MINISTRY OF EDUCATION AND TRAINING

VIETNAM ACADEMY

OF SCIENCE AND TECHNOLOGY

GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY

…………***…………

VU THI THU LE

RESEARCH ON CHEMICAL CONSTITUENTS AND

BIOLOGICAL ACTIVITIES OF CALLICARPA CANDICANS AND

CALLICARPA MACROPHYLLA GROWING IN VIETNAM

Major: Chemistry of natural compounds

Code: 9.44.01.17

SUMMARY OF CHEMICAL DOCTORAL THESIS

HA NOI - 2020

This thesis was completed at Graduate University of Science and Technology

- Vietnam Academy of Science and Technology.

Supervisors:

1. Assoc.Prof. Dr. Pham Thi Hong Minh

Institute of Natural Products Chemistry – Vietnam Academy of Science

and Technology

2. Prof. Dr. Pham Quoc Long

Institute of Natural Products Chemistry – Vietnam Academy of Science

and Technology

Examiner 1: Assoc.Prof. Dr. Phan Minh Giang

University of Science – Vietnam National University

Examiner 2: Dr. Đỗ Hữu Nghị

Institute of Natural Products Chemistry – Vietnam Academy of

Science and Technology

The thesis defense was monitored by the Graduate University level Board

of Examiners, held at: Graduate University of Science and Technology - 18

Hoang Quoc Viet - Cau Giay - Ha Noi.

At ……….. , ….………………….. 2020

The thesis is available in Vietnam National Library and Library of Graduate

University of Science and Technology.

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PREAMBLE 1. The urgency of the thesis

Vietnam is located in a tropical climate region with extremely rich natural

resources and biodiversity with many valuable medicinal herbs. The increasing

use of traditional medicinal herbs or from naturally occurring compounds has

taken an important position in medicine. Herbal remedies are the subject for

scientists to fully study the nature of the active ingredients in natural plants, the

results of the study will contribute to a better explanation of the effects.

Treatment of traditional medicinal plants is still often used in folk.

In the world, studies of the genus Callicarpa mainly focus on: botanical,

pharmacology, plant chemistry and clinical. Pharmacological studies

conducted on crude extracts or pure compounds provide the scientific basis for

traditional use. Biological activity studies mainly focused on activities: anti-

inflammatory, hemostasis, memory loss, oxidation, antibacterial. Studies of

chemical composition and biological activity in the world show that

diterpenoid and triterpenoid compounds have very good anticancer activity,

which is the most abundant chemical component in Callicarpa genus.

The research to find out the active ingredients of plants has been receiving

the attention of scientists from many countries around the world, including

Vietnam. This is also the reason for the topic "Studying the chemical

composition and biological activity of the Sheic species (Callicarpa candicans)

and the large-leafed Tuzhou (Callicarpa macrophylla) in Vietnam." be selected

for research.

.

2. The research objectives of the thesis

Isolate and determine the chemical structure of compounds from C.

candicans and C. macrophylla species collected in Vietnam.

Assessment of toxic activity on some cancer cell lines: liver (Hep-G2), lung

(Lu-1) and breast (MCF-7) of isolated clean compounds.

Researching the chemical composition of C. candicans and C. macrophylla

essential oils.

3. The main research content of the thesis

1.Determining the chemical structure of compounds isolated from leaves of C.

candicans and C. macrophylla in Vietnam

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2.Determining the essential chemical composition of leaves of C. candicans

and C. macrophylla in Vietnam by conventional steam entraining methods and

microwave-assisted distillation methods.

3.Evaluation of toxic activity on three cancer cell lines (liver - HepG2, prostate

- PC3, lung - A549) and in vitro antimicrobial testing of essential oils obtained

from C. candicans leaves and C. macrophylla.

4. Evaluation of toxic activity on three strains of liver cancer (Hep-G2), lung

(Lu-1) and breast (MCF-7) in vitro of clean compounds isolated

from leaves of C. candicans and C. macrophylla

Chapter 1. OVERVIEW

The literature review is a collection of national and international research on:

1.1. General characteristics of botany Callicarpa

1.2. Pharmacological effects of the genus Callicarpa

1.3. Chemical composition of plants genus Callicarpa

1.4. Biological activity of genus Callicarpa plants

Chapter 2. SUBJECTS AND METHODS OF THE STUDY

This section describes in detail the sample handling processes, the methods of

generating sediments, chromatography and the isolation of compounds;

determine the chemical composition of essential oils and methods of bioactive

test.

2.1. Materials and research methods

2.1.1. Plant samples

Leaves, stems, branches and fruits (Callicarpa candicans (Burm.f.) Hochr.)

And Tu Chau big leaves (Callicarpa macrophylla Vahl) collected in Tam Dao

district - Vinh Phuc in October 2015 and Dai Tu district, province Thai Nguyen

in October 2017, Dr. Nguyen Quoc Binh (Vietnam Museum of Nature - VAST)

identifies the scientific name and denominator model stored at the Vietnam

Museum of Nature.

2.1.2. Method of isolation of compounds from tree samples

The analysis and separation of plant extracts are made by different chromatographic

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methods such as thin layer chromatography (TLC), normal column chromatography

(CC) with static phase is silica gel (Merck), Inverted phase chromatography with

static phase is YMC RP 18 (Merck), molecular wire chromatography with static

phase is sephadex LH-20 (Merck) and stationary phase chromatography is silica

gel.

2.1.3. Methods of determining chemical structure

The structure of the compounds is determined by the combination of physical parameters with modern spectroscopic methods such as melting point (Mp), polarity ([α] D), mass gas chromatography. spectroscopy (GC-MS), electronic atomization mass spectrometry (ESI-MS), high resolution mass spectrometry (HR-ESI-MS), nuclear magnetic resonance spectroscopy including 1-dimensional spectrum (1H-, 13C -NMR and DEPT) 2-dimensional spectrum (COZY, HSQC, HMBC and NOESY). 2.2. Method of extracting essential oil 2.2.1. The steam-distillation distillation method uses a micro-essential oil distillator 2.2.2. The method of steam distillation involves the use of microwaves 2.2.3 Method of analyzing essential oil chemical composition

Determine the chemical composition of essential oil by combining GC –MS system with standard library and Mass Finder 4.0 retention time locking software. 2.3. Biological activity test method

The cytotoxic activity was tested by SRB method on three cancers: liver (Hep-G2), lung (LU-1) and breast (MCF-7) and MTT method [3- [4,5- dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide] on three lines of liver cancer (Hep-G2), prostate (PC3) and lung (A549).

