Summary of Curriculum Dissertation of Chemistry

MINISTRY OF EDUCATION AND VIETNAM ACADEMY OF TRAINING SCIENCE AND TECHNOLOGY

GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY ------

NGU TRUONG NHAN

STUDY ON CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF DALBERGIA TONKINENSIS PRAIN IN VIETNAM

Major : Chemistry of natural compounds Code : 9 44 01 17

SUMMARY OF CURRICULUM DISSERTATION OF CHEMISTRY

HA NOI - 2019

This research has been done at Graduate University of Science and Technology – Vietnam Academy of Science and Technology

Supervisor 1: Assoc. Prof. Nguyen Manh Cuong Supervisor 2: Dr. Do Huu Nghi

Reviewer 1: …

Reviewer 2: …

Reviewer 3: ….

The dissertation will be defensed before the Evaluation Council of the doctoral dissertation at the Academy, meeting at the Academy of Science and Technology - Vietnam Academy of Science and Technology at ... hours ... ', date ... month… 201 …...

The dissertation can be found at: - Library of the Graduate University of Science and Technology - National Library of Vietnam

INTRODUCTION 1. Urgency of the dissertation Vietnam has an area of about 330000 km2, stretching 1650 km through 15 latitudes, has tropical climate with two hot and humid seasons, relatively high humidity (over 80%), abundant annual rainfall (middle 1200-2800 mm). With such natural environment, a diverse and abundant flora in species has been, green around year, and has many uses to serve human life such as food, making processing interior and exterior decorations, landscaping, medicine,... According to statistics, Vietnam's vascular plants has more than 10,000 species, of which about 3,200 species of plants are used in traditional medicine to make medicine. The Dalbergia genus (Fabaceae) contains more than 300 recognised species that are widely distributed in tropical and sub-tropical regions. The wood of many species of Dalbergia is commonly called ‘rosewood’ due to its dark red colour and beautiful grain, and has been used for hundreds of years for high quality furniture and musical instruments. The Dalbergia tonkinensis Prain species (commonly called ‘Sua Do’ due to the heartwood of perennial plant is red-brown to red-black), is is a precious timber species with high economic value. It has been listed in Vietnam’s Red Book (2007), in IUCN Red List Categories (2015) as Vulnerable (VU), and in Government Decree No. 32/2006 / ND-CP under group IA. Phytochemical studies of the genus Dalbergia have indicated that its chemical constituents are various flavonoids (including flavones, isoflavones, neoflavones, flavanones, isoflavanones, flavanes, isoflavanes, neoflavans, flavonols, flavanonols, and isoflavanonols), 4-aryl-coumarins, phenols, sesquiterpenes, phytosterols, as well as coumestan and coumaronochromone derivatives. Dalbergia species were previously found to possess a wide spectrum of biological activities, including antimicrobial, antidiarrheal, anti-cancer, spermicidal, anti- inflammatory, antipyretic, analgesic, and cardiovascular activity. The heartwood of Dalbergia species are used in traditional Chinese medicines for treating cardiovascular diseases including heart failure, myocarditis, myocardial infarction, myocardial fibrosis, and coronary failure.

So far, the Dalbergia tonkinensis Prain in Vietnam has not been studied fully and comprehensively about botany, chemical composition and biological effects. Phytochemical studies, there is only a preliminary study in the country. However, the part of studied plant was not described. From the above scientific and practical bases, we chose the topic ‘‘Study on chemical constituents and biological activities of Dalbergia tonkinensis Prain in Vietnam’’. 2. Research purposes of the dissertation Studying on Dalbergia tonkinensis Prain systematicly and fully about: plants (microsurgery, gene sequences), chemical compositions and biological activity.

3. Main research contents of the dissertation In order to achieve the above objectives, the thesis has implemented the following contents: - Research on plants (microsurgery of leaves, stem, core powder and gene sequences) of Dalbergia tonkinensis Prain. - Isolate and determine the structure of compounds from Dalbergia tonkinensis Prain (heartwood) oriented biological effect (inhibiting enzyme a-glucosidase). - Surveying some biological activities: antibacterial and inhibition of a- glucosidase enzyme of total extraction, high extraction and some isolated clean compounds from Dalbergia tonkinensis Prain in Vietnam.

MAIN CONTENTS OF THE DISSERTATION CHAPTER 1. OVERVIEW The document overview is a collection of national and international studies on issues: 1.1. Overview of the legume Fabaceae and Dalbergia genus 1.1.1. Overview of the legume Fabaceae 1.1.1.1. Overview of the Dalbergia genus 1.1.1.2. Overview of Dalbergia tonkinensis Prain 1.2. Studying on chemical compositions and biological activity of the Dalbergia genus in the world

CHAPTER 2: PRACTICAL TEST AND STUDYING METHODS

This section describes in detail the process of material samples, microsurgical studying methods and gene sequences, making of extraction parts, chromatographic separation, compounds isolation and bioactive testing methods.

