Nghiên cứu phân lập và khảo sát hoạt tính kháng sinh, gây độc tế bào của các hợp chất thứ cấp từ ba chủng xạ khuẩn streptomyces g246, g261, g248 thu thập tại vùng biển miền trung việt nam tt tiếng anh

MINISTRY OF EDUCATION AND
TRAINING

VIETNAM ACADEMY
OF SCIENCE AND TECHNOLOGY

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

CAO DUC DANH

STUDY ON ISOLATION AND INVESTIGATION OF ANTIBIOTIC
AND CYTOTOXIC ACTIVITY OF SECONDARY COMPOUNDS
FROM THREE ACTINOMYCETES STRAINS STREPTOMYCES G246,
G261, G248 COLLECTED IN SOME CENTRAL EAST VIETNAM SEA

Major: Organic Chemistry
Code: 9 44 01 14

SUMMARY OF CHEMISTRY DOCTORAL THESIS

HA NOI- 2019


This thesis was completed at: Graduate University Science and Technology - Vietnam
Academy of Science and Technology

Advisor 1: Prof. Dr. Habil. Pham Van Cuong
Advisor 2: Dr. Tran Dang Thach

1st Reviewer:

2nd Reviewer:
3rd Reviewer:

The thesis will be defended at Graduate University of Science and Technology - Vietnam
Academy of Science and Technology, at hour … date … month … 2019

The thesis can be found in
- The library of the Graduate University of Science and Technology, Vietnam Academy of
Science and Technology.
- The National Library of Vietnam.


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INTRODUCTION
1. The urgency of the thesis
The ocean accounts for 70% of the earth's surface, where there is the largest biological
diversity on earth. It is home to 34 of the 36 animal and plant sectors on earth with more than
300,000 known species. The marine environment has been known as a rich source of natural
compounds, such as a huge pharmaceutical warehouse awaiting exploration and discovery.
Characteristics of harsh deep sea habitats are the conditions for forming organic compounds
with unique chemical structure characteristics and precious biological activity. The study of
secondary active ingredients produced from marine microorganisms in the world has achieved
many remarkable achievements. Among them are many secondary compounds with interesting
chemical structure and biological activity. At the same time many of these compounds are
being tested further for medical applications. Vietnam is located in the Pacific region (with
sovereignty with an area of about 1,000,000 km2) with a rich and diverse marine system, with
great potential for marine resources. The Government of Vietnam has oriented the development
of marine economy, exploitation of natural resources and the study of natural products from the
sea. However, the study of secondary compounds from Vietnam's marine microbial source has
only just begun, there are very few published studies, although the source of marine diversity of

our country is very large. Infectious diseases account for the majority of human and animal
diseases. Around the second half of the 19th century, it was discovered that microorganisms are
the cause of infectious diseases. Therefore, chemotherapy aims at pathogenic microorganisms
that have been developed into the main therapy. In 1928, Alexander Fleming discovered
Penicillin - a powerful antibiotic compound and purified by Abraham, Chain, and Florey in a
stable form that had a therapeutic effect in 1941. Penicillin antibiotics became famous because
It saved many lives in World War II. Since then, antibiotics have become an early miracle
pharmaceutical that occupies the world's leading position in the pharmaceutical field, with
increasingly new results, with increasing demand and increasing production volume. Currently,
there are more antibiotics that are extracted from fungi, bacteria, actinomycetes, which occupy
most of them with marine actynomycetes. But more and more microorganisms cause disease
resistance to existing antibiotics. Therefore, it is necessary to continue to research, find and
discover new antibiotics, active ingredients with tuberculosis and anti-cancer resistance.
Therefore, we carried out this thesis with the title: “Study on isolation and investigation of
antibiotic and cytotoxic activity of secondary compounds from three actynomycetes strains
Streptomyces G246, G261, G248 collected in some central East Vietnam sea”


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2. The aim of the thesis
- Study to isolate and determine the structure of secondary compounds from cultivation of 3
strains of microorganisms isolated from central East Viet Nam sea.
- Survey of cytotoxic activity and testing antimicrobial activity of isolated substances as a
scientific basis for the research and application of these compounds.
3. The main contents of the thesis