The method of assessing antimicrobial activity was conducted to evaluate the antibiotic activity of extracted samples by methods of Vander Bergher and Vlietlinck (1991), and McKane & Kandel (1996) on 8 strains: Vi Gram, mycelium and yeast.

The tests were conducted at the Department of Biologically active,

Institute of Natural Products Chemistry, VAST

Chapter 3. EXPERIMENTAL The empirical section described in detail the processes: Sample processing

and isolation of clean substances from the leaves of two species of Large-leafed Tuzhou (C. macrophylla) and Nang nang (C. candicans); Physical constants and spectral data of compounds isolated from 2 studied species.

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3.1. Big tree leaves of Zizhu (C. macrophylla)

3.1.1. Acquire extracts from the leaves of the Ziocuo tree

The process of processing the leaf sample of Tu Chau tree is large

Diagram 3.1. Extraction diagram of Tu Chau leaves (C. macrophylla) 3.1.2. Isolation and purification of substances from the leaves of the Tu Chau leaves The process of isolating compounds from n-hexane and ethyl acetate residues of the leaves of Chau Chau tree is as big as Figure 3.2

Diagram 3.2. Isolation of n-hexane and ethyl acetate residues of broadleaf

wattle tree

3.2. Her candlelight leaves (C. candicans)

3.2.1. Obtain extracts from the leaves of Nang nàng

The process of treating her leaves is as Figure 3.3

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Diagram 3.3. Diagram of extracting leaves of Nang (C. candicans) leaves

3.2.2. Isolate and refine substances from the extract of Nang leaves

The process of isolation of compounds from n-hexane residues and ethyl

acetate of Her leaves as Figure 3.4

Diagram 3.4. Diagram of isolation of n-hexane and ethyl acetate residues

from Her leaves

Chapter 4. RESULTS AND DISCUSSION

Chapter 4 presents how to determine the structure of the isolated

compounds, the chemical composition of essential oils, and the results of the bioactive test of the components

4.1. Chemical composition of the essential oil of Nang nang and Tu Chau

leaves

GC - MS analysis in the composition of dried leaves She has 39

components, accounting for 92.57% of the total content. 25 sesquiterpene

hydrocarbons (62.98%) and 11 sesquiterpenes contain oxygen (22.46%). The

main component identified in essential oil is α-Gurjunene (21.97%). In the

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essential oil of fresh leaves, there are 47 components discovered, accounting

for 93.17% of the total content of essential oils. Among them, there are 28

sesquiterpene hydrocarbons (69.84%), 12 sesquiterpen (16.50%). In particular,

the largest constituent content is α-gurjunene (21.31%)

The chemical composition of essential oil from the Nang Nang flower

said there are 47 components, accounting for 92.86% of the total content. The

high content constituents are determined in the essential oil of flower. The

constituents with the main content determined in the Nang essential oil of her

plant are E- caryophyllene (5.07%), α- selinene (5.66%), δ-cadinene (5.44%).

The chemical composition of the fresh leaves of Eucalyptus tree has 50

components, accounting for 90.59% of total content. The main component

identified in essential oil is Phytol (11.03%).

4.2. The compounds were isolated from the leaves of the Big Leaf and the

Taurus

From the residue of n-hexane and ethyl acetate extracts from the leaves of

the Tzu tree leaves, up to 10 compounds were isolated and chemically

structured, including 7 terpenoid compounds: callimacrophylla B (M8), 3β-

hydroxyolean-12-ene. (M2), β-amyrin (M3), ursolic acid (M6), oleanolic acid

(M10), callimacrophylla A (M1) and ent-1β-acetoxy-7β, 14α-dihydroxy-16-

kauren-15-on (M7) ) and 3 steroid compounds: spinasterol (M5), β-sitosterol

(M4) and daucosterol (M9). In particular, callimacrophylla A (M1) and

callimacrophylla B (M8) are two new compounds.

From the residue of n-hexane and ethyl acetate extracts of Nang Nang

leaves (C. candicans) 11 compounds were isolated and identified chemical

structure, including 4 flavonoid compounds: 5-hydroxy-7.4'-dimethoxyflavon

(C1), 5-hydroxy-3 ', 4', 7-trimethoxyflavon (C3), genkwanin compound (C9)

and cynaroside (C10); 5 terpenoid compounds: ursolic acid (M6), 2α-hydroxy-

ursolic acid (C7), 2α, 3β, 23-trihydroxyurs-12-en-28-oic acid (C8), seco-

hinokiol (C5) and methyl seco -hinokiol (C6) and 2 steroid compounds: β-

sitosterol (M4) and daucosterol (M9). In it, the compound methyl seco-hinokiol

was isolated for the first time from nature. The main chemical composition of the two genera Callicarpa species

studied are mainly flavonoid, diterpennoid and triterpenoid compounds. Several compounds were isolated in two species: ursolic acid, β-sitosterol and daucosterol.

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Triterpenoid compounds isolated from 2 research species show that the main chemical components are ursane and oleane frame compounds, suitable for the main chemical components of triterpenoid compounds in Callicarpa genus in the literature. Father.

The diterpene compounds isolated from two research species show that the main chemical components are ent-kaurane and abietane frame compounds, which are suitable for the main chemical components of diterpenoid compounds in Callicarpa in the literature. announced.