2.1. Subject

Dalbergia tonkinensis Prain includes branches, leaves and brack that are collected in Buon Ma Thuot city, Dak Lak province. The scientific name of the tree was determined by Dr. Nguyen Quoc Binh, Vietnam Natural Museum, Vietnam Academy of Science and Technology. The specimen sample of plants are stored in the plant's specimen room, Institute of Ecology and Biological Resources and at the Bioactive Department, Institute of Natural Products Chemistry, sample symbols are C-561 and C-612.

After being collected, Sua samples are washed, removed skin, only get red cores and dried to isolate the substance. Then the sample is minced and soaked several times with MeOH at temperature room. After removing solvent, residue is fractionated with solvents with increasing polarity such as: n-hexane, chloroform or dichloromethane, ethyl acetate and water. 2.1. Subject The plant and heartwood of Dalbergia tonkinensis Prain (over ten-year old) was collected in Buon Ma Thuot city, Daklak province, Vietnam. The plant was identified by botanist Dr. Nguyen Quoc Binh, Vietnam National Museum of Nature, VAST, Hanoi, Vietnam. A voucher specimen (C-561) and (C-612) was deposited in Department of Bioactive Products, Institute of Natural Products Chemistry, VAST, Hanoi, Vietnam

Dried powdered heartwoods (3.7 kg) of Dalbergia tonkinensis Prain were extracted with hot methanol (5 x 7.0 L) under reflux, filtered, and then concentrated under decreased pressure giving a black crude methanol residue (120 g). The suspension of the methanol residue in hot methanol-water (1:1, v/v) was successively 3

partitioned with n-hexane, dichloromethane and ethyl acetate to give n-hexane (29.0 g, MH), dichloromethane (45.1 g, MD), ethyl acetate (11.1 g, ME) and water (12.0 g, MW) fractions, respectively. And then we describe the isolation of the compounds from the fractions of the methanol extract of Dalbergia tonkinensis Prain.

Heartwood (3,7 kg)

Soaked MeOH (5 x 7,0L)

Residue after Methanol attraction extracting (120 g, M)

Boiling water (4 x Allocated attraction 3,0L) n-hexane dichloromethane ethyl acetate water

W MH MD ME MW (4,2 g) (29,0 g) (45,1 g) (11,1 g) (12,0 g)

Figure 2.1. Processing sample and extracting from heartwood of Dalbergia tonkinensis Prain 2.2. Methodology

2.2.1. Microsurgical study: Performed at the Department of Plant - Hanoi University of Pharmacy. 2.2.2. Genetic sequence study: Performed at the Institute of Ecology and Biological Resources - Vietnam Academy of Science and Technology. 2.2.3. Method of separating compounds obtained from the extraction The methods have been used such as: Column chromatography was carried out on silica gel (Si 60 F254, 40–63 mesh, Merck, St. Louis, MO, USA), or reverse phase (ODS, YMC (30-50 μm)), column chromatography Dianion HP-20, Sephadex LH-20. All solvents were redistilled before use. Besides, using crystallization method to collect clean substances. Pre-coated thin layer chromatography (TLC) plates (Si 60 F254) were used for analytical purposes. Compounds were visualized under UV radiation (254, 365 nm) and by spraying plates with 10% H2SO4 followed by heating with a heat gun.

2.2.4. Methods of determining chemical structure of the compounds The general method for determining the chemical structure of substances is the combination of physical parameters with modern spectral methods including: 1H-NMR (500 MHz) and 13C-NMR (125 MHz) were measured on a Bruker Avance 500 MHz spectrometer. HR-ESI-MS was obtained from a Varian FT-MS spectrometer and MicroQ-TOF III (Bruker Daltonics, Ettlingen, Germany). IR spectroscopy was fulfilled on Nicolet Impact 410 spectrometer. UV was performed in spectroscopic V- 630 UV-VIS instrument. The CD data was obtained from a Chirascan spectrometer. 2.2.5. Methods of testing biological activity 2.2.5.1. Method of testing anti microorganism activity In vitro antimicrobial on some species (with a significant minimum inhibitory concentration [MIC] value: Bacillus subtillis, Staphylococcus aureus, Aspergillus niger, Fusarium oxyporum, Saccharomyces cerevisiae, Candida albicans. They performed at the Institute of Natural Products Chemistry - Vietnam Academy of Science and Technology 2.2.5.2. Method of testing enzyme -glucosidase inhibitory activity The inhibitory activity against α-glucosidases was done according to the assay described by Nguyen, et al. 2018.