- Review of previous studies on secondary compounds as well as biological activity from
marine microbial strains.
- Finding procedures for handling culture fluid to create extracts. Purify these extracts on

column chromatography to obtain fractions.
- Refining substances in the segments to obtain compounds.
- Determine the chemical structure of isolated compounds.
- Testing antimicrobial activity to test isolated substances.
- Testing anticancer activity in vitro for some cancer cell lines such as KB, MCF-7, Hep-G2,
Lu-1 of compounds isolated.
CHAPTER 1. OVERVIEW
1.1. The distribution and diversity of marine microorganisms
1.2. Bacterial radiation and the formation of secondary compounds from actinomycetes
1.3. Common marine bacteriophage families
1.4. Secondary compounds with biological activity from marine bacteriophages
1.4.1. Finding in the world
1.4.1.1. The compounds have antibiotic activity
1.4.1.2. The compounds are active against tuberculosis
1.4.1.3. The compounds with cytotoxic activity
1.4.2. Finding in our country
CHAPTER 2. MATERIALS AND METHODS
2.1. Materials and research equipment
2.1.1. Material
2.1.3. Device
2.2. Research Methods
2.2.1. Sample collection method
2.2.2. Marine bacteriophage isolation method
2.2.3. Methods for cleaning strains with loop implants
2.2.4. Method to keep the actinomycetes after isolation


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2.2.5. Activation and culture methods
2.2.6. Method of extraction from cultured fluid for biological activity screening

2.2.7. Method of identifying bacteriophages
2.2.8. Mass birth method
2.2.9. Methods of isolation of secondary compounds.
2.2.10. Methods for determining the chemical structure of clean compounds can be extracted
2.2.11. Method of testing antibiotic antimicrobial activity
2.2.12. Test method of cytotoxic activity
2.2.13. Test method for anti-tuberculosis activity.
CHAPTER 3. EXPERIMENT AND RESULTS
3.1. Sample collection results
25 samples have been collected from central East Vietnam sea, namely:
- In Thanh Hoa sea area, 2 sediment samples were collected.
- In Quang Binh sea area, 2 sediment samples, 1 sea form, 2 seaweed samples and 1 coral
sample were collected. In the Quang Tri sea area, there were 2 sediment samples, 1 sea pattern,
1 seaweed sample.
- In the water of Son Tra peninsula, Da Nang obtained 1 sample of sediment, 2 samples of
seaweed, 1 sample of seaweed, 1 sample of thorny skin, 1 sample of bag.
- In Cu Lao Cham coastal area, Quang Nam obtained 3 sediment samples, 1 model of thorny
skin, 1 soft body sample, 1 sea rabbit model, 1 solid tail pattern.
3.2. Result of isolation of actinomycete strains
- From 25 samples collected in the central waters of Vietnam, 32 bacteria strains with different
morphology and colors were isolated.
- From 12 samples collected in the waters of Thanh Hoa, Quang Binh and Quang Tri, 12
bacteria strains were isolated.
- From 13 samples collected in Da Nang and Quang Nam waters, 20 bacteria strains were
isolated.
3.3. Results of bioactivity test of actinomycete strains
Antiretroviral resistance results: 29/32 active strains (see details in table 3.1).
Results of anti-tuberculosis screening were performed at UIC University for
Mycobacterium tuberculosis H37Rv strain. The results showed that 21/32 strains showed antituberculosis activity, in which 1 strain showed very good activity with> 90% inhibitory value, 7
strains showed good activity with inhibitory%> 50% (see details in table 3.2).

4
Coarse residues of cultured extracts of 32 studied strains were tested for cytotoxic activity
against human cancer cell line (KB - carcinoma (CCL-17). Toxic cancer of KB epithelial cells
(see details in Table 3.3)
3.4. Results of identification of bioactive antibiotic strains
3.4.1. Observe morphological characteristics of studied strains
3.4.2. Multiplication of 16S RNA riboxom gene
3.4.3. Genome sequencing
The nucleotide sequence of the 16S ARN riboxom gene section of strains G246, G261,
G248 showed that the studied strains belong to genus Streptomyces sp. (see PLIII.1.4)
3.5. The results of high biomass of strains have good biological activity.
The strains of G246, G261, G248 have good biological activity, they are fermented in
large quantities of 50 liters in suitable conditions to conduct further studies.
3.6. Extraction and chemical structure of secondary compounds from Streptomyces sp.
G246
3.6.1. Sample processing, creating a residue
3.6.2. Isolate substances from the extract