The compounds isolated from leaves of two studied species

Table 4.26. The compounds were isolated from 2 studied species

Tên hợp chất Lớp chất Loài phân lập KL

(mg) Tính mới

callimacrophylla A (M1) Diterpneoid C. macrophylla 10,8 M ent-1α-acetoxy-7β,14 α-dihydroxy-kaur-16-en-15-on (M7)

Diterpenoid C. macrophylla 12,5 H

seco-hinokiol (C5) Diterpenoid C. candicans 22,8 H methyl seco-hinokiol (C6) Diterpenoid C. candicans 31,0 H

3β-hydroxyolean-12-ene (M2) Triterpenoid C. macrophylla 10,3 L β-amyrin (M3) Triterpenoid C. macrophylla 12,7 L

ursolic acid (M6) Triterpenoid C. macrophylla C. candicans

15,5 15,7

L L

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callimacrophylla B (M8) Triterpenoid C. macrophylla 10,1 M 2α-hydroxy-ursolic acid (C7) Triterpenoid C. candicans 12,5 L 2α,3β,23-trihydroxyurs-12-en-28-oic acid (C8)

Triterpenoid C. candicans 11,5 L

oleanolic acid (M10) Triterpenoid C. macrophylla 8,5 spinasterol (M5) Steroid C. macrophylla 11,2 L

β–sitosterol (M4) Steroid

C. macrophylla C. candicans

20,0 19,0

daucosterol (M9) Steroid C. macrophylla C. candicans

16,5

5-hydroxy-7,4’-dimethoxyflavon (C1) Flavonoid C. candicans 8,5 L 5-hydroxy-3’,4’,7-trimethoxyflavon (C3) Flavonoid C. candicans 11,2 L genkwanin (C9) Flavonoid C. candicans 13,0 L cynaroside (C10) Flavonoid C. candicans 10,8 L

M: New compound; L: For the first time isolated from species; H: First

isolated from them

The two newly obtained compounds are 1 substance belonging to the

class of diterpenoid and triterpenoid. They are structured based on the following spectral data

4.2.1.1. Callimacrophylla A (M1) - New compound Compound 1 was obtained as a white crystal and its molecular

formula was deduced as C20H28O3 by HR-ESI mass spectrum (found m/z 387.1940 [Mþ35Cl]-, calcd. For C20H32O535Cl: 387.1938; m/z 389.1918 [Mþ37Cl]-, calcd. for C20H32O537Cl: 389.1909).).

Hình 4.7. Phổ ESI-MS của M1

1H-NMR spectrum of 1 exhibited two methyl singlet signals at dH 0.65

and 1.02, one oxygenated methine at dH 3.66 (dd, J ¼ 10.5, 5.5 Hz), the doublet

of doublet signals oftwo oxygenated methylene at dH 2.85/3.18 and 3.45/3.54,

and other signals from dH 0.93 to 4.78 ppm. (Bảng 4.3).

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Hình 4.8. Phổ 1H-NMR của M1

Hình 4.9. Phổ 13C-NMR của M1

The 13C-NMR spectrum of 1 showed signals of 20 carbon atoms, which

were sorted by DEPT spectra into one ketone group at dC 219.3; one

oxygenated methine at dC 69.5; two oxygenated methylenes at dC 61.4 and

69.9; two methylgroups at dC 17.4 and 17.7, four quaternary carbons at dC

36.9, 38.5, 58.4, and 78.4; six methylene and three methine groups. The above

results suggested that compound 1 to be a diterpene with the ent-kaurane

skeleton similar to ent-7a,16b,17-trihydroxy-18- acetoxykaur-15-one except

for the disappearance of acetoxy group at C-18 instead of the addition of

hydroxy group at this position in 1 (Giang et al. 2013). Assignments of

1H- and 13C-NMR data of 1 were completed by HSQC and HMBC spectra in

comparison with the corresponding data of ent-7a,16b,17-trihydroxy-18-

acetoxykaur-15-one (Giang et al. 2013) 101].

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Hình 4.10. Phổ DEPT của M1

In the HSQC spectra, protons at dH 0.65, 1.02, and 3.62 had cross peaks

with carbons at dC 17.4, 17.7, and 69.5, respectively; while protons at dH

2.85/3.18 and 3.45/3.54 had cross peaks with carbons at dC 69.9 and 64.1,

respectively. Furthermore, four proton signals at dH 4.30, 4.43, 4.49, and 4.78

did not have HSQC cross peaks with any carbons suggesting that compound 1

had four hydroxy groups (hình 4.6 và bảng 4.3).

Hình 4.11. Phổ HSQC của M1

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Hình 4.12. Phổ HMBC của M1

In the HMBC spectra of 1, the correlations from protons at dH 0.65 and

2.85/3.18 to carbons C-3 (dC 34.7), C-4 (dC 36.9), C-5 (dC 44.9), from H-20

(dH 1.02) to C-1 (dC 38.6), 2 D. T. LAM ET AL. C-5 (dC 44.9), C-9 (dC 52.6),

C-10 (dC 38.5), and from H-7 (dH 3.45/33.54) to C-13 (dC 38.7), C-15 (dC

219.3), C-16 (dC 78.4) were observed, confirming that three hydroxy groups

were at C-18, C-6 and C-17, and the assignments of NMR data of the concerned

positions. The last hydroxy group was at C-7 confirming by the HMBC

correlations from hydroxy proton at dH 4.30 to C-6 (dC 26.9), C-7 (dC 65.9),

C-8 (dC 58.4). In

addition, HMBC correlations from 18-OH (dH 4.49) to C-18 and C-4, from 16-

OH (dH 4.78) to C-13, C-15, C-16, and from 17-OH (dH 4.43) to C-17 and C-

16 were observed. On the other hand, the carbon chemical shifts of C-18 (dC

69.9) and C-19 (dC 17.4) of 1 were similar to the corresponding values of

siderone (18-hydroxy derivative, dC-18 ¼ 71.0 and dC-19 ¼ 16.9) (Venturella

et al. 1983), and difference from the corresponding values of diterpene SL-II

(19-hydroxy derivative, dC-19 64.3 and dC-18 22.8) (Piozzi et al.1980). Above

evidence further confirmed that the hydroxy group was at C-18. The large

coupling constant of H-7 (J ¼ 10.5 Hz) of 1 comparing to the broad singlet of

H-7 of 7b,16a,17-trihydroxy-ent-kauran-19-oic acid (Nhiem et al. 2015)

confirmed equatorial orientation (a-configuration) of 7-OH group in 1. The

carbon chemical shifts of C-16 (dC 78.4) and C-17 (dC 61.4) of 1 were

consistent with the corresponding values of ent- 7a,16b,17-trihydroxykaur-18-

acetoxy-15-one (dC-16 77.4 and dC-17 63.1) (Giang et al.2013) and difference

from 16a,17-dihydroxy-15-oxo-ent-kaur-19-oic acid (dC-16 83.0 and dC-17

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65.3) (Braca et al. 2004) suggesting the b-configuration of 16-OH group. From

the above evidence, compound 1 was determined to be ent-7a,16b,17,18-tetrahydroxykaur-15-one, a new compound and named as callimacrophylla A.