Rice α-glucosidase (Type 4) was purchased from Sigma Aldrich (St. Louis City, MO, USA). Enzyme α-glucosidases from Saccharomyces cerevisiae, Bacillus stearothermophilus and from rice were provided by Sigma Aldrich, Singapore. Acarbose and p-nitrophenyl glucopyranoside (pNPG) were purchased from Sigma Aldrich, 3050 Spruce Street, St. Louis, MO, USA.

All these tests were performed at the Pharmacy Department, Tam Kang University, Taiwan. 2.2.6. Physical and chemical parameters and spectral data of isolated compounds This section shows details of the spectral data as well as the physical constants of 18 compounds were isolated from heartwood of Dalbergia tonkinenis Prain.

CHAPTER 3. RESULTS AND DISCUSSIONS

This chapter shows the study results of microsurgery, DNA, the isolation result and determination of chemical structure of compounds, antibacterial activity and inhibition of a-glucosidase enzyme. 3.1. Research results on microsurgery and gene sequences of Dalbergia tonkinenis Prain This section describes in detail the microscopic morphology results of leaves, stems and heartwood of Dalbergia tonkinensis Prain.

3.1.1. Results of microsurgery research We had conducted research on microscopic leaves, stems and core powder of Dalbergia tonkinensis Prain collected in Buon Ma Thuot. Its microcharacters were described to support morphological classification as well as supplement genetic database for this species. Some results are shown in the following figure:

Figure 3.1. Microscopy of leaves Figure 3.2. Microscopy of core powder 3.1.2. Research results on gene sequences In this study, we have identified the sequence of 3 chloroplast genome rbcL, rpoB and rpoC of 2 collected samples in Buon Ma Thuot City (C-561-L) and Hanoi (C-564-L) and compared the genetic distance of each genetic region with other 5 Dalbergia species that have announced their sequences on GenBank. The results showed that (Figure 3.3), the three regions of rbcL, rpoB and rpoC are suitable for the taxonomy study of species of the Sua genus (Dalbergia) with 99%,

98% and 97%. The sequence of three chloroplast genome rbcL, rpoB and rpoC of Dalbergia tonkinenis Prain in Vietnam has been registered on gene banks with the numbers of KY283103, KY287755 and KY287750 respectively.

0.016 0.014 0.012 0.01 0.008 0.006 0.004 0.002 0

rpoB rpoC rbcL

Figure 3.3. Comparison of the ability to distinguish between Dalbergia of three studied gene regions 3.2. Isolation results and determination of chemical structure of compounds from heartwood of Dalbergia tonkinensis Prain

This section details the isolation work, spectrum analysis and determination of chemical structure of Dalbergia tonkinensis Prain, including two new compounds and the rest are known compounds. 18 compounds from heartwood of Dalbergia tonkinensis Prain were isolated and determined their chemical structure. They include: 08 compounds from dichloromethane extraction, 07 compounds from ethyl acetate extraction, and 03 compounds from water extraction. The structure of 18 isolated compounds are in Table 3.1 below.

Table 3.1. Chemical structure of isolated compounds from the heartwood of Dalbergia tonkinensis Prain

Pinocembrin Naringenin

3'-hydoxy-2,4,5-trimethoxydalbergiquinol Medicarpin

Buteaspermanol Daltonkin A (2S)-8-carboxyethylpinocemprin (New)

Daltonkin B Dalbergin (2S)-2,6-dicarboxyethylnaringenin (New)

Liquiritigenin 7,3',5'-trihydroxyflavanone

Sativanone 3'-O-methylviolanone

Vestitone Calycosin

Formononetin 7,3',4'-trihydroxyaurone (Sulfuretin)

Isoliquiritigenin 4',7-dihydroxy-3-methoxyflavone

3.2.1. New compound daltonkin A Daltonkin A was isolated as a white amorphous solid. In negative high- resolution electrospray ionisation mass spectrometry (HR-ESI-MS), the quasi- molecular ion peak at m/z = 327.0868 [M – H]– suggested that the molecular formula

of compound daltonkin A is C18H16O6. Its infrared (IR) spectrum showed absorption bands at 3354 and 1701 cm-1, which can be attributed to hydroxyl and carbonyl groups, respectively. In addition to the phenyl rings A and B, a methylene carbon at δC 44.3

(C-3), an oxymethine carbon at δC 80.5 (C-2), and a carbonyl carbon at δC 197.5 (C-4) of ring C in the 13C-NMR spectrum indicated that compound daltonkin A is a flavanone. The 1H and 13C-NMR spectra of compound daltonkin A are similar to that of the flavanone pinocembrin isolated from Dalbergia odorifera, except for the replacement of the aromatic proton H-8 in pinocembrin by a carboxyethyl group

(CH2CH2COOH) (H-9 [δH 2.86, t, 8.0 Hz]/C-9 [δC 18.7], H-10 [δH 2.49, t, 8.0

Hz]/C-10 [δC 34.1], and C-11 [δC 177.7]) (Figure 3.43.6 and Table 3.2).

