m, H-3b), 1.16
(3H, d, 5.5 Hz, 5'''-CH3); 13C-NMR (125 MHz, MeOD, δ ppm): 198.5 (s, C-4), 166.6 (s, C-7),
165.0 (s, C-5), 164.6 (s, C-8a), 159.1 (s, C-4'), 130.9 (s, C-1'), 129.1 (d, C-2', C-6'), 116.4 (d,C-3', C-5'), 104.9 (s, C-4a), 102.5 (d, C-1'''), 99.4 (d, C-1''), 97.9 (d, C-6), 96.8 (d, C-8), 80.7

101

(d, C-2), 79.2 (d, C-2'''), 79.1 (d, C-3''), 78.1 (d, C-5''), 73.9 (d, C-4'''), 72.2 (d, C-2''', C-3'''),
71.3 (d, C-4''), 70.0 (d, C-5'''), 62.2 (t, C-6''), 44.1 (t, C-3), 18.2 (q, 5'''-CH3).
2.3. Phytochemical investigation of leaf extract and structural elucidation
2.3.1. Isolated procedure
The leaf (2.0 kg) was extracted with MeOH (10.0 L) for 4h using the Soxhlet extractor,
to produce the crude MeOH extract (300.0 g). This extract was then suspended in hot MeOHH2O (1:1, v/v) and successively partitioned with CHCl3 and EtOAc to yield the corresponding
fr.s. Using column chromatography filled with silica gel and CHCl3-MeOH (99:1, v/v), theCHCl3 extract (60.7 g) was separated to give 8 fr.s (C1-C8). Applying silica gel columns withmobile phases of CHCl3-CH3COCH3 (30:1, v/v), and n-hexan-CH3COCH3 (6:1, v/v),
compounds 11 (45.7 mg) and 29 (34.6 mg) were precipitated out of the fr. C2 (5.1 g) and the
fr. C3 (4.3 g), respectively. Compound 30 (15.6 mg) was isolated from the fr. C6 (5.7 g) byusing a silica gel column with CHCl3-MeOH (3:1, v/v).
The H2O-soluble residue (fr. W, 95.5 g) was subjected to Diaion HP-20
chromatography with gradient solvents of H2O-MeOH (1:0→0:1, v/v) to afford 3 fr.s (W1W3). The 25% MeOH fraction (fr. W2, 4.57 g) was separated by a normal silica gel CC,
eluting with a gradient of CHCl3-MeOH-H2O (3:1:0.1, v/v/v) to yield compound 32 (20.6 mg)
and 9 fr.s (W21-W29). The fr. W27 (0.9 g) was then purified by HPLC (Cosmosil 5C18-ARII column (10x250 mm)) with MeOH-H2O (7:3, 1 mLmin-1) to give compound 33 (1.2 mg),while compound 34 (4.6 mg) were obtained from the fr. W28 (1.9 g) using the same manner.
Finally, ultilizing HPLC (Cosmosil 5C18-AR-II column (10x250 mm)) with MeOH-H2OHCOOH (3:1: 0.1%, 2 mLmin-1), compound 35 (1.6 mg) was obtained from the fr. W29 (0.6
g).

102

2.3.2. Chemical and physical data of isolated compounds
Compound 29: Genistein

White crystal, EI-MS (+): m/z 270 [M]+, C15H10O5; 1H-NMR (500 MHz, CD3OD, δppm): 8.01 (1H, s, H-2), 7.32 (2H, d, 8.5 Hz, H-2', H-6'), 6.80 (2H,d , 8.5 Hz, H-3', H-5'), 6.30
(1H, d, 2.0 Hz, H-8), 6.17 (2H, d, 2.0 Hz, H-6); 13C-NMR (125 MHz, CD3OD, δ ppm): 182.1(s, C-4), 164.6 (s, C-7), 162.5 (s, C-5), 158.3 (s, C-4' ), 157.4 (s, C-8a), 153.4 (d, C-2), 123.3(s, C-1' ), 121.9 (s, C-3), 130.0 (d, C-2', C-6' ), 114.9 (s, C-3', C-5'), 104.9 (s, C-4a), 99.7
(d,C-6), 93.4 (d, C-8).
Compound 30: Orobol

Yellow amorphous powder, EI-MS (+): m/z 286 [M]+, C15H10O6; 1H-NMR (500 MHz,
CD3OD, δ ppm): 8.02 (1H, s, H-2), 7.01 (1H, d, 2.0 Hz, H-2'), 6.84 (1H, dd, 8.0, 2.0 Hz, H-6'),
6.84 (1H, d, 8.0 Hz, H-5'), 6.32 (1H, d, 2.0 Hz, H-8), 6.20 (2H, d, 2.0 Hz, H-6); 13C-NMR(125 MHz, CD3OD, δ ppm): 180.9 (s, C-4), 164.6 (s, C-7), 162.5 (s, C-5), 158.3 (s, C-8a),
153.4 (d, C-2), 145.4 (s, C-4'), 144.8 (s, C-3'), 123.4 (s, C-1'), 122.4 (s, C-3), 120.3 (d, C-6'),
116.0 (d, C-5'), 114.9 (d, C-2'), 104.9 (s, C-4a), 98.7 (d, C-6), 93.4 (d, C-8).

103

Compound 31: 3-(1H-indol-3-yl)-2-methoxypropanamide

White powder;25[α] D +60.0 (c 0.43, MeOH);
IR (film): 3416 (sh), 3045, 2980, 1629 (sh), 1548, 1514, 1453, 1355, 1199, 1146 cm-1;
UV (MeOH) λmax nm (log ε): 223 (4.43), 281 (3.79), 288 (3.72);
HR-ESI-MS: m/z 241.0953 [M+Na]+ (calcd. for C12H14N2O2Na 241.0952), APCI-MS:
m/z 188 [M+H-OCH3]+, 144 [M+H-OCH3-CONH2]+, 118 [M+H-methoxypropanamide]+;1

H-NMR (500 MHz, CD3OD, δ ppm) and 13C-NMR (125 MHz, CD3OD, δ ppm): See

Table 4.
Compound 32: Dalsissooside

White amorphous powder, EI-MS (+): m/z 668 [M]+, C30H36O17; 1H-NMR (500 MHz,
MeOD, δ ppm): 8.11 (1H, s, H-2), 6.93 (1H, s, H-6'), 6.91 (1H, s, H-8), 6.77 (1H, s, H-3'),104