Figure 4.6. Chemical structure, the main interaction HMBCHC) of M1

Table 4.3. Spectrum data of 1H- and 13C-NMR of M1 and reference substance

TT Hợp chất M1 [103]

δC δH (mult., J Hz) #δC #δH (mult., J Hz)

1 38,6 0,57 (1H, m)/ 1,66 (1H, m) 41,7 3,57 (m)

2 17,3 1,38 (1H, m)/ 1,57 (1H, m) 20,3 2,03 (m)

1,85 (overlap)

3 34,7 1,11 (1H, m)/ 1,40 (1H, m) 39,2 2,01 (m) 1,42 (m)

4 36,9 - 44,2

5 44,9 - 48,1 1,63 (dd, 11,9, 1,7)

6 26,9 1,75 (1H, m)/ 1,66 (1H, m) 30,5 1,75 (m) 1,40 (m)

7 69,5 3,62 (1H, dd, 10,5; 5,5) 78,1 2,28 (dt, 13,3, 4,1)

1,33 (overlap)

8 58,4 - 49,0 -

9 52,6 0,94 (1H, d, 9,0) 51,1 1,85 (overlap)

10 38,5 1,20 (1H, m)/ 1,53 (1H, m) 40,4 -

11 17,9 1,20 (1H, m)/ 1,53 (1H, m) 19,1 3,66 (dd, 10,7, 6,0)

1,68 (m)

12 28,0 1,18 (1H, m)/ 1,60 (1H, m) 27,6 1,98 (m) 1,57 (m)

13 38,7 2,21 (1H, br d, 3,5) 46,1 2,95 (br s)

14 25,3 1,66 (1H, m)/ 2,38 (1H, m) 37,5 2,45 (d, 11,8) 1,33 (overlap)

15 219,3 - 219,3 -

16 78,4 - 82,9 -

17 61,4 3,45 (1H, dd, 6,5; 12,0) 3,54 (1H, dd, 4,5; 12,0)

66,7 6,02 (br s) 5,16 (br s)

18 69,9 2,85 (1H, dd, 5,0; 10,5) 3,18 (1H, dd, 5,0; 10,5)

71,0 3,64 (d, 10,5) 3,32 (d, 10,5)

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19 17,4 0,65 (3H, s) 16,8 0,88 (s)

20 17,7 1,02 (3H, s) 16,1 1,44 (s)

7-OH 4,30 (1H, d, 5,0)

16-OH 4,78 (1H, s)

17-OH 4,43 (1H, dd, 4,5; 6,5)

18-OH 4,49 (1H, dd, 5,0; 10,0)

#δH và #δC của scopariusol L (1H: 500 MHz, 13C: 125 MHz, pyridine-d5) [102]

4.2.2.1. Callimacrophylla B compound (M8) - New compound Compound 2 was obtained as a white crystal and its molecular formula was

deduced as C32H50O4 by HR-ESI mass spectrum (found m/z 499.3786 [M þ H]þ, calcd. 499.3786 for C32H50O4

1H-NMR spectrum of compound M8 appeared 6 groups of methyl singlet at H 0.83 (3H, s, H3-28); 1.17 (3H, s, H3-26); 1,18 (3H, s, H3-25); 1,34 (3H, s, H3-27); 0.89 (6H, s, H3-23 and H3-24), 2 methyl groups as doublet at 0,H 0.80 (3H, J = 6.5 Hz, H3-29) and 0.93 (3H, J = 6.5 Hz, H3-30) and 1 methyl group at H 2.05 (3H, s, CH3CO) of acetoxy group. The 1H-NMR spectrum of M8 also showed the presence of 6 methane groups [H 4,53 (1H, dd, J = 11.5; 4,5, H-3); 0.82 (1H, m, H-5); 2.50 (1H, s, H-9), 2.43 (1H, dd, J = 11.5; 1,5, H-18); 1.42 (1H, m, H-19) and 1.08 (1H, m, H-20)] and 8 methine groups have a chemical shift between 5H 0.95-2.75 (H- 1, H-2, H-6, H-7, H-15, H-16, H-21 and H-22), a proton singlet at H 6.27 (1H, s, 12-OH) ( Table 4.7)

Hình 4.17. Phổ HR-ESI-MS của M8

Hình 4.18. Phổ 1H-NMR của M8

13C-NMR spectra combined with DEPT spectroscopy showed that compound M8 has 32 carbon atoms, including 8 quaternary carbon [C 38,0 (C-4); 45.5 (C-8); 37.0 (C-10); 195.2 (C-11); 144.5 (C-12); 134.4 (C-13); 41.7

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(C-14) and 33.4 (C-17)], 6 methine groups [C 80.5 (C-3); 55.0 (C-5); 59.7 (C-9); 48.9 (C-18); 39.3 (C-19) and 40.8 (C-20)], 8 methylene groups [C 38.9 (C-1); 23.5 (C-2); 17.4 (C-6); 32.9 (C-7); 27.3 (C-15); 27.5 (C-16); 31.2 (C-21) and 41.2 (C-22)] and 8 methyl groups [C 28.0 (C-23); 16.6 (C-24); 16.7 (C-25); 18.6 (C-26); 21.0 (C-27); 28.8 (C-28); 16.6 (C-29) and 20.9 (C-30)]. In addition, there are signals of 01 acetoxy group at 170C 170.9 (CH3CO) and 21.3 (CH3CO) also obtained from 13C-NMR spectrum.