Figure 3.4. The 1H-NMR of compound daltonkin A

Figure 3.5. 13C-NMR of compound daltonkin A

Figure 3.6. DEPT of compound daltonkin A This was confirmed by cross-peaks between the protons H-9 and H-10 in correlation spectroscopy (COSY), as well as between protons H-9 and H-10, and the carbonyl carbon C-11 in the heteronuclear multiple-bond correlation (HMBC) spectrum (Figure 3.7-3.8).

Figure 3.7. COSY spectrum of Figure 3.8. HMBC spectrum of compound compound daltonkin A daltonkin A

Moreover, correlations between carboxyethyl proton H-9 and carbons C-7, C-8, and C-8a in the HMBC spectrum indicated that the position of the carboxyethyl group was at C-8 of ring A.

Table 3.2. 1H -NMR and 13C- NMR spectroscopic data for daltonkin A daltonkin A (*) Position

δH (ppm) (J in Hz) δC (ppm) 2 5.47, dd, 13.0; 3.0 80.5, d

2.80, dd, 17.0; 3.0, H-3eq 3 44.3, t 3.11, dd, 17.0; 13.0, H-3ax 4 197.5, s 4a 103.3, s 5 162.8, s 6 6.02, s 95.6, d 7 166.1, s 8 108.5, s 8a 162.8, s 9 2.86, t, 8.0 18.7, t 10 2.49, t, 8.0 34.1, t 11 177.7, s

9' 10' 11' 1' 140.5, s 2',6' 7.52, d, 7,5 127.3, d 4' 7.39, t, 7,5 129.6, d 3',5' 7.44, d, 7,5 129.7, d 1 13 (*) H-NMR (500 MHz, methanol-d4); C-NMR (125 MHz, methanol-d4) Flavanones with a 2S-configuration exhibit a positive Cotton effect at ~330 nm (n–* transition), and a negative Cotton effect at 270–290 nm (–* transition) in the CD spectrum. The CD (c 0,4, MeOH) curve of compound daltonkin A showed a

* positive Cotton effect at 330 + 2.10 (n– transition), and a negative Cotton effect at

* 288 – 10.43 (– transition); this suggested a 2S-configuration for compound daltonkin A.

Figure 3.9. CD spectrum of compound daltonkin A Based on these spectroscopic results, compound named daltonkin A was determined to be (2S)-8-carboxyethylpinocembrin, a new monocarboxyethylflavanone isolated from the heartwood of Dalbergia species.

Figure 3.10. Chemical structure of compound daltonkin A

Figure 3.11. Selected HMBC (H→C) and COSY (H→H) correlations of compound daltonkin A 3.2.2. New compound daltonkin B Daltonkin B was isolated as a white amorphous solid, with a suggested molecular formula of C21H20O9 according to a quasi-molecular ion peak at an m/z of 415.1014 [M – – H] (calculated for C21H19O9 415.1024) in negative HR-ESI-MS. The IR spectrum indicated bands at 3369 cm-1 (hydroxyl groups) and 1751 cm-1 (carbonyl groups). Similar to daltonkin A, daltonkin B has characteristic signals for flavanone compounds; 13

specifically, a methylene carbon at δC 44.0 (C-3), an oxymethine carbon at δC 80.3 (C- 13 2), and a carbonyl carbon at δC 198.6 (C-4) in the C-NMR spectrum. The 1H and 13C-NMR spectra of daltonkin B was similar to that of the flavanone naringenin isolated from Dalbergia odorifera, except for the replacement of two aromatic protons (H-8 and H-6) in compound naringenin by two carboxyethyl groups

(H-9 [δH 2.85, m]/C-9 [δC 18.8], H-10 [δH 2.55, m]/C-10 [δC 34.4], and C-11 [δC 179.1]) and (H-9' [δH 2.81, m]/C-9' [δC 19.4], H-10' [δH 2.51, m]/C-10' [δC 34.8], and C-11' [δC 179.2]) in daltonkin B (Figure 3.11-3.14 and Table 3.3)