5.04 (1H, d, 7.5 Hz, H-1''), 4.95 (1H, d, 2.5 Hz, H-1'''), 3.35-4.06 (13H, sugar protons), 3.90
(3H, s, 4'-OCH3), 3.89 (3H, s, 6-OCH3), 3.80 (3H, s, 2'-OCH3), 3.78 (3H, s, 5'-OCH3); 13CNMR (125 MHz, MeOD, δ ppm): 180.6 (s, C-4), 156.7 (s, C-7), 156.2 (d, C-2), 153.3 (s, C-5),
152.8 (s, C-8a), 152.4 (s, C-2'), 150.3 (s, C-4'), 142.9 (s, C-5'), 132.9 (s, C-6), 120.4 (s, C-3),
116.3 (s, C-6'), 111.0 (s, C-1'), 109.9 (d, C-1'''), 107.1 (s, C-4a), 100.7 (d, C-1''), 98.3 (d, C-3'),
94.6 (d, C-8), 79.0 (d, C-3'''), 76.8 (d, C-2'''), 76.7 (d, C-3''), 76.0 (d, C-5''), 73.7 (t, C-4'''),
73.4 (d, C-2''), 70.3 (d, C-4''), 67.9 (t, C-6''), 64.2 (t, C-5'''), 60.2 (6-OCH3), 56.3 (q, 2'-OCH3),
55.9 (q, 5'-OCH3), 55.5 (q, 4'-OCH3).
Compound 33: Daltonkinensiside

White amorphous powders;25[α] D −266.9 (c 0.24, EtOH);
IR (film): 3510, 3378, 2903, 1654, 1617, 1581, 1525, 1506, 1467, 1371, 1282, 1211,
1148, 1065, 980, 905, 853, 783, 678 cm-1;
UV (EtOH) λmax nm (log ε): 295 (3.61), 263 (3.87), 205 (3.96);
HR-FAB-MS: m/z 707.1577 [M+K]+ (100%) (calcd. for C30H36O17K 707.1576), FABMS: m/z 707 [M+K]+, 669 [M+H]+, 667 [M+H]−;

105

1

H-NMR (500 MHz, C5D5N, δ ppm) and 13C-NMR (125 MHz, C5D5N, δ ppm): See

Table 5.
Acid hydrolysis of 33
Compound 33 (0.85 mg) was added into 1 N HCl (2 mL) and then heated to 80°C for 3.0 h.
The acidic solution was extracted with chloroform (2 mL×2 times). The organic layer waswashed with distilled water (2 mL×3 times), dried over Na2SO4, and evaporated to give anaglycone (0.45 mg) as a white amorphous powder. The aglycone was identiﬁed as isocaviunin
by NMR spectroscopy techniques and HR-ESI-MS: m/z 397.0872 (100%) [M+Na]+ (calcd.
for C18H19O8Na 397.0899); Rf (TLC): 0.4 (CH2Cl2–MeOH, 6:1).
Compound 34: Ambocin

White amorphous powder, EI-MS (+): m/z 564 [M]+, C26H28O14; 1H-NMR (500 MHz,
MeOD, δ ppm): 8.09 (1H, s, H-2), 7.34 (1H, d, 8.5 Hz, H-2', H-6'), 6.80 (1H, d, 8.5 Hz, H-3',
H-5'), 6.66 (1H, d, 2.0 Hz, H-8), 6.46 (1H, d, 2.0 Hz, H-6), 4.94 (1H, d, 8.0 Hz, H-1''), 4.91
(1H, d, 2.5 Hz, H-1'''), 4.01 (1H, m, H- H-6''a), 3.99 (1H, d, 10.0 Hz, H-4'''a), Hz, 3.90 (1H, d,
2.5 Hz, H-2'''), 3.73 (1H, d, 10.0 Hz, H-4'''b), 3.63 (1H, t, 7.5 Hz, H-5''), 3.58 (1H, m, H-6''b),
3.56 (2H, d, 1.5 Hz, H-5'''), 3.43 (2H, m, H-2'', H-3''), 3.34 (1H, m, H-4''); 13C-NMR (125MHz, MeOD, δ ppm): 181.1 (s, C-4), 163.3 (s, C-7), 162.1 (s, C-5), 157.8 (s, C-8a), 157.5 (s,C-4'), 154.0 (d, C-2), 130.0 (d, C-2', C-6'), 123.6 (s, C-3), 121.7 (s, C-1'), 114.9 (d, C-3', C-5'),106109.8 (d, C-1'''), 106.6 (s, C-4a), 100.2 (d, C-1''), 99.8 (d, C-6), 94.6 (d, C-8), 79.1 (d, C-3'''),
76.8 (d, C-2'''), 76.8 (d, C-3''), 76.5 (d, C-5''), 73.7 (t, C-4'''), 73.3 (d, C-2''), 70.3 (d, C-4''),67.7 (t, C-6''), 64.4 (t, C-5''').
Compound

35:

BiochaninA

7-β-D-apiofuranosyl-(1→6)-β-D-glucopyranosside(Lanceolanrin)

White amorphous powder, EI-MS (+): m/z 578 [M]+, C27H30O14; 1H-NMR (500 MHz,
MeOD, δ ppm): 8.18 (1H, s, H-2), 7.49 (1H, d, 10.0 Hz, H-2', H-6'), 6.99 (1H, d, 10.0 Hz, H3', H-5'), 6.74 (1H, s, H-8), 6.53 (1H, s, H-6), 4.97 (1H, d, 10.0 Hz, H-1''), 4.96 (1H, d, 1.5 Hz,
H-1'''), 3.83 (3H, s, 3'-OCH3), 3.37-4.06 (13H, sugar protons); 13C-NMR (125 MHz, MeOD, δppm): 181.3 (s, C-4), 163.3 (s, C-7), 162.1 (s, C-5), 159.9 (s, C-4'), 157.8 (s, C-8a), 154.2 (d,C-2), 129.9 (d, C-2', C-6'), 123.3 (s, C-3), 123.0 (s, C-1'), 113.5 (d, C-3', C-5'), 109.8 (d, C1'''), 106.6 (s, C-4a), 100.2 (d, C-1''), 99.8 (d, C-6), 94.6 (d, C-8), 79.1 (d, C-3'''), 76.8 (d, C2'''), 76.5 (d, C-3''), 75.8 (d, C-5''), 73.8 (t, C-4'''), 73.3 (d, C-2''), 70.2 (d, C-4''), 67.7 (t, C-6''),64.4 (t, C-5'''), 54.4 (q, 5'-OCH3).
2.4. Phytochemical investigation of root extract and structural elucidation
2.4.1. Isolation procedure
The CH2Cl2 root fr. (45.5 g) was chromatographed on a normal silica gel column,
using a gradient of CH2Cl2-CH3COCH3 (3:1, v/v) to afford 6 fr.s (D1-D6). Compounds 39
107