Hình 4.19. Phổ 13C-NMR của M8

All of the above data suggests that compound M8 is a ursane triterpene

containing an acetoxy group and has a structure similar to 3β-acetoxy-urs-12-ene-

11-one [94], except for differences. on the chemical displacement of carbon atoms

at C-11, C-12 and C-13. When comparing NMR data of M8 and 3β-acetoxy-urs-12-

ene-11-one compounds (TLTK), we can see in TLTK of proton signal of olefin

group at H 5,54 (1H, s) directly linked to C-12 is a quaternary carbon (not linked to

hydrogen) with a chemical shift C of 144.5 ppm (C-12). Also at the C-12 position

associated with the hydroxy group is also shown by high resolution mass

spectrometry. In addition, it was also determined by the proton nuclear interaction of

protons at H 6.27 (1H, s, 12-OH) with carbon atoms at C 195.2 (C-11); 144.5 (C-

12) and 134.4 (C-13). In addition, links on the HMBC spectrum between H-18 (2,4H

2.43) and C-12 (144.5) / C-13 (134C 134.4) indicate the position of the double bond

at C-12 / C-13 and ketones group at C-11 (Figure 4.16)

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Hình 4.20. HSQC của M8

Hình 4.21. Phổ HMBC của M8

Besides, the link between methyl protons at H-23 / H-24 (H 0.89) and C-3 (C 80.5) / C-4 (C 38.0) and C- 5 (C 55,0) as well as the link between H-3 (H 4,53) / CH3CO (H 2,05) with carbon atoms at C 170,9 (CH3CO), combined with constant the large coupling number of H-3 (J = 11.5 Hz) on the 1H-NMR spectrum confirms that the acetoxy group bound at C-3 has direction có. In addition, the H-3 proton has a α direction determined by the bonds from H-2α (H 1.65) and H3-23 (H 0.89) to H-3 (H 4.53). as well as from H3-25 (H 1.18) to H-2β (H 1.72) on the ROESY spectrum. On the other hand, links between H-20 and H-29 / H-19 and between H-2 protons and H-1 / H-3 were also found on the 1H-1H COZ spectrum.

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Figure 4.22 Spectrum 1H-1H Cozy and NOESY of M8 Combine spectral data with HMBC, 1H-1H COZY and ROESY and

compare with spectral data of 3β-acetoxy-urs-12-ene-11-one compounds [94] in the reference document that allows confirmation Compound M8 is 3β-acetoxy-urs-12-ene-11-one-12-ol. This is a new compound and is named callimacrophylla B

Figure 4.16. Chemical structure, the main interaction HMBC (HC) of M8 Table 4.7. Spectrum data 1H- and 13C-NMR of M8 and reference substance

Vị trí M8 [94]

δC δH (mult., J Hz) #δC #δH (mult., J Hz)

1 38,9 2,75 (1H, dt, 6,5; 3,5)

1,13 (1H, m) 38,9 2,75(lH,ddd, 3,5;3,5;13,5)

2 23,5 1,65 (1H, m) / 1,72 (1H, m) 23,6 3 80,5 4,53 (1H, dd, 11,5; 4,5) 80,7 4,51 (lH, dd, 4,6; 11,7) 4 38,0 - 38,1 5 55,0 0,82 (1H, m) 55,1 6 17,4 1,43 (1H, m)/ 1,58 (1H, m) 17,5 7 32,9 1,43 (1H, m)/ 1,68 (1H, m) 32,8 8 45,5 - 45,2 9 59,7 2,50 (1H, s) 61,5 2,34 (1H, s)

10 37,0 - 36,7

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11 195,2 - 199,5 12 144,5 - 130,5 5,54 (1H, s) 13 134,4 - 164,8 14 41,7 - 43,7 15 27,3 0,95 (1H, m)/ 2,10 (1H, m) 27,2 16 27,5 1,17 (1H, m)/ 1,90 (1H, m) 27,3 17 33,4 - 33,9 18 48,9 2,43 (1H, dd, 11,5; 1,5) 59,1 19 39,3 1,42 (1H, m) 39,2 20 40,8 1,08 (1H, m) 39,3 21 31,2 1,25 (1H, m)/ 1,43 (1H, m) 30,9 22 41,2 1,39 (1H, m)/ 1,47 (1H, m) 40,9 23 28,0 0,89 (3H, s) 28,1 0,87 (3H, s) 24 16,6 0,89 (3H, s) 16,7 0,88 (3H, s) 25 16,7 1,18 (3H, s) 16,5 1,16 (3H, s) 26 18,6 1,17 (3H, s) 18,6 1,18 (3H, s)

27 21,0 1,34 (3H, s) 20,5 1,29 (3H, s)

28 28,8 0,83 (3H, s) 28,9 0,81 (3H, s) 29 16,6 0,80 (3H, d, 6,5) 17,5 0,80 (3H,d, 6,0) 30 20,9 0,93 (3H, d, 6,5) 21,2 0,94 (3H, d, 6,0)

CH3CO 170,9 - 170,9 - CH3CO 21,3 2,05 (3H, s) 21,1 2,04 (3H, s) 12-OH 6,27 (1H, s)

#δH và #δC của 3β-acetoxy-urs-12-ene-11-one (1H: 500 MHz,13C:125MHz,CDCl3) [94]

4.3. Evaluate the biological activity of Nang nang and Chau Tu large leaves 4.3.1. Evaluate the biological activity of the essential oil of Nang nang leaves and Tu Chau leaves

The results showed that dried her leaf essential oil showed weak activity

on Hep-G2 liver cancer cell line (IC50 = 94.5 µg / mL). Fresh leaf oil extracted

by conventional steam-distillation distillation method and Zenzi leaves were

not shown to be active on test cancer cell lines Table 4.20. Results of in vitro cytotoxic activity of Lady and Tu Chau leaf oil

on large cell lines: liver (Hep-G2), prostate cancer (PC3) and lung cancer (

A549)

Form name IC50 (µg/mL)

Hep-G2 PC3 A549

Dry leaf essential oil 94,53 >100 >100

Ordinary fresh leaves essential oil >100 >100 >100

Fresh leaves essential oil uses a microwave

14,65 23,87 56,21

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Essential oil of fresh Tuzhou leaves >100 >100 >100

Control (paclitaxel) 4,03 3,48 3,69

The results also indicate that the microwave-assisted distillation method has obtained the constituents or mixture of constituents in the leaves essential oil. ordinary water. That has important implications for the next research direction of the essential oils of Her in particular and essential oils from other plants in general.