Figure 3.12. HR-ESI-MS high resolution spetrum of compound daltonkin B

Figure 3.13. 1H-NMR spectrum of compound daltonkin B

Figure 3.14. 13C-NMR spectrum of compound daltonkin B Cross-peaks between H-9/H-10 and H-9'/H-10' in the COSY spectrum, as well as correlations between protons H-9 and H-10 to C-11, and H-9' and H-10' to C-11', further confirmed the existence of two carboxyethyl groups. Moreover, the correlations between proton H-9' to C-5/C-6 and C-7, as well as proton H-9 to C-7/C-8 and C-8a, were observed in the HMBC spectrum. This suggests that two carboxyethyl groups were located. Moreover, the correlations between proton H-9' to C-5/C-6 and C-7, as well as proton H-9 to C-7/C-8 and C-8a, were observed in the HMBC spectrum. This suggests that two carboxyethyl groups were located at C-6 and C-8 of ring A (Figure 3.15-3.16)

Figure 3.15. COSY spectrum of Figure 3.16. HMBC spectrum of compound daltonkin B compound daltonkin B

Table 3.3. 1H NMR and 13C NMR spectroscopic data for daltonkin B daltonkin B (*) Position δH (ppm) (J in Hz) δC (ppm) 2 5.39, dd, 13.0, 3,0 80.3, d

2.79, dd, 17.0, 3.0, H-3eq 3 44.0, t 3.13, dd, 17.0, 13.0, H-3ax 4 198.6, s 4a 103.4, s 5 159.8, s 6 109.0, s 7 163,9, s 8 108.3, s 8a 161.1, s 9 2.85, m 18.8, t 10 2.55, m 34.4, t 11 179.1, s

9' 2.81, m 19.4, t 10' 2.51, m 34.8, t 11' 179.2, s 1' 131.4, s 2',6' 7.36, d, 8.5 128.8, d 4' 158.9, s 3',5' 6.85, d, 8.5 116.4, d

1 13 (*) H-NMR (500 MHz, methanol-d4); C-NMR (125 MHz, methanol-d4) Similar to daltonkin A, daltonkin B was determined to be (2S). Thus, compound daltonkin B was determined to be (2S)-6,8-dicarboxyethylnaringenin, a new dicarboxyethylflavanone.

Figure 3.17. Chemical structure of compound daltonkin B 16

Figure 3.18. Selected HMBC and COSY correlations of daltonkin B 3.2. The result of testing biological activity 3.2.1. Antimicrobial activity of isolated compounds In vitro antimicrobial results are displayed in Table 2. Among the tested compounds, pinocembrin (3) exhibited inhibitory effects on filamentous fungi (with a significant minimum inhibitory concentration [MIC] value of 50 µg/mL), while compounds pinocembrin (3) and naringenin (4) showed moderate MIC values of 100 µg/mL against yeast and the Gram-positive bacterium Staphylococcus aureus. Compound 3'-hydroxy-2,4,5-trimethoxydalbergiquinol (5) failed to show any activity. Table 3.4. Antimicrobial activity of isolated compounds from (Dalbergia tonkinensis Prain)

Antimicrobial activity (MIC: µg/mL) N Gram-positive bacteria Filamentous fungi Yeast o Bacillus Staphylococcus Aspergillus Fusarium Saccharomyces Candida subtillis aureus niger oxyporum cerevisiae albicans 3 (-) 100 50 50 100 100 4 (-) 100 100 (-) (-) (-) 5 (-) (-) (-) (-) (-) (-)

(-): IC50 ≥ 200 µg/mL 3.2.2. The α-glucosidase inhibitory activity 3.2.2.1. New records of Dalbergia tonkinensis Prain extracts To determine which part used is the most active, the heartwood, trunk bark, and leaves of Dalbergia tonkinensis were collected and extracted with methanol; their activity was then tested. As shown in Figure 1A, all the parts used of Dalbergia tonkinensis show stronger activity (98–100%) than that of positive control (62%). These results highlighted the promising in vitro antidiabetic property of the extracts from the heartwood, trunk bark and leaves of Dalbergia tonkinensis. To clarify the 17

results, the activity was also expressed as IC50 value. The heartwood extract demonstrated the strongest activity among the Dalbergia tonkinensis parts used due to its smallest IC50 value of 0.17 mg/mL; therefore, it was used in conducting subsequent experiments, including stability tests and purification.