(15.0 mg) and 40 (5.5 mg) were separated from the fr. D1 (2.1 g) by CC with n-hexaneCH3COCH3 (3:1, v/v). The fr. D2 (3.2 g) was rechromatographed on a Sephadex LH-20,
eluting with MeOH-H2O (1:1, v/v) to yeild compounds 36 (4.2 mg), 41 (3.1 mg), 43 (2.1 mg),and 44 (3.99 mg). Compounds 45 (2.0 mg) and 46 (2.5 mg) were separated from fr. D3 (2.7 g),
by using CHCl3-MeOH (3:1, v/v). Compound 47 (1.1 mg) was found in a small subfr. D31(20 mg), by using HPLC [Cosmosil 5C18-AR-II column (10x250 mm) with MeOH-H2OHCOOH (7:3:0.1%, 2 mLmin-1)].
The EtOAc root fr. (30.5 g) was purified by CC with a gradient of CHCl3-MeOH-H2O(3:1:0.1, v/v/v) to afford seven fr.s. The E2 fr. (2.35 g) was then purified by HPLC [Cosmosil
5C18-AR-II column (10x250 mm) with MeOH-H2O (7:3, 2 mLmin-1)] to give compounds 2(1.3 mg), 6 (0.7 mg), 13 (1.2 mg), 14 (2.3 mg), 22 (1.5 mg), 26 (0.5 mg), and 42 (1.3 mg).
Compounds 4 (2.4 mg), 8 (1.8 mg), and 15 (4.0 mg) were precipitated out of the E3 fr. (1.2 g),
obtained after CC with CHCl3-MeOH (3:1, v/v). The E2 fr. (0.85 g) was also subjected toSephadex LH-20 with MeOH-H2O (1:1, v/v), to produce compounds 37 (5.1 mg) and 38 (25.2mg). Compounds 1 (7.2 mg) and 9 (2.1 mg) were obtained from the fourth fraction using
Sephadex LH-20 with MeOH-H2O (1:1, v/v).
2.4.2. Chemical and physical data of isolated compounds
Compound 36: Tectorigenin

Yellow amorphous powder, EI-MS (+): m/z 300 [M]+, C16H12O6; 1H-NMR (500 MHz,CDCl3, δ ppm): 13.10 (1H, s, 5-OH), 7.87 (1H, s, H-2), 7.40 (2H, d, 8.5 Hz, H-2', H-6'), 6.91

108

(2H, d, 8.5 Hz, H-3', H-5'), 6.51 (1H, s, H-8), 4.04 (3H, s, 6-OMe); 13C-NMR (125 MHz,CDCl3, δ ppm): 182.0 (s, C-4), 155.8 (s, C-4'), 155.2 (s, C-7), 153.5 (s, C-5), 152.9 (d, C-2),
152.6 (d, C-8a), 130.4 (s, C-2', C-6'), 123.1 (s, C-3), 123.0 (s, C-1'), 115.6 (s, C-3', C-5'),
106.5 (s, C-4a), 93.2 (d, C-8), 60.9 (q, 6-OMe).
Compound 37: Tectoridin

Yellow amorphous powder; EI-MS (+): m/z 462 [M]+, C22H22O11; 1H-NMR (500 MHz,
CD3OD, δ ppm): 9.18 (1H, s, H-2), 8.18 (2H, d, 8.5 Hz, H-2', H-6'), 7.67 (1H, s, H-8), 7.62
(2H, d, 8.5 Hz, H-3', H-5'), 5.88 (1H, d, 7.0 Hz, H-1''), 4.57 (3H, s, 6-OMe), 3.92-4.53 (6H,
sugar protons); 13C-NMR (125 MHz, CD3OD, δ ppm): 182.1 (s, C-4), 158.8 (s, C-4'), 157.9 (s,C-7), 156.0 (d, C-2), 154.2 (s, C-5), 153.1 (s, C-8a), 133.7 (s, C-6), 131.5 (s, C-2', C-6'), 123.3(s, C-3), 122.3 (s, C-1'), 116.4 (s, C-3', C-5'), 107.7 (s, C-4a), 101.4 (d, C-1''), 95.3 (d, C-8),
78.6 (d, C-5''), 78.0 (d, C-3''), 74.4 (d, C-2''), 70.9 (d, C-4''), 61.9 (t, C-6''), 61.6 (q, 6-OMe).
Compound 38: Tectorigenin 7-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside109

Yellow amorphous powder; EI-MS (+): m/z 594 [M]+, C27H30O15; 1H-NMR (500 MHz,
CD3OD, δ ppm): 8.18 (1H, s, H-2), 7.39 (2H, d, 8.5 Hz, H-2', H-6'), 6.89 (1H, s, H-8), 6.84
(2H, d, 8.5 Hz, H-3', H-5'), 5.03 (1H, d, 7.5 Hz, H-1''), 4.95 (1H, d, 2.5 Hz, H-1'''), 3.93 (3H, s,
6-OMe), 3.48-4.03 (13H, sugar protons); 13C-NMR (125 MHz, CD3OD, δ ppm): 181.1 (s, C4), 158.2 (s, C-4'), 157.5 (s, C-7), 154.3 (d, C-2), 153.3 (s, C-5), 153.1 (s, C-8a), 132.7 (s, C6), 130.0 (s, C-2', C-6'), 123.1 (s, C-3), 121.7 (s, C-1'), 114.9 (s, C-3', C-5'), 109.8 (d, C-1'''),
107.1 (s, C-4a), 100.6 (d, C-1''), 94.4 (d, C-8), 79.0 (d, C-3'''), 76.7 (d, C-3''), 76.6 (d, C-2'''),
75.9 (d, C-5''), 73.6 (t, C-4'''), 73.3 (d, C-2''), 70.2 (d, C-4''), 67.7 (t, C-6''), 64.3 (t, C-5'''),
60.1 (q, 6-OMe).
Compound 39: 1-MonolaurinWhite amophour powder; EI-MS (+): m/z 274 [M]+ C15H30O4; 1H-NMR (500 MHz,
CD3Cl3, δ ppm): 4.20 (1H, dd, 11.0, 6.0 Hz, H-1'a), 4.14 (1H, dd, 11.0, 6.0 Hz, H-1'b), 3.93
(2H, m, H-2'), 3.66 (1H, m, H-3'a), 3.60 (1H, m, H-3'b), 2.35 (2H, t, 7.0 Hz, H-2), 1.63 (2H, t,
7.0 Hz, H-3), 1.26 (16H, brs, H-4−H-11), 0.88 (3H, t, 7.0 Hz, 12-Me); 13C-NMR (125 MHz,
CD3Cl3, δ ppm): 174.0 (C-1), 70.3 (d, C-2'), 65.2 (t, C-1'), 63.3 (t, C-3'), 34.2 (t, C-2), 22.729.7 (8C, C-4−C-11), 24.9 (t, C-3), 14.1 (q, 12-Me).