Figure 3. 1. Diagram of isolation of Streptomyces sp. G246


5
3.6.3. Physical parameters and spectral data of compounds isolated from Streptomyces sp.
G246
3.7. Extraction and chemical structure of secondary compounds from Streptomyces sp.
G261
3.7.1. Sample processing, creating a residue
3.7.2. Isolate from Streptomyces sp. G261

Figure 3. 2. Diagram of isolation of Streptomyces sp. G261
3.7.3 Physical parameters and spectral data of secondary compounds from the Streptomyces
sp. G261
3.8. Extraction and chemical structure of secondary compounds from Streptomyces sp.
G248
3.8.1. Sample processing, creating a residue
3.8.2. Isolate substances from the extract

3.8.3. Physical parameters and spectral data of secondary compounds from the Streptomyces
sp. G248


6

Figure 3. 3. Diagram of isolation of Streptomyces sp. G248
3.9. Results of bioactivity test of compounds isolated from actinomycete strains
3.9.1. Performance test results for test microorganisms of some isolated compounds
Of the 26 substances isolated, we have tested antimicrobial activity. As a result, there are
13 substances with antimicrobial antibiotic activity, many of which have very strong activity
such as G246-2, G248-11, G248-12, G248-9 and many substances with good antifungal
activity as G246-6, G261-11, G248-11 (see details in table 3.5).
3.9.2. The results of anti-tuberculosis activity test of some isolated compounds
The results of tuberculosis resistance testing were performed at UIC University. Some
substances tested for anti-tuberculosis activity with Mycobacterium tuberculosis H37Rv, in
which G246-1 exhibited the best activity with MIC value of 6.00 µg / ml, G248-10 showed


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good activity with MIC value = 11.17 µg / ml, while G248-9 showed weak activity with MIC

value of 48.02 µg/ ml The remaining substances did not show activity at the study
concentration (50 µg/ ml) (see details table 3.6).
3.9.3. Test results of cytotoxic activity of some isolated compounds
14 compounds with tested antimicrobial activity, tuberculosis resistance from three
research strains (G246, G261 and G248) were tested for cytotoxic activity with four cancer cell
lines in: KB - Schedule cancer tissue (CCL - 17TM); Hep G2 - liver cancer (HB - 8065TM);
MCF-7 - breast cancer (HTB - 22TM) and LU-1 - lung cancer (HTB-57TM). The results are
detailed in Table 3.5. The result has 5 compounds with cytotoxic activity, including 4 active
substances with KB cell line, 5 active compounds with liver cancer cell line Hep-G2, 4 active
compounds calculated with Lu-1 cell lines, 4 active compounds with MCF7 cell lines (see
details in table 3.7)
CHAPTER 4. DISCUSSION OF RESULTS
4.1. Sample collection results
A total of 25 samples included: 12 samples in the waters of Thanh Hoa, Quang Binh and
Quang Tri; 13 samples in Da Nang and Quang Nam waters. Specifically, there are 10 samples
of sediment, 4 samples of seaweed, 4 samples of seaweed, 1 sample of soft corals, 2 samples of
echinoderms and 4 other samples.
4.2. Result of isolation of actinomycete strains
From 25 samples, 32 isolates with different morphology and colony color were isolated
from the collected sample (details are described in Appendix PL III.1.2).
4.3. Results of bioactivity test of strains obtained.
- The results of the test of antimicrobial activity with crude scale showed that 29/32 isolated
strains are resistant to the strains of test microorganisms.
- The results showed that 21/32 strains exhibited anti-tuberculosis activity.
- The results showed that 20 strains had toxic activity of KB epithelial cancer cells (IC50 <128
µg / ml). From the results of screening of anti-microbial antimicrobial activity, anti-tuberculosis
activity as well as cytotoxic activity (with KB line) in this thesis, I selected 3 isolates with the
best activity. for follow-up studies (including actinomycete strains G246, G261, G248).
4.4. Results of identification of bioactive antibiotic strains
4.4.1. Observe morphological characteristics of studied strains