Table 4.21. Test results of the antimicrobial activity of the essential oil of Nang nang leaves and Tu Chau leaves

Tên mẫu MIC (g/mL)

EC PA BS SA AN FO SC CA

Dry leaf essential oil

>200 >200 >200 >200 >200 >200 >200 >200

Ordinary fresh leaves essential oil

>200 >200 >200 >200 >200 200 >200 200

Fresh leaves essential oil uses a microwave

>200 >200 >200 >200 >200 100 >200 200

Zingzhou essential oil

>200 >200 >200 >200 >200 >200 >200 >200

EC: Escherichia coli, PA: Pseudomonas aeruginosa, BS: Bacillus subtillis, SA: Staphylococcus aureus, AN: Aspergillus niger, FO: Fusarium oxysporum, SC: Saccharomyces cerevisiae, CA: Candida albicans 4.3.2 Evaluation of in vitro cytotoxic activity of magnetic extract from

species tu chau la to and Nang nang The data from Table 4.22 shows that the ethanol residue from leaves, fruits,

stems of large leaves of Chau Chau and She exhibited good inhibitory activity on 3 cancer cell lines tested with CS values (%) from 29.69 0.8 to 89.62 1.2%. In particular, the ethanol residue of the leaves of Big Chau and Tu Chau (L.CM and L.CC) has a good inhibitory effect with CS values (%) of 47.84 2.1 and 56, respectively. 28 2,6 (for Hep-G2 cancer cell line); 39.40 2.2 and 29.69 0.8 (for Lu-1 cancer cell line) and 30.23 1.5 and 35.18 1.0 (for cancer cell line) letter MCF-7). Preliminary results of the cytotoxic activity of the three cancer cell lines Hep-G2, Lu-1 and MCF-7 showed that the leaves of the Chau Chau leaves and the Lady were more effective than the fruits. and the trunk of their branches. Therefore, the next chemical composition research focused on the leaves of the Big tree and the tree Table 4.22. In vitro cytotoxic activity total ethanol residues from leaves, berries and stems of Big tree leaves

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TT KH mẫu Nồng độ

đầu

(g/mL)

Giá trị CS (%) Dòng tế bào

Hep-G2 Lu-1 MCF-7

DMSO - 100 100 100

Chứng (+) 5 1,34 0,8 2,66 0,9 1,21 0,71

1 Q.CC 20 89,29 1,1 33,04 1,4 40,43 2,79

2 T.CC 20 89,62 1,2 33,53 1,6 91,29 0,32

3 L.CC 20 56,28 2,6 29,69 0,8 35,18 1,0

4 Q.CM 20 60,02 2,3 47,31 1,5 38,86 2,26

5 T.CM 20 64,66 2,2 55,64 2,8 90,22 0,15

6 L.CM 20 47,84 2,1 39,40 2,2 30,23 1,5

Q: fruit, T: stems, L: leaves, CC: C. candicans, CM: C. macrophylla) Continue to test the activity of the n-hexane, EtOAc and methanol residues

of big leaf and conifer tree leaves for cancer cell lines Hep-G2, Lu-1 and MCF-7. With the exception of methanol fractional sludge, which hardly shows activity, both residual fractional residues of the leaves of these two species have different effects, in which the n-hexane segment (L. CM.H) of Big tree and EtOAc (L.CC.E) of Her leaves showed the strongest activity with low CS (%) and low CS (%) values from 12.49 1,4 - 30,17 0,1%. This result is explained by the presence of strong cytotoxic activity groups such as terpenoids and flavonoids in the segments of weak and moderate polarization Table 4.23. The cytotoxic activity of the residues extracted from the leaves of

the Big tree and the leafy tree

TT KH mẫu Nồng độ đầu

(g/mL)

Giá trị CS (%)

Dòng tế bào

Hep-G2 LU-1 MCF-7

DMSO - 100 100 100

Chứng (+) 5 1,34 0,8 2,66 0,9 1,21 0,71

1 L.CM.H 20 20,18 0,8 12,49 1,4 11,61 2,11

2 L.CM.E 20 98,03 0,9 18,20 1,3 40,43 2,79

3 L.CM.M 20 100 100 91,29 0,32

4 L.CC.H 20 98,84 0,9 73,04 1,5 38,86 2,26

5 L.CC.E 20 30,17 0,1 15,69 2,3 19,93 0,11

6 L.CC.M 20 100 100 90,22 0,15

(L: lá, H: n-hexane, E: ethyl acetate, M: methanol, CC: C. candicans, CM: C.

macrophylla)

4.3.3. Evaluation of in vitro cytotoxic activity of clean compounds isolated from Big leaf and Tui Chau leaves

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Table 4.24. In vitro cytotoxic activity test of substances on cancer cell lines (Hep-G2), lung (Lu-1) and breast (MCF-7)