110

Compound 40: 1-Monomyristin

White amophour powder; EI-MS (+): m/z 288 [M]+ C16H32O4; 1H-NMR (500 MHz,
CD3Cl3, δ ppm): 4.20 (1H, dd, 11.0, 6.0 Hz, H-4'a), 4.14 (1H, dd, 11.0, 6.0 Hz, H-4'b), 3.93
(2H, m, H-2'), 3.67 (1H, m, H-3'a), 3.61 (1H, m, H-3'b), 2.35 (2H, t, 7.0 Hz, H-2), 1.63 (2H, t,
7.0 Hz, H-3), 1.27 (20H, brs, H-4−H-13), 0.88 (3H,t, 7.0 Hz, 14-Me); 13C-NMR (125 MHz,
CD3Cl3, δ ppm): 174.4 (C-1), 70.3 (d, C-2'), 65.2 (t, C-1'), 63.3 (t, C-3'), 34.2 (t, C-2), 31.9 (t,C-11), 29.1-29.7 (10C, C-4−C-13), 24.9 (t, C-3), 22.7 (t, C-12), 14.1 (q, 13-Me).
Compound 41: (3R)-Vestitol

Yellow amophour powder; EI-MS (+): m/z 272 [M]+ C16H16O4; 1H-NMR (500 MHz,
CD5N5, δ ppm): 7.22 (1H, d, 8.5 Hz, H-6'), 7.11 (1H, d, 8.5 Hz, H-5), 6.90 (1H, d, 2.0 Hz, H5'), 6.91 (1H, d, 2.0 Hz, H-3'), 6.88 (1H, dd, 2.5 Hz, 8.5 Hz, H-6), 6.62 (1H, d, 2.5 Hz, H-8);
13

C-NMR (125 MHz, CD5N5, δ ppm): 159.9 (s, C-4'), 158.2 (s, C-8a), 157.4 (s, C-2'), 155.9 (s,C-7), 130.7 (d, C-5), 128.3 (d, C-6'), 121.0 (s, C-1'), 113.6 (s, C-4a), 109.0 (d, C-6), 104.5 (d,C-5'), 103.9 (d, C-8), 102.5 (d, C-3').

111

Compound 42: Melilotolcarpan D

Yellow amorphous solid, EI-MS (+): m/z 316 [M]+, C17H16O6; 1H-NMR (500 MHz,
CD3Cl3, δ ppm): 7.14 (1H, d, 8.5 Hz, H-1), 6.76 (1H, d, 8.5 Hz, H-6), 6.66 (1H, d, 8.5 Hz, H2), 6.46 (1H, d, 8.5 Hz, H-7), 5.60 (1H, d, 7.0 Hz, H-1b), 4.35 (2H, dd, 11.0, 5.0 Hz, H-5a),3.91 (3H, s, 3-OCH3), 3.88 (3H, s, 8-OCH3), 3.62 (1H, m, H-5a); 13C-NMR (125 MHz,
CD3Cl3, δ ppm): 148.1 (s, C-8), 147.4 (s, C-3), 146.1 (s, C-9a), 143.3 (s, C-4a), 133.9 (s, C-4),
130.7 (s, C-9), 121.6 (d, C-1), 121.4 (s, C-6a), 114.8 (d, C-6), 113.8 (s, C-1a), 105.3 (d, C-2),
103.8 (d, C-7), 79.2 (d, C-1b), 66.8 (t, C-5), 56.5 (q, 3-OCH3), 56.3 (q, 8-OCH3), 40.2 (d, C5a).
Compound 43: Daidzein

Yellow amorphous solid, EI-MS (+): m/z 254 [M]+, C15H10O4; 1H-NMR (500 MHz,
CD3OD, δ ppm): 8.12 (1H, s, H-2), 8.05 (1H, d, 8.5 Hz, H-5), 7.36 (2H, d, 8.5 Hz, H-2', H-6'),
6.92 (1H, dd, 2.0, 8.5 Hz, H-6), 6.85 (1H, s, H-8), 6.82 (1H, d, 8.5 Hz, H-3', H-5'); 13C-NMR(125 MHz, CD3OD, δ ppm): 176.8 (s, C-4), 163.8 (s, C-7), 158.2 (s, C-4a), 157.3 (s, C-4'),112

153.2 (d, C-2), 130.0 (d, C-2', C-6'), 127.1 (d, C-5), 124.5 (s, C-1'), 123.0 (s, C-3), 116.6 (s,C-4a), 114.9 (d, C-6), 114.8 (d, C-3', C-5'), 101.8 (d, C-8).
Compound 44: 3'-Hydroxyldaidzein

Yellow amorphous solid, EI-MS (+): m/z 270 [M]+, C15H10O5; 1H-NMR (500 MHz,
CD3OD, δ ppm): 8.12 (1H, s, H-2), 8.04 (1H, d, 8.5 Hz, H-5), 6.96 (1H, d, 2.0 Hz, H-2'), 6.93
(1H, dd, 2.0, 8.5 Hz, H-6), 6.83 (1H, dd, 2.0, 8.5 Hz, H-5'), 6.82 (1H, d, 8.5 Hz, H-6'), 6.79
(1H, d, 2.0 Hz, H-8); 13C-NMR (125 MHz, CD3OD, δ ppm): 176.6 (s, C-4), 163.5 (s, C-7),
158.5 (s, C-8a), 153.5 (d, C-2), 147.7 (s, C-3'), 146.0 (s, C-4'), 127.1 (d, C-5), 124.8 (s, C-3),
124.4 (s, C-1'), 120.2 (d, C-5'), 116.7 (s, C-4a), 116.0 (d, C-2'), 115.2 (d, C-6), 114.8 (d, C-6'),
101.9 (d, C-8).
Compound 45: Formononetin-7-O-β-D-glucopyranoside

White needle, EI-MS (+): m/z 430 [M]+, C22H22O9; 1H-NMR (500 MHz, DMSO, δppm): 8.45 (1H, s, H-2), 8.08 (1H, d, 9.0 Hz, H-5), 7.55 (2H, d, 8.5 Hz, H-2', H-6'), 7.25 (1H,d, 2.0 Hz, H-8), 7.15 (1H, dd, 9.0, 2.0 Hz, H-6), 7.01 (2H, d, 8.5 Hz, H-3', H-5'), 5.11 (1H, d,
7.5 Hz, H-1''), 3.80 (3H, s, 4'-OCH3), 3.72 (1H, dd, 5.5, 10.5 Hz, H-6''a), 3.47 (2H, m, H-6''b,
H-5''), 3.37 (Overlap, H-2'', H-3''), 3.18 (1H, m, H-4''); 13C-NMR (125 MHz, DMSO, δ ppm):
113

175.5 (s, C-4), 161.9 (s, C-7), 159.1 (s, C-4'), 157.5 (s, C-8a), 154.1 (d, C-2), 130.6 (d, C-2',
C-6'), 127.5 (d, C-5), 124.5 (s, C-1'), 123.8 (s, C-3), 118.9 (s, C-4a), 116.1 (d, C-6), 114.1 (d,C-3', C-5'), 103.9 (d, C-8), 77.7 (d, C-5''), 76.9 (d, C-3''), 73.6 (d, C-2''), 70.1 (d, C-4''), 61.1 (t,C-6''), 55.6 (q, 4'-OCH3).
Compound 46: Killitol (4-Hydroxy methylbenzoate)