4.4.2. Multiplication of 16S RNA riboxom gene
4.4.3. Solving the gene sequence to identify the name of the actinomycetes


8
Identified the actinomycetes strains G246, G261, G248 all belong to genus Streptomyces
sp. (See details at PLIII.1.4)
4.5. The results of high biomass of strains have good biological activity.
Successful fermentation of 50 liters for strains G246, G261, G248 to continue the next
study.
4.6. Chemical structure of secondary compounds from Streptomyces sp. G246
From the culture fluid of the microorganism strain Streptomyces sp. G246, we isolated
and determined the chemical structure of 10 compounds, including 2 new compounds G246-1
and G246-2. The structure of the compounds was determined by NMR, MS spectroscopy
methods and compared with reference materials, the compounds were identified as (2S, 2 ″ S) 6-lavandulyl-7-methoxy-5 , 2 ′, 4′-trihydroxylflavanone (G246-1), (2 ″ S) -5′-lavandulyl-4′methoxy-2,4,2 ′, 6′-tetrahydroxylchalcone (G246-2), cyclo- ( Pro-Gly (G246-3), cyclo- (L-ProL-Tyr) (G246-4), cyclo- (D-Pro-L-Tyr) (G246-5), cyclo- (Pro-Ala) ) (G246-6), norharman
(G246-7), tryptophan (G246-8), 3-indol carboxylic acid (G246-9), phenyl alanine (G246-10),
including 2 new substances G246-1 and G246-2. The absolute configuration of the two new
compounds G246-1 and G246-2 is determined based on the method of calculating quantum
circular electron chemistry (ECD) based on Gaussian 09 software.

Figure 4.28. Chemical structure of compound G246-1 to G246-10
4.6.1. Compound G246-1: (2S,2″S) -6-lavandulyl-7-methoxy-5,2′,4′-trihydroxylflavanone
(new compound)
Compound 1 was isolated as an amorphous solid, and optically active [a]25D -28(c, 0.8,
MeOH). Its positive HR-ESI mass spectrum showed the proton adduct ion [M+H]+ at m/z
439.2115 (calcd. for C26H31O6, 439.2121) which, together with

13

C-NMR data, are consistent

9
with the molecular formula of C26H30O6. The IR spectrum showed the presence of hydroxyl
(3299 cm-1) and carbonyl (1670 cm-1) functionalities.
In the 1H-NMR spectrum of 1, the presence of an ABX system at δH 6,35 (1H, d, J =

2.0 Hz, H-3′), 6,37 (1H, dd, J = 2.0, 8.5 Hz, H-5′), 7.31 (1H, d, J = 8.5 Hz, H-6′), and a
singlet proton at δH 6.13 was observed in the aromatic region. Additionally, the resonances of
protons at dH 5.56, 4.98, 4.54 and 4.59, a methoxy group at dH 3.84, three singlet methyls at δH
1.49, 1.58 and 1.65, and a number of aliphatic protons were noted. Analysis of the

13

C-NMR

data with the aid of HSQC experiment revealed 26 carbon resonances for G246-1, including
one ketone group (δC 193.9), ten sp2 quaternary carbons, five sp2 methines, one sp2 methylene,
two sp3 methines, three sp2 methylenes, three methyls and one methoxy group. Beside the
aromatic ABX system, two other spin-spin coupling systems were revealed from the COSY
spectrum: H-2/CH2-3 (I) and CH2-1”/H-2”/CH2-3”/H-4” (II)). The chemical shifts of carbons at

δC 75.5 (C-2), 156.6 (C-20), 159.5 (C-400), 164.9 (C-5), 161.9 (C-7) and 164.9 (C-9) suggested
their linkage to oxygen (Table 4.1). In the HMBC spectrum, cross-peaks of C-200 (δC 48.2)
with CH3-10” (δH 1.65) and CH2-9” (δH 4.54), and those of C-8” (δC 149.8) with protons of
CH2-1” (δH 2.64) indicated the linkage of C-2” of the coupling system II with the isopropenyl
group. Furthermore, the HMBC correlations of H-4” (δH 4.98) with C-6” (δC 17.8) and C-7”
(δC 25.9), and CH2-3” (δH 2.02) with C-5” (δC 132.0) indicated the lavandulyl group in the
structure of G246-1. The presence of the B-ring was confirmed by cross-peaks of H-3’ (δH
6.35) with C-1’ (δC 118.5), C-2’ (δC 156.6), C-4’ (δC 159.5) and C-5’(δC 107.6). Similarly, the