A B 1.6 100 1.25 IC50 1.4 mg/ 80 ( 1.2 mL) 1.0 60 0.78 0.8 0.57

aGI (%) aGI 40 Heartwood 0.6

Trunk bark (mg/mL) EC50 0.4 20 Leaves 0.17 Acarbose 0.2 0 0.0 0 1 2 3 4 5 Heartwood Bark Leaves Acarbose Concentration (mg/mL) Part used Figure 3.19. The inhibition α-glucosidase of different of Dalbergia tonkinensis Prain; aGIs: α-glucosidase inhibitors 3.2.2.2. Primary partitioned separation of methanol extract from heartwood of Dalbergia tonkinensis Prain The methanol extract of heartwood of Dalbergia tonkinensis (HDT) was successively partitioned with n-hexane, dichloromethane, ethyl acetate and water to obtain 04 fractions. The yield and activity of HDT and its fractions are recorded in Table 3.5. HDT-3 partitioned by ethyl acetate demonstrated the strongest activity due to its greatest inhibition (%) and lowest EC50 value of 95% and 0.069 mg/mL, respectively. Thus, this fraction was chosen for the isolation of active compounds. The water fraction (HDT-4) showed acceptable activity. Since it had the good profile of TLC separation, it was also considered for subsequent experiments of purification. Table 3.5. α-glucosidase inhibition of HDT and its fractions after partition Yield α-Glucosidase Inhibition Samples (g) IC50 (mg/mL) Inhibition (%) * HDT (crude MeOH extract) 60.3 0.172 ± 0.011 98 ± 3.2 HDT-1 (Hexane Fr.) 1.6 1.712 ± 0.210 73 ± 4.1 HDT-2 (Dichloromethane Fr.) 27.2 0.124 ± 0.003 90 ± 2.5 HDT-3 (Ethyl acetate Fr.) 11.1 0.069 ± 0.001 95 ± 3.7 HDT-4 (Water Fr.) 12.0 0.513 ± 0.051 82 ± 2.3 Acarbose (positive control) 1.357 ± 0.03 62 ± 1.8 *: the inhibition of acarbose and fractions were detected at their concentration range of 0.1–5 mg/mL; results are means ± SD of multi tests (n = 3). 3.2.2.3. Sub-fractionation of the extract, and identification of active compounds 18

The most active fraction (HDT-3) was separated by a silica open column to obtain 12 sub-fractions. The activity of these sub-fractions was tested and expressed as %. As shown in Figure 3.20, HDT-3.1 showed the best activity (98%) while the others demonstrated weak inhibition (2–36%) at the same tested concentration of 0.1 mg/mL. Further separation of HDT-3.1 was applied to the most active sub-fraction (HDT- 3.1.2), showing inhibition of 99% (Figure 3.20).

A B C

100 100 100

80 80 80

60 60 60

aGI (%) aGI (%) aGI 40 40 (%) aGI 40

20 20 20

0 0 0

3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 3.10 3.11 3.12 3.1.1 3.1.2 3.1.3 3.1.4 3.1.5 3.1.6 3.1.7 3.1.8 Sub-fractions of HDT-3 Components separated from HDT-3.1 Sub-fractions of HDT-4 D E

100 100

80 80

60 60

aGI (%) aGI

40 (%) aGI

20 Compound 1 (purified HDT-3.1.2) 20 Compound 2 (purified HDT-4.3.3) Acarbose (positive control) 0 0 0 1 2 3 4 5

4.3.1 4.3.2 4.3.3 4.3.4 4.3.5 4.3.6 Components separated from HDT-4.3 Concentration (mg/mL) Figure 3.20. Inhibitory activity (%) of sub-fractions and purified compounds; aGIs: α-glucosidase inhibitors 3.2.2.4. Specific inhibitory activity of the purified compounds Specific inhibition of the purified compounds was detected to characterize them as active antidiabetic drugs; a total of 04 enzymatic sources, including α-glucosidases from rat, yeast, bacterium, and rice were conducted to test the inhibition. The activity was expressed as IC50 value and presented in Table 3.6 Table 3.6. Specific inhibitory activity of sativanone, formononetin, and acarbose

Inhibition Expressed as IC50 (mg/mL) No. Enzyme Source Sativanone Formononetin Acarbose 1 Yeast α-glucosidase 0.23 ± 0.012 0.06 ± 0.002 1.321 ± 0.048 2 Rat α-glucosidase 0.37 ± 0.022 0.23 ± 0.037 0.121 ± 0.001 3 Bacterial α-glucosidase 0.07 ± 0.001 0.03 ± 0.002 0.001 ± 0.000 4 Rice α-glucosidase 0.81 ± 0.023 0.98 ± 0.029 0.031 ± 0.005 All tests were performed in triplicate; results are means ± SD of multi tests (n = 3)

In this study, both sativanone and formononetin demonstrated much stronger inhibition against yeast α-glucosidase than did acarbose, and showed their effects on 19

rat α-glucosidase comparable to that of acarbose. The inhibitory activity of sativanone and formononetin against rat α-glucosidase was newly investigated in the current study, based on our current references review. Therefore, they were determined as new mammalian.

3.2.2.5. The rat α-glucosidase inhibitory activity of crude extracts, fractions, sub- fractions and isolated compounds from Dalbergia tonkinensis Prain All the parts used extracts (the heartwood, trunk bark, and leaves) of Dalbergia tonkinensis Prain, the fractions, sub-fractions, and purified compounds separated from HDT, as well as acarbose, were tested their inhibition against α-glucosidase from rat.