Yellow amorphous powder, EI-MS (+): m/z 152 [M]+, C8H8O3; 1H-NMR (500 MHz,
CD3OD, δ ppm): 7.86 (2H, d, 8.5 Hz, C-2, C-6), 6.81 (2H, d, 8.5 Hz, C-3, C-5), 3.84 (3H, s,
OCH3); 13C-NMR (125 MHz, CD3OD, δ ppm): 167.3 (s, CO), 162.1 (s, C-4), 131.3 (d, C-2,
C-6), 120.8 (s, C-1), 114.7 (d, C-3, C-5), 50.8 (q, OCH3).
Compound 47: 2,5'-Dimethyl-5'-vinylhexahydro-2,2'-bifuran-5(2H)-one

Yellowish amorphous powders;25[α] D = –1.2 (c 1.1, MeOH);
IR (film): 2973.7, 1771.3, 1607.4, 1452.1, 1271.8, 1163.8, 1084.8, 944.9, 914.1, 829.2, 715.5,
617.1, 598.8, 564.1 cm-1;

114

UV λmax (MeOH) nm (log ε): 206.5 (2.462), 257.5 (1.339);
HR-EI-MS: m/z [M]+ 210.1254 calcd. for C12H18O3; found: 210.1253), EI-MS: m/z [M]+ 210,
1

H-NMR (500 MHz, CD3OD, δH ppm) and 13C-NMR (125 MHz, CD3OD, δC ppm) are given

in Table 6.

115

Chapter 3 Chemical constituents from Cratoxylum formosum
3.2. Phytochemical investigation of root extract and structural elucidation
3.2.1. Isolated procedure
Dried leaf (2.0 kg) was extracted with MeOH (10 L, 3 times), to produce the crude
MeOH extract (220.0 g). This extract was then subjected to normal silica gel CC, eluting withincreased mobile phase to yield the corresponding fr.s 1-7. Using silica gel CC with CHCl3MeOH (8:1, v/v), the fr.4 (16.7 g) was separated to give 4 fr.s (Fr41-Fr44). Compounds 48(45 mg) and 49 (15 mg) were obtained from fr.1 (30.5 g) using n-hexane-CH3COCH3 (3:1,
v/v) by silica gel CC. Applying Sephadex LH-20 with MeOH-H2O (1:1, v/v), compound 50(37.5 mg) was isolated from the fr.42 (2.1 g), meanwhile compound 51 (200 mg) was
obtained from the fr.44 (2.3 g) by silica gel CC with CHCl3-MeOH-H2O (3:1:0.1, v/v/v).
Similarly, the fr.6 (20.5 g) was separated by Sephadex LH-20 with MeOH-H2O (2:1, v/v) toafford compound 52 (15.0 mg) and the fr.61 (150 mg). the fr.61 was further subjected to
HPLC [MeOH-H2O (7:3, v/v), UV 210 nm, 1.0 mL/min] to yield compounds 53 (5.4 mg), 55(4.2 mg) and 56 (3.2 mg). From the fr.7 (30.1 g), the purified powders were obtained as asingle compound 54 (240 mg) by washing with MeOH.
3.2.2. Chemical and physical data of isolated compounds
Compound 48: β-Sitosterol116

White needles, EI-MS (+): m/z 414 [M]+; C29H50O; 1H-NMR (500 MHz, CDCl3): 5.36
(1H, d, 5.5 Hz, H-6), 3.51 (1H, m, H-3), 1.01 (3H, s, H-29), 0.93 (3H, d, 8.0, H-19), 0.85 (3H,d, 8.5 Hz, H-24), 0.83 (3H, d, 9.0 Hz, H-26), 0.82 (3H, d, 9.0 Hz, H-27), 0.68 (3H, d, 9.5 Hz,
H-18); 13C-NMR (125 MHz, CDCl3): 140.8 (s, C-5), 121.7 (d, C-6), 71.8 (d, C-3), 56.8 (d, C14), 56.1 (d, C-17), 50.1 (d, C-9), 45.8 (d, C-22), 42.4 (s, C-13), 42.3 (t, C-4), 39.8 (t, C-12),37.3 (t, C-1), 36.5 (s, C-10), 36.1 (d, C-18), 34.0 (t, C-20), 31.9 (t, C-7) 31.9 (d, C-8), 31.7 (t,C-2), 29.1 (d, C-25), 28.3 (t, C-16), 26.1 (d, C-21), 24.3 (t, C-15), 23.1 (t, C-23), 21.1 (t, C11), 19.8 (q, C-26), 19.4 (q, C-27), 19.0 (q, C-19), 18.8 (q, C-28), 12.0 (q, C-24), 11.8 (q, C29).
Compound 49: Stigmasterol

White needles, EI-MS (+): m/z 412 [M]+; C29H48O; 1H-NMR (500 MHz, CDCl3): 5.36
(1H, d, 5.5 Hz, H-6), 5.14 (1H, dd, 9.0, 15.5 Hz, H-21), 5.01 (1H, dd, 9.0, 15.5 Hz, H-20),
3.51 (1H, m, H-3), 1.01 (3H, s, H-29), 0.93 (3H, d, 8.0, H-19), 0.85 (3H, d, 8.5 Hz, H-24),
0.83 (3H, d, 9.0 Hz, H-26), 0.82 (3H, d, 9.0 Hz, H-27), 0.68 (3H, d, 9.5 Hz, H-18); 13C-NMR(125 MHz, CDCl3): 140.8 (s, C-5), 138.3 (d, C-20), 129.2 (d, C-21), 121.7 (d, C-6), 71.8 (d,C-3), 56.8 (d, C-14), 56.1 (d, C-17), 50.1 (d, C-9), 45.8 (d, C-22), 42.4 (s, C-13), 42.3 (t, C-4),
39.8 (t, C-12), 37.3 (t, C-1), 36.5 (s, C-10), 36.1 (d, C-18), 31.9 (t, C-7) 31.9 (d, C-8), 31.7 (t,C-2), 29.1 (d, C-25), 28.3 (t, C-16), 24.3 (t, C-15), 23.1 (t, C-23), 21.1 (t, C-11), 19.8 (q, C26), 19.4 (q, C-27), 19.0 (q, C-19), 18.8 (q, C-28), 12.0 (q, C-24), 11.8 (q, C-29).
117

Compound 50: (+)-Catechin-3-O-(3,4-dihydroxybenzoyl)