A-ring was established by HMBC correlations of H-8 (δH 6.13) with C-6 (δC 109.6), C-7 (δC
161.9), C-9 (δC 164.9) and C-10 (δC 105.7) (Figure 4.1).

Figure 4.1. Chemical structure and interactions HMBC (H → C), COSY (H─H) of compound
G246-1 and chemical structure of reference compounds.
The spectral features of coupling systems I and the carbonyl group deduced from HMBC
signals indicated structural similarity of G246-1 to flavanone compounds. The connection of


10
the lavandulyl group to the A-ring at C-6 via C-6/C-1” linkage was revealed by HMBC crosspeaks of the protons of CH2-1” with C-5, C-6 and C-7.
Table 4. 1. NMR spectral data of compound G246-1 and reference compound

C

C#,b

Ca

H (multi, J, Hz)

2

75,3

75,5

5,56 (dd, 2,5; 13,0)

3

42,8

48,2

2,89 (dd, 13,0; 16,5)
2,71 (dd, 2,5; 16,5)

4

198,2

193,9

5

163,0

164,9

6

96,2

109,6

7

165,3

161,9

8

107,8

93,4

9

162,1

164,9

10

103,2

105,7

1′

117,9

118,5

2′

156,1

156,6

3′

103,4

103,4

4′

159,4

159,5

5′

107,8

107,6

6,37 (dd, 2,0; 8,5)

6′

128,6

128,5

7,31 (d, 8,5)

1′′

27,7

28,2

2,64 (m)

2′′

47,8

48,2

2,52 (m)

3′′

31,9

32,4

2,02 (m)

4′′

124,5

124,8

5′′

131,6

132,0

6′′

17,9

17,8

1,97 (s)

7′′

25,8

25,9

1,98 (s)

8′′

149,1

149,8

9′′

111,2

111,2

6,13 (s)

6,35 (d, 2,0)

4,98 (t, 6,5)

4,54 (s)
4,59 (s)

10′′
OMe

19,1

19,2

1,65 (s)

55,9

3,84 (s)

a) measured in CD3OD; b) measured in CD3COCD3; δC # data of sophoraflavanone G compound [89]


11

Finally, the methoxy group at δH 3.85 was attached to C-7 as indicated by their
correlation in the HMBC spectrum. Complete analyses of 2D-NMR spectra established the
structure of G246-1 as 6-lavandulyl-7-methoxy-5,2’,4’-trihydroxylflavanone. Despite extensive
effort, attempts to produce suitable crystals of 1 and 2 for X-ray diffraction analysis were
unsuccessful. Alternatively, the absolute configurations of G246-1 were resolved by
comparison of their experimental and calculated electronic circular dichroism (ECD) spectra.
The ECD quantum chemical calculations were performed using the Gaussian 09 software. To
obtain minimum energy conformers, geometry optimization of each possible isomer of these
compounds was conducted. The calculated ECD spectra of compound G246-1 were generated
using the time-dependent density functional method at the B3LYP/6-31++G(d,p) level. Since,
the CD spectrum of compound G246-1 displayed a positive Cotton effect at 336nm (Δ +2.98
mdeg) and a negative Cotton effect at 292nm (Δ -9.08 mdeg), the S configuration was thus
assigned for carbon C-2 of G246-1 [89]. To determine the absolute configuration of C-2” of
G246-1, the ECD spectra of the two isomers, 2S,2”S-1 and 2S,2”R-G246-1, were performed in
gas phase. The experimental CD spectrum of G246-1 showed excellent agreement with the
calculated ECD of 2S,2”S-G246-1 (Figure 4.1). Thus, the S-configuration was suggested for
both C-2 and C-2” of compound G246-1.
4.6.2. Compound G246-2: (2″S)-5′-lavandulyl-4′-methoxy-2,4,2′,6′-tetrahydroxylchalcone
(new compound)