The results were expressed as IC50 and max inhibition then summarized in Table 3.7. Table 3.7. Rat α-glucosidase inhibitory activity of crude extracts, fractions, sub- fractions, and isolated compounds from Dalbergia tonkinensis Prain extract Rat α-glucosidase inhibitory activity Components IC50 (mg/mL) Max Inhibition (%) Heartwood Extract (HDT) 1.72 ± 0.116b 61 ± 3.46e Trunk bark extract 2.91 ± 0.289a 51 ± 4.62f Leaves extract 2.78 ± 0.173a 54 ± 4.60f HDT-3 1.31 ± 0.057c 68 ± 5.77d HDT-3.1 1.13 ± 0.058cd 75 ± 5.20c HDT-3.1.2 0.92 ± 0.023d 77 ± 5.18c Sativanone 0.357 ± 0.006fg 91 ± 4.61a HDT-4 1.43 ± 0.115bc 67 ± 2.89d HDT-4.3 0.87 ± 0.035de 78 ± 4.61c HDT-4.3.3 0.55 ± 0.012ef 84 ± 4.62b Formononetin 0.251 ± 0.006fg 94 ± 5.11a Acarbose 0.119 ± 0.005g 93 ± 2.5a Coefficient of variation 12.50026 1.853018 The samples and acarbose were tested at their concentration range of 0.05–3.2 mg/mL;

results are means ± SD of multi tests (n = 3); the means of IC50 and max inhibition values Among the 3 parts used extracts, HDT also demonstrated strongest inhibitory activity due to its smallest IC50 and greatest inhibition values of 1.72 µg/mL, and 61%, respectively. The activity was gradually increased via steps of partial purification. The 02 purified compounds showed their much stronger activity than that of the crude sample (HDT) and its fractions (HDT-3, HDT-4), and sub-fractions (HDT-3.1, HDT- 3.1.2, HDT-4.3, and HDT-4.3.3).

CONCLUSION AND SUGGESTION

 The results of plant research -The microsurgical characteristics of leaves, stems and heartwood powder of the Dalbergia tonkinensis Prain have been described in detail, contributing to standardize this precious wood species. - Sequenced 3 chloroplast gene rbcL, rpoB and rpoC sequences of 02 Dalbergia species samples and compared the genetic distance of each gene regions with 5 other Dalbergia species that have been sequenced on GenBank. - 03 chloroplast gene rbcL, rpoB and rpoC are capable of distinguishing the species of the Sua (Dalbergia) with rate 99%, 98% and 97% respectively. - The sequence of three regions rbcL, rpoB and rpoC of (Dalbergia tonkinensis Prain) in Vietnam has been registered on gene banks in the numbers of KY283103, KY287755 and KY287750 respectively.  The chemical composition - The first time from heartwood of Dalbergia tonkinensis Prain has isolated and determined the chemical structure of 18 flavonoid compounds: pinocembrin, naringenin, 3'-hydroxy-2,4,5-trimethoxydalbergiquinol, medicarpin, buteaspermanol, daltonkin A [(2S)- 8-carboxyethylpinocembrin], daltonkin B [(2S)-2,6-dicarboxyethylnaringenin], dalbergin, isoliquiritigenin, 7,3',5'-trihydroxyflavanone, vestitone, calycosin, 4',7-dihydroxy-3- methoxyflavone, liquiritigenin, sativanone, 3'-O-methylviolanone, 7,3',4'-trihydroxyaurone and formononetin. - 02 compounds daltonkin A and daltonkin B are new mono- and di- carboxyethylflavanones. 3. Biological effects of extracts and compounds were isolated 3.1. In vitro antimicrobial: pinocembrin compound exhibits inhibitory activity on mildew (with a minimum inhibitory concentration [MIC] of 50 µg/mL), whereas for yeast and Gram-positive bacteria staphylococcus aureus, pinocembrin and naringenin showed moderate inhibition with MIC value of 100 µg/mL. 3.2. Inhibition effect of enzyme α-glucosidase

- Methanol extraction from heartwood, cover and leaves of Dalbergia tonkinensis Prain has been evaluated for inhibiting enzyme a-glucosidase activity, in which methanol extraction of heartwood has the strongest effect

with IC50value of 0.17 mg/mL, compared with positive control is acabose

(IC50 of 1.21 mg/mL). - Isolating oriented inhibition of enzyme α-glucosidase has identified two compounds of sativanone and formononetin have more potent than acarbose

(positive control) with inhibition rate and IC50 respective values of sativanone (90%, 0.23 mg/mL) and formononetin (98%, 0.06 mg/mL) compared to acarbose (62%, 1,321 mg/mL). - Methanol extraction from heartwood (HDT) has effectted inhibition enzyme

α-glucosidase on mice stronger than cover and leaves with IC50 value of 1.72 mg / mL. - Sativanone and formononetin have effectted to inhibit enzyme α-glucosidase

of mouse with inhibitory rate and IC50 values respectively (91%, 0.357 mg / mL), (94%, 0.251 mg / mL).