Yellow amorphous powder, EI-MS (+): m/z 426 [M]+; C22H18O9; 1H-NMR (500 MHz,
CD3OD): 7.42 (1H, d, 2.0 Hz, H-2''), 7.39 (1H, dd, 8.5, 2.0 Hz, H-6''), 6.96 (1H, d, 2.0 Hz, H2'), 6.78 (1H, dd, 8.5, 2.0 Hz, H-6'), 6.76 (1H, d, 8.5 Hz, H-5''), 6.76 (1H, d, 8.5 Hz, H-5'),
5.93 (1H, d, 2.5 Hz, H-6), 5.90 (1H, d, 2.5 Hz, H-8), 4.80 (1H, s, H-2), 4.17 (1H, m, H-3),
2.86 (1H, dd, 17.0, 4.5 Hz, H-2a), 2.72 (1H, dd, 17.0, 3.0 Hz, H-2b); 13C-NMR (125 MHz,
CD3OD): 170.9 (s, CO), 156.6 (s, C-7), 156.3 (s, C-8a), 156.0 (s, C-5), 149.1 (s, C-4''), 144.6(s, C-3''), 144.4 (s, C-3'), 144.3 (s, C-3'), 130.9 (s, C-1'), 124.3 (s, C-1''), 122.2 (d, C-6''),118.0 (d, C-6'), 116.4 (d, C-2''), 114.5 (d, C-5''), 114.2 (d, C-5'), 113.9 (d, C-2'), 98.7 (s, C-4a),98.0 (d, C-6), 94.5 (d, C-8), 78.5 (d, C-2), 66.1 (d, C-3), 27.9 (t, C-4).
Compound 51: Quecertin

Yellow amorphous powder, EI-MS (+): m/z 302 [M]+; C15H10O7; 1H-NMR (500 MHz,
CD3OD): 7.69 (1H, d, 2.0 Hz, H-2'), 7.60 (1H, dd, 8.5, 2.0 Hz, H-6'), 6.83 (1H, dd, 8.5 Hz, H5'), 6.34 (1H, d, 2.0 Hz, H-8), 6.13 (1H, d, 2.0 Hz, H-6); 13C-NMR (125 MHz, CD3OD):

118

177.1 (s, C-4), 165.3 (s, C-7), 162.0 (s, C-5), 157.4 (s, C-8a), 149.0 (s, C-4'), 148.1 (s, C-2),
146.3 (s, C-3'), 137.0 (s, C-3), 123.2 (s, C-1'), 121.2 (d, C-6'), 116.9 (d, C-5'), 116.3 (d, C-2'),
104.2 (s, C-4a), 99.5 (d, C-6), 94.6 (d, C-8).
Compound 52: Quercitrin

Yellow amorphous powder, EI-MS (+): m/z 448 [M]+; C21H20O11; 1H-NMR (500 MHz,
CD3OD): 7.31 (1H, d, 1.5 Hz, H-2'), 7.30 (1H, dd, 8.0, 1.5 Hz, H-6'), 6.90 (1H, dd, 8.0 Hz, H5'), 6.36 (1H, s, H-6), 6.19 (1H, s, H-8), 5.34 (1H, d, 1.5 Hz, H-1''), 4.21 (1H, brd, 1.5 Hz, H2''), 3.75 (1H, dd, 9.0, 3.0 Hz, H-3''), 3.41 (1H, m, H-5''), 3.34 (1H, brd, 9.0 Hz, H-4''), 0.93
(3H, d, 6.0 Hz, 5''-CH3); 13C-NMR (125 MHz, CD3OD): 178.2 (s, C-4), 164.5 (s, C-7), 161.8(s, C-5), 157.9 (s, C-8a), 157.1 (s, C-2), 148.4 (s, C-4'), 145.0 (s, C-3'), 134.8 (s, C-3), 121.6(s, C-1'), 121.5 (d, C-6'), 115.5 (d, C-5'), 115.0 (d, C-2'), 104.5 (s, C-4a), 102.1 (d, C-1''), 98.4
(d, C-6), 93.3 (d, C-8), 71.9 (d, C-4''), 70.7 (d, C-3''), 70.6 (d, C-2''), 70.5 (d, C-5''), 16.3 (q,5''-CH3).
Compound 53: Hyperin (Quercetin-3-O-β-D-galactopyranoside)119

Yellow amorphous powder, EI-MS (+): m/z 464 [M]+; C21H20O12; 1H-NMR (500 MHz,
CD3OD): 7.83 (1H, s, H-2'), 7.58 (1H, d, 8.5 Hz, H-6'), 6.85 (1H, d, 8.5 Hz, H-5'), 6.39 (1H, s,H-6), 6.20 (1H, s, H-8), 5.15 (1H, d, 8.5 Hz, H-1''), 3.84 (1H, d, 2.5 Hz, H-4''), 3.82 (1H, dd,
5.0, 9.5 Hz, H-2''), 3.64 (1H, dd, 6.0, 10.0 Hz, H-6''a), 3.55 (1H, dd, 3.5, 10.0 Hz, H-6''b),
3.53 (1H, m, H-3''), 3.44 (1H, m, H-5''); 13C-NMR (125 MHz, CD3OD): 178.0 (s, C-4), 164.7(s, C-7), 161.6 (s, C-5), 157.4 (s, C-8a), 157.1 (s, C-2), 148.6 (s, C-4'), 144.0 (s, C-3'), 134.4(s, C-3), 121.5 (d, C-1'), 121.4 (s, C-6'), 116.4 (d, C-5'), 115.7 (d, C-2'), 104.0 (s, C-4a), 102.1(d, C-1''), 98.5 (d, C-6), 93.3 (d, C-8), 75.8 (d, C-5''), 73.7 (d, C-3''), 71.8 (d, C-2''), 68.6 (d, C4''), 60.5 (t, C-6'').
Compound 54: Mangiferin

White amorphous powder, EI-MS (+): m/z 422 [M]+; C19H18O11; 1H-NMR (500 MHz,
CD3OD, δ ppm): 7.43 (1H, s, H-8), 6.80 (1H, s, H-5), 6.35 (1H, s, H-4), 4.89 (1H, d, 10 Hz,H-1'), 4.17 (1H, t, 5.5 Hz, H-2'), 3.86 (1H, dd, 12.5, 2.5 Hz, H-6'a), 3.73 (1H, dd, 12.5, 5.5 Hz,
H-6'b), 3.46 (2H, m, H-3', H-4'), 3.41 (1H, m, H-5'); 13C-NMR (125 MHz, CD3OD, δ ppm):
180.1 (s, C-9), 163.8 (s, C-3), 161.9 (s, C-1), 157.4 (s, C-4a), 154.0 (s, C-6), 151.7 (s, C-4b),
143.6 (s, C-7), 112.5 (s, C-8a), 107.9 (d, C-8), 106.1 (s, C-2), 102.2 (d, C-5), 101.9 (s, C-8b),93.5 (d, C-4), 81.3 (d, C-5'), 78.8 (d, C-3'), 73.9 (d, C-1'), 71.2 (d, C-2'), 70.5 (d, C-4'), 61.5 (t,C-6').