Figure 4.2. Chemical structure and interactions HMBC (H → C), COSY (H─H) of compound
G246-2 and chemical structure of reference compounds.
Compound G246-2 was isolated as an optically active [a]25D -8.6 (c, 0.25, CH2Cl2). Its
HR-ESI-MS showed the proton adduct ion [M+H]+ at m/z 439.2115 (calcd. For C26H31O6,
439.2121). A long with the

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C-NMR data, a molecular formula of C9H8O4 was suggested for

G246-2. Comparison of the 1D-NMR spectra with those of G246-1 revealed the same
substructures, lavandulyl group, A- and B-ring systems for compound G246-2. The differences
were noted for the signals of the CH=CH system [characterized from protons at H 7.82 (d, J =


12
16.0 Hz, H-a) and 7.96 (d, J = 16.0 Hz, H-b)] of G246-2 instead of the resonances of the
coupling system CH-2/CH2-3 in the structure of G246-1.
Table 4. 1. NMR spectral data of compound G246-2 and reference compound

C#

C#,b

C

Ca

H (multi, J, Hz)

1′

118,5

1

116,5

2′

156,6

2

156,9

3′

103,4

3

106,2

4′

159.5

4

159,1

5′

107,6

5

109,1

6,43 (br d, 8,0)

6′

128,5

6

131,0

7,39 (d, 8,0)

10

105,7

1′

106,2

9

164,9

2′

161,0

8

93,4

3′

90,9

7

161,9

4′

161,0

6

109,6

5′

107,4

5

164,9

6′

165,8

1′′

28,2

1′′

29,7

6,37 (br,s)

5,92 (s)

2,70 (dd, 6,0; 14,0)
2,71 (dd, 8,0; 14,0)

2′′

48,2

2′′

46,5

2,39 (m)

3′′

32,4

3′′

31,9

2,14 (m)

4′′

124,8

4′′

122,8

5′′

132,0

5′′

132,9

6′′

17,8

6′′

18,0

1,60 (s)

7′′

25,9

7′′

25,8

1,69 (s)

8′′

149,8

8′′

150,5

9′′

111,2

9′′

110,7

10′′

19,2

10′′

20,7

OH-6′

5,11 (t, 6,5)

4,76 (s)/ 4,79 (s)
1,72 (s)
14,46 (s)

OMe

55,9

OMe

55,7

3,85 (s)

3

48,2

α

126,0

7,82 (d, 16,0)

2

75,5

β

137,5

7,96 (d, 16,0)

4

193,9

γ

193,2

a) measured in CDCl3; b) measured in CD3OD; δC# data of G246-1 compound


13
This observation suggested a chalcone skeleton for G246-2 which was confirmed by
cross-peaks of H-b with C-2 (C 156.9), C-6 (dC 131.0) and C- γ (C 193.2) in the HMBC
spectrum of G246-2. The location of the lavandulyl at C-5’ was indicated by the HMBC
correlations of CH2-1” (H 2.70 and 2.71) with C-5’ (C 164.9) and C-6’ (C 109.6), and H-2”
(H 2.39) with C-6’ (C 109.6) (Figure 4.2). Similarly, HMBC cross-peaks of C-4’ (C 161.0)

with the protons of the methoxy at H 3.85 assigned the connection of C-4’ with the methoxy
group. Since, a strong coupling constant (J = 16.0 Hz) was noted for H-a and H-b, a transconfiguration was thus assigned for CH-a/CH-b double bond. Complete analyses of the 2DNMR spectra identified the structure of H246-2 as 5’-lavandulyl-4’-methoxy-2,4,2’,6’tetrahydroxylchalcone.
Despite extensive effort, attempts to produce suitable crystals of G246-2 for X-ray
diffraction analysis were unsuccessful. Alternatively, the absolute configurations of G246-2
were resolved by comparison of their experimental and calculated electronic circular dichroism
(ECD) spectra. The ECD quantum chemical calculations were performed using the Gaussian 09
software. To obtain minimum energy conformers, geometry optimization of each possible
isomer of these compounds was conducted. The calculated ECD spectra of compound G246-2
were generated using the time-dependent density functional method at the B3LYP/631++G(d,p) level. Similarly, the S-configuration was also assigned for compound G246-2 by
comparison of its experimental CD spectrum with the calculated ECD spectra (in gas phase) of
S- and R-configuration of G246-2.