SUGGESTION

The results of our study on Sua (Dalbergia tonkinensis Prain), have led to the isolation and identification a lot of interesting structural compounds and strong inhibitory activity on enzymes α-glucosidase together with the abundant data in plants (microsurgery and DNA). - Studying chemical composition and biological effects of wood parts, and root of Dalbergia tonkinensis Prain to create complete data and system of this species in Vietnam.

- Studying to develop effect of enzyme α-glucosidase inhibitory and treat diabetes of two sativanone and formononetin compounds and methanol

extraction of heartwood.

NEW CONTRIBUTION OF THE THESIS

Chemical constituent from heartwood of Dalbergia tonkinensis Prain grown in Vietnam and their biological activities were reported for the first time. - 18 flavonoids: pinocembrin (DT.01), naringenin (DT.02), 3'-hydroxy-2,4,5- trimethoxydalbergiquinol (DT.03), medicarpin (DT.04), buteaspermanol (DT.05), daltonkin A [(2S)-8-carboxyethylpinocembrin] (DT.06), daltonkin B [(2S)-2,6- dicarboxyethylnaringenin] (DT.07), dalbergin (DT.08), isoliquiritigenin (DT.09), 7,3',5'- trihydroxyflavanone (DT.10), vestitone (DT.11), calycosin (DT.12), 4',7-dihydroxy-3- methoxyflavone (DT.13), liquiritigenin (DT.14), sativanone (DT.15), 3'-O- methylviolanone (DT.16), 7,3',4'-trihydroxyaurone (DT.17) and formononetin (DT.18) were isolated and elucidated. - 02 compounds: daltonkin A (DT.06) and daltonkin B (DT.07) are new mono- and di-carboxyethylflavanones, respectively. - Methanol extraction from heartwood of Dalbergia tonkinensis Prain demonstrated stronger inhibitory activity against α-glucosidase from rat than that of the trunk bark and leaves with IC50 value of 1.72 mg/mL. - Sativanone and formononetin demonstrated much stronger activity against yeast α-glucosidase than that of the acarbose (positive control) with the maximum activity and IC50 values of , 90%, 0.23 mg/mL, 98 %, 0.06 mg/mL, and 62%, 1.32 mg/mL, respectively. - The specific inhibitory activity tests showed that sativanone and formononetin also have good effects on rat α-glucosidase with IC50 values of 91%, 0.36 mg/mL, and 94%, 0.25 mg/mL, respectively.

LIST OF THE PUBLISHED WORKS

1. Nguyen Manh Cuong, Ngu Truong Nhan, Ninh The Son, Do Huu Nghi, To Dao Cuong, Daltonkins A and B, Two New Carboxyethylﬂavanones from the Heartwood of Dalbergia tonkinensis, Korean Chemical Society, 2017, 38 (12), 1511–1514 (SCI, IF: 0.76). 2. Van Bon Nguyen, San-Lang Wang, Ngu Truong Nhan, Thi Hanh Nguyen, Nguyen Phuong Dai Nguyen, Do Huu Nghi, Nguyen Manh Cuong, New Records of Potent In-Vitro Antidiabetic Properties of Dalbergia tonkinensis Heartwood and the Bioactivity-Guided Isolation of Active Compounds, Molecules, 2018, 23 (7), 1589- 1600 (SCI-E, IF: 3.098).

3. Ngu Truong Nhan, Ninh The Son, To Dao Cuong, Nguyen Phuong Dai Nguyen, Pham Ngoc Khanh, Tran Thu Huong, Nguyen Manh Cuong, Further study on chemical constituents from the heartwood of Dalbergia tonkinensis, Vietnam Journal of Science and Technology, 2018, 56 (4A), 252-258 (ACI). 4. Nguyen Manh Cuong, Ngu Truong Nhan, Nguyen Phuong Dai Nguyen, Pham Ngoc Khanh, Ninh The Son, Tran Thu Huong, To Dao Cuong, Nguyen Thi Hong Mai, Nguyen Thi Phuong Trang, Comparison of ability to identify vietnam dalbergia tonkinensisspecies of several chloroplast DNA markers, National Biotechnology Conference, Hanoi, Natural Science and Technology Publishing House, 2018, 100-

106, ISBN: 978-604-913-759-4. 5. Ngu Truong Nhan, Nguyen Manh Cuong, Do Huu Nghi, A document survey on the natural flavonoids of the genus Dalbergia (Fabaceae), Pharmaceutical Journal, 2018, 505 (58), 16-21.