120

Compound 55: Afzelin

Yellow amorphous powder, EI-MS (+): m/z 471 [M]+; C21H20O10; 1H-NMR (500 MHz,
CD3OD): 7.72 (2H, d, 9.0 Hz, H-2', H-6'), 6.89 (2H, d, 9.0 Hz, H-3', H-5'), 6.33 (1H, brs, H-8),
6.16 (1H, brs, H-6), 5.33 (1H, brs, H-1''), 3.68 (1H, m, H-2''), 3.44 (1H, brs, H-3''), 3.16 (1H,
brs, H-4''), 3.12 (1H, m, H-5''), 0.87 (3H, d, 5.5 Hz, 5''-CH3); 13C-NMR (125 MHz, CD3OD, δppm): 178.2 (s, C-4), 164.8 (s, C-7), 161.0 (s, C-5), 160.3 (s, C-4'), 157.9 (s, C-8a), 157.1 (s,C-2), 134.8 (s, C-3), 130.5 (d, C-2', C-6'), 121.2 (s, C-1'), 115.1 (d, C-3', C-5'), 102.3 (s, C-4a),102.1 (d, C-1''), 98.5 (d, C-6), 93.4 (d, C-8), 71.8 (d, C-4''), 70.7 (d, C-3''), 70.6 (d, C-2''), 70.5
(d, C-5''), 16.2 (q, 5''-CH3).
Compound 56: Isoquercetin (Quercetin-3-O-β-D-glucopyranoside)

Yellow amorphous powder, EI-MS (+): m/z 464 [M]+; C21H20O12; 1H-NMR (500 MHz,
CD3OD): 7.70 (1H, s, H-2'), 7.58 (1H, d, 8.5 Hz, H-6'), 6.85 (1H, d, 8.5 Hz, H-5'), 6.39 (1H, s,H-6), 6.20 (1H, s, H-8), 5.24 (1H, d, 8.5 Hz, H-1''), 3.71 (1H, m, H-6''a), 3.59 (1H, m, H-6''b),
3.49 (1H, m, H-3''), 3.44 (1H, m, H-2''), 3.35 (1H, overlap, H-4''), 3.23 (1H, m, H-5''); 13CNMR (125 MHz, CD3OD): 178.3 (s, C-4), 164.7 (s, C-7), 161.6 (s, C-5), 157.4 (s, C-8a),121157.2 (s, C-2), 148.7 (s, C-4'), 144.4 (s, C-3'), 134.4 (s, C-3), 121.7 (d, C-1'), 121.5 (s, C-6'),
116.4 (d, C-5'), 114.7 (d, C-2'), 104.0 (s, C-4a), 102.9 (d, C-1''), 98.5 (d, C-6), 93.3 (d, C-8),
75.8 (d, C-5''), 73.7 (d, C-3''), 71.8 (d, C-2''), 68.6 (d, C-4''), 61.1 (t, C-6'').

122

Chapter 4 Biological activities
4.1. Plant materials
The studied materials include the various extracts, fractions, and isolated compoundsof D. tonkinensis species. The dried powdered leaf, stem bark, and root, 2.0 kg of each partwas administered using methanol (10.0 L) for 4h in a Soxhlet extractor, to produce the crude
extracts (300.0 g, DB1), (198.5 g, DB3), and (200.0 g, DB6), respectively. Similar to theprocedure for heartwood, the powders after methanol extraction were then subjected todecoction in hot water (4.0 L) for 6h to afford the extracts leaf (25.8 g, DB2), stem bark (20.4
g, DB4), and root (19.7 g, DB9). Furthermore, the crude extract of the root (DB5) was
obtained using ethanol (10.0 L) for 4h in a Soxhlet extractor, was resuspended in hot
methanol-water (1:1, v/v) and then was successively partitioned with dichloromethane andethyl acetate to yield the corresponding fractions dichloromethane (DB7), and ethyl acetate(DB8). Besides that, the experimental crude raws also contained the hot water decoction
extract of powers after methanol extraction DB10, and the chloroform fraction of heartwood
DB11. They were mentioned above.
4.2. Anti-microbacterial experiments
4.2.1. The first model
P. gingivalis (ATCC33277) was anaerobically incubated at 37oC to an optical densityof 1.0 at 620 nm. Precultured P. gingivalis was anaerobically incubated with both the extracts
DB1-DB11 at concentration of 1.0 mg/mL, the isolated compounds 1-21 and a mixture ofcompounds 2+15 at concentration of 0.1 mg/mL and in 200 µL of Gifu anaerobic medium(GAM, Nissui, Tokyo, Japan) at 37 oC for 100 h in a 96-well plate (BD Falcon, NJ, USA).
The isolated compounds and the extracts of D. tonkinensis were dissolved in DMSO (Nacalai123Tesque, Kyoto, Japan). The degree of turbidity in the broth culture was measured at anabsorbance of 600 nm using a microplate reader (Thermo Scientific, MA, USA). Similarly,
the remainning bacteria were incubated at 37oC to an optical density of 1.0 at 620 nm.
Precultured Bacillus cereus (ATCC27522), Escherichia coli (JM109), Staphylococcus aureus(ATCC25923), Staphylococcus epidermidis (ATCC14990), Streptococcus pneumonia(ATCC49619),

Streptococcus

pyogenes((ATCC12344),

Vibrioparahaemolyticus(RIMD2210010), Streptococcus mutans (MT8148), Streptococcus mitis (ATCC903), andStreptococcus sobrinus (MT10186) were incubated with both the extracts DB1-DB11 and theisolated compounds 1-21 and a mixture of compounds 2+15 at concentration 0.1 mg/mL in
200 µL of brain heart infusion medium (BHI, Gibco) at 37 oC for 24 h in a 96-well plate (BDFalcon, NJ, USA). The isolated compounds and the extracts were dissolved in DMSO(Nacalai Tesque, Kyoto, Japan). The degree of turbidity in the broth culture was measured at
an absorbance of 600 nm using a microplate reader (Thermo Scientific, MA, USA).
Ampicillin sodium salt and DMSO were used as the positive, and negative controls,
respectively.
4.2.2. The second model
The antimicrobial assays were performed as described by Vanden and Vlietlinck andXiong et al. [93,94]. Briefly, heart wood methanol extract, and its chloroform fraction,
compounds 4, 8, 11, and 21 were diluted with DMSO 10%. The in vitro antimicrobial
experiments were carried out by the 96-well microtiter plate. The plates were then incubatedat 37oC in 24 hours with bacterial strains and 30oC in 48 hours with fungal strains. The result
was shown by MIC values (Minimum Inhibitory Concentration).