4.7. Chemical structure of secondary compounds from Streptomyces sp. G261
From the culture fluid of the microorganism strain Streptomyces sp. G261 isolated and
determined the chemical structure of 13 compounds: norharman (G261-1), 2,3-butanediol
(G261-2), 1H-pyrrole-2-carboxylic acid (G261-3), 2- oxo-2,3-dihydrobenzo [d] oxazole-4carboxylic acid (G261-4), 3-hydroxy-4-methoxybenzoic acid (G261-5), 2-acetamidobenzamide
(G261-6), phenyl alanine (G261-7), cyclo-(Pro-Gly) (G261-8), cyclo- (Pro-Ala) (G261-9),
cylo-(Pro-Leu) (G261-10), cyclo-(Pro-Tyr) (G261-11), cyclo-trans-4-OH-(Pro-Phe) (G26112), cyclo-(Leu-Tyr) (G261-13), in which 1 substance was first isolated from nature are 2-oxo2,3-dihydrobenzo [d] oxazole-4-carboxylic acid (G261-4).
Below is a detailed description of the method of determining the structure of compounds
G261-4 which is the first compound isolated from nature.


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Figure 4. 2. Chemical structure of compound G261-1 to G261-13
4.7.1. Compound G261-4: 2-oxo-2,3-dihydrobenzo [d] oxazole-4-carboxylic acid

Figure 4.32. Chemical structure and interactions on HMBC spectrum of G261-4
ESI-HRMS mass spectra of G261-4 for the molecular mimic ion peak at m/z 178,0152
[M-H]- (theoretical calculation for molecular formula C8H4NO4 m/z 178,0140). 1H-NMR

spectra show signal appearance of aromatic ring protons in δH 7.24 (1H, d, J = 8.0 Hz, H-7);
7.23 (1H, dd, J = 8.0; 8.0 Hz, H-6); 7.49 (1H, dd, J = 2.0; 7.0 Hz, H-5). 13C-NMR spectrum in
combination with HSQC showed 3 aromatic methine groups at δC 118.1 (C-5); 123.6 (C-7);
126.4 (C-6); and 3 carbon quaternary sp2 at δC 116.7 (C-4); 144,2 (C-3); 146.5 (C-8); In
addition, the presence of a cabonyl group in the low-field region associated with nitrogen
allergens at δC 149,2 and a carbonyl group at δC 163.4 was also observed. In HMBC spectrum,
there is a long-term interaction of proton H-5 (δH 7.49) with C-4 (δC 116.7); C-3 (δC 144.2) and
C = O (δC 163.4) interaction of protons H-7 (δH 7.24) with C-8 ((C 146,5), C-6 (δC 123, 6).
Combining spectral data and comparing references [102] allowed the determination of G261-4
as 2-oxo-2,3-dihydrobenzo [d] oxazole-4-carboxylic acid. This is the first compound isolated
from nature [103].


15
4.8. Chemical structure of secondary compounds from Streptomyces sp. G248
From the cultured extract of the Streptomyces sp. G248, by chromatography and
spectroscopic methods MS, 1D-NMR, 2D-NMR, isolated and determined the chemical
structure of 13 symbol compounds from G248-1 to G248-13 including cyclo-(Pro-Leu) (G2481), cyclo-(Pro-Phe) (G248-2), norharman (G248-3), cyclo-(Pro-Tyr) (G248-4), cyclo-(Pro-Gly)
(G248-5), cyclo-(Pro-Trp) (G248-6), Adenine (G248-7), 2-(4-hydroxyphenyl) acetic acid
(G248-8), (2S,2″S)-6-lavandulyl-7,4′-dimethoxy-5,2′-dihydroxylflavanone (G248-9), (2S) -6prenyl-4′-methoxy-5,7-dihydroxylflavanone
tetrahydroxylflavanone(G248-11),
tetrahydroxylchalcone

(G248-12),

G248-10,
(2″S)

(2S,2″S)-6-lavandulyl-5,7,2′,4′-5′-lavandulyl-2′-methoxy-2,4,4′,6′-

(2S,2″S)-6-lavandulyl-7-methoxy-5,2′,4′-

trihydroxylflavanone (G248-13). Including 3 new compounds are G248-9, G248-11, G248-12
of flavonoids compound.