TT Hợp chất

Giá trị IC50 (g/mL) Dòng tế bào

HepG2 Lu1 MCF7

Ellipticine 0,29 0,51 0,48

1 methyl seco-hinokiol (C6) 8,65 8,53 2,20

2 seco-hinokiol (C5) 8,25 9,13 2,46

3 callimacrophylla A (M1) 2,72 2,68 1,57

4 ent-1β-acetoxy-7β,14α-dihydroxy-16-kauren-15-on (M7) 0,31 1,55 0,23

5 callimacrophylla B (M8) 0,32 1,87 0,28

6 ursolic acid (M6) 0,25 0,31 0,21

7 2α,3β,23-trihydroxyurs-12-en-28-oic acid (C8) - - -

8 β-amyrin (M3) - - -

9 3β-hydroxyolean-12-ene (M2) 5,85 - 2,36

10 spinasterol (M5) 8,22 8,29 1,82

11 5-hydroxy-7,4’-dimethoxyflavon (C1) - - -

12 5-hydroxy-3’,4’,7-trimethoxyflavon (C3) - - -

13 genkwanin (C9) - - -

14 cynaroside (C10) - - -

Assessment of toxic activity of tested cancer cell lines: Table 4.24 shows that the anti-cancer activity of liver cancer (Hep-G2),

lung (Lu-1) and breast (MCF-7) of terpenoid compounds is stronger than that of steroid and flavonoid compounds. The compounds showed a more cytotoxic activity against breast cancer cells (MCF-7) than the liver cancer cell lines (Hep-G2) and lungs (Lu-1). Anti-cell activity against liver cancer (Hep-G2), lung (Lu-1) and breast (MCF-7) among 14 active compounds showed 6 active inactive compounds with IC50> 100 ( g / mL) include: 5-hydroxy-7.4'-dimethoxyflavon (C1), 5-hydroxy-3 ', 4', 7-trimethoxyflavon (C3), 2α, 3β, 23-trihydroxyurs-12-en- 28-oic acid (C8), Genkwanin (C9) and cynaroside (C10) and β-amyrin (M3). The remaining compounds showing strong activity are methyl seco-hinokiol (C6), seco-hinokiol (C5), ent-1 α-acetoxy-7β, 14α-dihydroxy-16-kauren-15-on (M7) , callimacrophylla B (M8), ursolic acid (M6), spinasterol (M5), 3β-hydroxyolean-12-ene (M2), callimacrophylla A (M1) with 0.2 g / mL

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with IC50 = 0.23 and 0.21 g / mL, stronger than the Ellipticine control with IC50 = 0.48 g / mL

Two compounds abietane (methyl seco-hinokiol (C6), seco-hinokiol (C5)) and a steroid compound (spinasterol (M5) exhibit potent cytotoxicity against breast cancer cell lines (MCF-7) with IC50 values in the range of 1.82 g / mL

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(MCF-7) with IC50 value = 0.25; 0.31 and 0.21 g / mL, while compounds 2α, 3β, 23-trihydroxyurs-12-en-28-oic acid (C8) did not show activity. This may be due to the presence of two hydrochloric groups at sites C-2 and C-23 that reduced the toxic activity against liver cancer cells (Hep-G2), lungs (Lu-1) and breast. (MCF-7). Another ursane-triterpenoid compound, callimacrophylla B (M8) has strong anti-cancer activity against liver cancer (Hep-G2), lung (Lu-1) and breast (MCF-7) with IC50 value = 0, 32; 1.87 and 0.28 g / mL, equivalent to ursolic acid, may be related to the ketone group at C-11.

CONCLUDE

Chemical composition of clean substances isolated 1. The thesis for the first time studies the chemical composition of two species of Her plant (C. candicans) and the giant leafy plant (C. macrophylla) of Callicarpa genus in Vietnam. 21 compounds have been isolated, including 2 new compounds, one for the first time isolated from nature. 2. From n-hexane and ethyl acetate residues from the leaves of the Tzhou tree leaves, up to 10 compounds have been isolated and chemically determined, including 7 terpenoid compounds: callimacrophylla B (M8), 3β-hydroxyolean-12. -ene (M2), β-amyrin (M3), ursolic acid (M6), oleanolic acid (M10), callimacrophylla A (M1) and ent-1β-acetoxy-7β, 14α-dihydroxy-16-kauren-15-on (M7) and 3 steroid compounds: spinasterol (M5), β-sitosterol (M4) and daucosterol (M9). In particular, callimacrophylla A (M1) and callimacrophylla B (M8) are new compounds. 3. From the residue of n-hexane and ethyl acetate extracts of the leaves of the tree (Callicarpa candicans) 11 compounds have been isolated and identified chemical structure, including 4 flavonoid compounds: 5-hydroxy-7.4'- dimethoxyflavon (C1), 5-hydroxy-3 ', 4', 7-trimethoxyflavon (C3), genkwanin (C9) and cynaroside (C10); 5 terpenoid compounds: ursolic acid (M6), 2α-hydroxy-ursolic acid (C7), 2α, 3β, 23-trihydroxyurs-12-en-28-oic acid (C8), seco-hinokiol (C5) and methyl seco -hinokiol (C6) and 2 steroid compounds: β-sitosterol (M4) and daucosterol (M9). In particular, the compound methyl seco-hinokiol is isolated for the first time from nature

Chemical composition in essential oils 4.Chemical composition of essential oils of her leaves and flowers Follow the normal distillation method. GC - MS analysis in the composition of dried leaves She has 39

components, accounting for 92.57% of the total content. The main component identified in essential oil is α-Gurjunene (21.97%). In the essential oil of the fresh leaves, she has 47 components accounting for 93.17% of the total content of essential oils. The main component identified in essential oil is α-Gurjunene

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(21.31%). The composition of her essential oil has 47 components, accounting for 92.86% of the total content. The main content of the constituents identified in her flower essential oil is E-caryophyllene (5.07%), α-selinene (5.66).

Under the method of steam distillation with microwave support GC - MS analysis in the composition of fresh leaves She had 46

components accounting for 93.4% of the total content. The main component identified in fresh lady's leaf oil is β-caryophyllene (10.45%), δ - cadinene (10.28%).

5. Chemical composition of essential oil of Zenzi leaves The composition of the fresh leaves of Tu Chau leaves has 50 components,

accounting for 90.59% of the total content. The main component identified in essential oil is phytol (11.03%).

Biological activity 6. Results of toxic activity test on 3 lines of liver cancer (Hep-G2), lung

cancer (Lu-1) and breast cancer (MCF-7) of some clean substances isolated, said 2 The new compound (callimacrophylla A and callimacrophylla B) exhibits strong activity, with most of the rest exhibiting fairly good activity. In particular, ursolic acid (M6) has anti-cancer activity against liver cancer (Hep-G2), lung (Lu-1) and breast (MCF-7) very strong with IC50 value = 2.5; 0.31 and 0.21 g / mL; compounds ent-1β-acetoxy-7β, 14α-dihydroxy-16-kauren-15-on (M7) and callimacrophylla B (M8) express strong anti-breast cell activity of CF-7 breast with IC50 = 0.23 and 0.21 g / mL, less than the ellipticine reference concentration.