124

4.3. Antioxidant activity
Free radical scavenging activity of different extracts of several isolated compounds
were measured by 1,1-diphenyl-2-picryl hydrazyl (DPPH). In brief, 0.1 mM solution of
DPPH in ethanol was prepared. This solution (180 µL) was added to 20 µL of different
samples in ethanol at different concentration (100, 50, 10, 5, 1 µg/mL). The mixture wascarried out by 96-well plate at room temp for 20 min. Then, absorbance was measured at 517
nm by using spectrophotometer (UV-VIS Shimadzu). The percent DPPH scavenging effectwas calculated by using following equation: DPPH scavenging effect (%) or percent
inhibition = [(AO - A1)/ AO] × 100. Where AO was the absorbance of control reaction and A1
was the absorbance in presence of test or standard sample.
4.4. Enzyme active screenings
4.4.1. Serine protease enzyme activities
The aqueous decoction extracts were diluted by deionized water whereas DMSO
(dimethyl sulfoxide) was used for the remaining samples. 80-µL aliquots of the test samples(extracts: 3.125, 31.25, and 312.5 ng/mL, compounds: 1.25, 12.5, and 125 µM) with 12.5 mM
Tris-HCl (pH 8.0) were mixed with 10-µL aliquots of serine proteases (trypsin(CALBIOCHEM): 2.5 ng/mL, chymotrypsin (CALBIOCHEM): 50 µg/mL, and elastase(ELASTIN PRODUCTS CO. INC.): 25 µg/mL) in 96-well microplates and incubated at room
temperature for 20 min. After preincubation at 37oC for 10 min, the reaction was initiated byadding 10 µL of individual fluorogenic synthetic peptide substrates (for trypsin: Boc-Glu-AlaArg-methylcoumarin amide (MCA), chymotrypsin: Glt-Ala-Ala-Phe-MCA and elastase: SucAla-Pro-Ala-MCA (PEPTIDE INSTITUTE, INC), 100 µM) in a total volume of 100 µL per
well and incubated at 37oC for 15 min. Reactions were stopped by adding 50 µL of 30%
acetic acid. The amount of 7-amino-4-methylcoumarin liberated from the substrates

125determined fluorometrically with excitation and emission wavelengths at 370 and 460 nm,
respectively, with fluorescence spectrophotometer (Infinite 200Pro, TECAN, Tokyo, Japan).
Data present mean ± SD values of 12 independent determinations. Differences were
determined using student t-test and considered to be significant when p-value was less than
0.05.
4.4.2. Casein cleavage activity
25 ng/mL of trypsin and 25 ng/mL of extracts DB1-DB2, DB4-DB6, and DB10 or 10µM of compounds 6, and 17 were mixed and incubated at room temperature for 30 min. 0.8
mg/mL of casein was added and incubated at 37

for 3hrs. Samples were separated by poly-acrylamide gel electrophoresis (SDS-PAGE) in 12% tris-glycine gel under reducing condition
using the method of Laemmli [95] and the proteins were revealed by silver stain according to
the manufacture’s instructions (Silver Stain Kit; Wako Pure Chemical Industries Ltd., Tokyo,
Japan).

126

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Aknowledgements

I myself express my sincere gratitude to Japan Society for the Promotion of Scienceorganization-JSPS RONPAKU scholarship 2017 programme, for supporting finance.
I offer my deepest gratitude to my supervisor Prof. Dr. Yoshiyasu Fukuyama, for accepting
me as a member of his group, for providing me helpful working facilities, for his interest in
my work as well as his willingness to proofread my thesis. I am also thankful to Assoc. Prof.
Dr. Nguyen Manh Cuong for providing the herbal raw materials.
I would like to thank Assoc. Prof. Dr. Miwa Kubo and Dr. Kenichi Harada for their
willingness to help me when I lived in Tokushima. My sincere thanks also go to Dr. Yasuko
Okamoto for mass spectral measurements.
I would especially like to thank Professor Masataka Oda, Professor Midori Suenaga,
Professor Yoiichi Matsunaga and their partners for enthusiastically helping me in biologicalexperments.
I thank my labmates for their kind help and enjoyable time we had together. I neverforget our nice memories. Particlularly, I highly appreciate the undergraduate students, Mr.
Tsuyoshi Yamamoto and Mrs. Mari Kamiji, who helped me to do several chemical
experiments carefully.
I thank the rest of my thesis committee: Professor Yoshinori Asakawa, and Professor
Akemi Umeyama, for ther insightful reviews, comments and encouragements, but also for
hard question which incented me to widen my research from various perspectives.

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Last but not the least, I would like to send my great thank to my family, relatives andfriends for understanding, encouraging and supporting me spiritually throughout my life.
With the most belief, I express my deepest gratitude to God for all blessings.

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Publications based on this dissertation
1. Ninh The Son, Mari Kamiji, Tran Thu Huong, Miwa Kubo, Nguyen Manh Cuong,
and Yoshiyasu Fukuyama, Chemical constituents of Vietnamese plants Dalbergia
tonkinensis Prain and Cratoxylum formosum (Jack) Dyer in Hook and theirantioxidative activities. Medicinal Chemistry Research, 2019, 28, 1441-1447.
https://doi.org/10.1007/s00044-019-02383-9 (SCI-E).
2. Ninh The Son, Midori Suenaga, Yoiichi Matsunaga, Luu Van Chinh, Miwa Kubo,
Kenichi Harada, Nguyen Manh Cuong, and Yoshiyasu Fukuyama, Serine Protease
Inhibitors and Activators from Dalbergia tonkinensis species: Structural and Bioactive
Highlights. Journal of Natural Medicines, 2019, https://doi.org/10.1007/s1141 8-01901347-y. (SCI-E).
3. Ninh The Son, Tsuyoshi Yamamoto, Yoshiyasu Fukuyama, Chemotaxonomic aspectsof the constituents of the plant Dalbergia tonkinensis. Biochemical Systematics andEcology, 2018, 78, 98-101 (SCI).
4. Ninh The Son, Masataka Oda, Naoki Hayashi, Daiki Yamaguchi, Yu Kawagishi,
Fumi Takahashi, Kenichi Harada, Nguyen Manh Cuong, and Yoshiyasu Fukuyama,
Antimicrobial activity of the constituents of Dalbergia tonkinensis and structuralbioactive highlights. Natural Product Communications, 2018, 13, 157–161 (SCI-E).
5. Ninh The Son, Kenichi Harada, Nguyen Manh Cuong, Yoshiyasu Fukuyama, Two
new carboxyethylflavanones from the heartwood of Dalbergia tonkinensis and theirantimicrobial activities. Natural Product Communications, 2017, 12, 1721–1723(SCI-E).

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6. Ninh The Son, Yoshiyasu Fukuyama, Nguyen Manh Cuong, chemical constituent
from heartwood of Dalbergia tonkinensis. Chemistry of Natural Compounds, in press(SCI).

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