7. Fresh leaves essential oil She obtained from the method of using a microwave shows good activity on all 3 cell lines of liver cancer (Hep-G2), prostate cancer (PC3), lung cancer (A549) tested with corresponding IC50 values of 14,65; 23.87 and 56.21 μg / mL and exhibited good Fusarium oxysporum inhibition activity with MIC value of 100 μg / mL. The remaining samples: dried leaves oil and fresh flowers Her, fresh leaves of Chauzhou big leaves distilled by conventional steam entraining method do not show activity.

REQUEST

The research results in the thesis show that the species Callicarpa is a rich source of natural compounds with interesting chemical structure and biological activity. Therefore, in the future continue to study the chemical composition, anticancer activity of the remaining parts of the above two species and other genus Callicarpa species in search of compounds with anticancer and activity. biology of other species from the genus Callicarpa of Vietnam, to further

24

confirm the scientific value of these species, contributing to the creation of products for public health care

NEW CONTRIBUTIONS OF THE THESIS The thesis has studied the chemical composition of two plants for the

first time: C. candicans and C. macrophylla of Callicarpa in Vietnam. 21 isolated compounds were isolated. In particular, callimacrophylla A and callimacrophylla B are two new compounds and methyl seco-hinokiol compounds first isolated from nature.

The first time the essential oils of two species of Big Lady and Tu Chau have been studied in Vietnam. Particularly her essential oils in the world have not had any research work.

Đâu The first time in Vietnam has studied toxic effects on a number of cell lines of liver cancer (Hep-G2), lung cancer (Lu-1) and breast cancer (MCF-7) of 14 clean compounds isolated from large leafy Tuzhou (C. macrophylla) and her (C. Candicans). In which 2 new compounds show strong activity,

Most of the remaining substances exhibited fairly active levels. Fresh leaves essential oil She obtained from the method of using the

microwave shows good activity on all three cell lines of liver cancer (Hep-G2), prostate cancer (PC3), lung cancer ( A549) tested with corresponding IC50 values of 14,65; 23.87 and 56.21 μg / mL and exhibited good Fusarium oxysporum inhibition activity with MIC value of 100 μg / mL. The remaining samples: dried leaf oil and fresh flowers Her, fresh Tu Chau leaves distilled by conventional steam entraining method do not show activity

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PUBLISHED SCIENTIFIC WORKS

International articles (ISI/Scopus):

1. Vũ Thị Thu Lê, Lại Phương Phương Thảo, Phạm Thị Hồng Minh, Hoàng

Thị Bích, Đỗ Tiến Lâm, Đinh Thị Thu Thủy, Phạm Minh Quân, Phạm Quốc

Long, Đào Việt Hùng, Trần Quốc Toàn, Essential oil of Callicarpa candicans

(Burm.f.) Hochr, Asian Journal of Chemistry, 2019 (chấp nhận đăng) (Scopus).

2. Do Tien Lam, Vu Thi Thu Le, Pham Minh Quan, Pham Thi Hong Minh,

Bui Huu Tai, and Phan Van Kiem, Two new terpenoids from the leaves of

Callicarpa macrophylla, Natural Product Research, 2019, 34, 688 (SCIE).

3. Quoc Toan Tran, Thu Le Vu Thi , Tien Lam Do, Hong Minh Pham Thi,

Bich Hoang Thi, Quang Truyen Chu, Phuong Thao Lai Phuong, Huu Nghi Do

, Hoai Thu Hoang Than , Thu Thuy Ta Thi, Van Huyen Luu, Mai Duong

Phuong Thi Thu Huong Phung Thi, Optimization of Microwave-Assisted

Extraction Process of Callicarpa candicans (Burm.f.) Hochr Essential Oil and

Its Inhibitory Properties against Some Bacteria and Cancer Cell Lines,

Processes, 2020, 8(2):173 (SCIE).

Domestic articles:

4. Vũ Thị Thu Lê, Luân Thị Thu, Phạm Thị Hồng Minh, Đỗ Tiến Lâm, Nguyễn

Phi Hùng, Đoàn Lan Phương, Nguyễn Thị Hồng Vân, Phạm Quốc Long, Một số

kết quả nghiên cứu ban đầu về dịch chiết n-hexan của cây Nàng nàng

(Callicarpa candicans (Burm.F) Hochr) ở Việt Nam, Tạp chí Khoa học và

Công nghệ, 2016, 54 (2B), 251-257.

5. Vũ Thị Thu Lê, Đỗ Tiến Lâm, Cầm Thị Ính, Đoàn Lan Phương, Phạm Quốc

Long, Trần Đăng Thạch, Tạ Thị Thu Thủy, Phạm Thị Hồng Minh, nghiên cứu

thành phần hóa học cặn chiết etyl axetat của cây Nàng nàng (Callicarpa

candicans (burm.f) hochr.) ở Việt Nam, Tạp chí Khoa học và công nghệ, 2018,

178(2), 1859-2171.

6. Vũ Thị Thu Lê, Đỗ Tiến Lâm, Cầm Thị Ính, Phạm Quốc Long, Trần Đăng

Thạch, Nguyễn Thị Hồng Vân, Phạm Thị Hồng Minh, Triterpenoid và

flavonoid từ dịch chiết etyl axetat của cây Nàng nàng (Callicarpa candicans

(Burm. f. Hochr.) Ở Việt Nam, Tạp chí Hóa học, 2018, 56(3), 341-345.

7. Do Tien Lam, Pham Minh Quan, Vu Thi Thu Le, Nguyen Thi Hong Van,

Cam Thi Inh, Ta Thi Thu Thuy, Pham Thi Hong Minh, Terpene compounds

isolated from Callicarpa macrophylla in Vietnam Journal of Science and

Technology, 2018, 56 (4A), 213-220

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