Figure 4.81. Chemical structure of compound G248-1 to G248-13
4.8.1. Compound G248-9: (2S,2″S)-6-lavandulyl-7,4′-dimethoxy-5,2′-dihydroxylflavanone

Figure 4. 3. Chemical structure and interactions HMBC (H → C), COSY (H─H) of G248-9
compound and chemical reference structure.


16
Compound G248-9 was isolated as an amorphous solid, and optically active [α]25D = - 5
(c 0.176, MeOH). Its positive HR-ESI mass spectrum showed the proton adduct ion [M+H]+ at
m/z 453.2270 (calcd for C27H33O6, 453.2277) which, together with 13C NMR data, is consistent
with the molecular formula of C27H31O6. The IR spectrum indicated the presence of hydroxyl
groups at 3366 cm-1, and a carbonyl functionality at 1696 cm-1. In the 1H-NMR spectrum, the
presence of an ABX system at δH 6.47 (d, J = 2.5 Hz, H-3’), 6.47 (dd, J = 2.0, 8.5 Hz, H-5’)
and 7.38 (d, J = 8.5 Hz, H-6’), and a singlet proton at δH 6.12 (s, H-8) was observed at the
aromatic region. Additionally, the resonances of protons at H 5.55, 4.96, 4.52 and 4.59, two
methoxy groups at H 3.82 and 3.83, three singlet methyls at H 1.48, 1.57 and 1.64, and a
number of aliphatic protons were noted. Analysis of 13C-NMR spectrum with the aid of HSQC
experiment revealed 27 carbon resonances for G248-9, including one ketone group (C 193.6),
three methyls, one sp2 methylene, three sp2 methylenes, five sp2 methines, two sp3 methines,
two methoxy groups and ten sp2 quaternary carbons (Table 1). Beside the aromatic ABX
system, two other spin-spin coupling systems were revealed from the COSY spectrum: H2/CH2-3 (I), and CH2-1’’/H-2’’/CH2-3’’/H-4’’ (II). The chemical shifts of carbons at C 75.0
(C-2), 160.1 (C-2’), 159.0 (C-4’’), 164.7 (C-5), 161.9 (C-7) and 165.4 (C-9) suggested their
linkage to oxygen. In the HMBC spectrum, cross-peaks of C-2’’ with CH3-10’’ and CH2-9’’,
and those of C-8’’ with protons of CH2-1’’ indicated the linkage of C-2’’ of the coupling

system II with the isopropenyl group. Additionally, HMBC correlations of H-4’’ with C-6’’ and
C-7’’, and CH2-3’’ with C-5’’ indicated the lavandulyl group in the structure of G248-9. The
presence of the A-ring was confirmed by cross-peaks of H-8 with C-6, C-7, C-9 and C-10.
Similarly, the B-ring was established by HMBC correlations of H-3’ with C-1’, C-2’, C-4’ and
C-5’. The spectral features of coupling systems I and the carbonyl group deduced from HMBC
signals indicated structural similarity of G248-9 to flavanone compounds. Furthermore, the
connection of the lavandulyl group to the A-ring at C-6 via C-6/C-1’’ linkage was revealed by
HMBC cross-peaks of the protons of CH2-1’’ with C-5, C-6 and C-7. Finally, two methoxy
group at H 3.82 and 3.83 was attached to C-7 and C-4’ as indicated by their correlation in the
HMBC spectrum (Figure 2). Complete analyses of 2D-NMR spectra established the planar
structure of G248-9 as 6-lavandulyl-7,4’-dimethoxy-5,2’-dihydroxylflavanone.
The G248-9 CD spectrum gives a positive Cotton effect at 336 nm (Δ +2.7) and a
negative Cotton effect at 292 nm (Δ - 8.5), allowing for absolute configuration at C- 2 is 2S
[89]. The absolute configuration at the C-2 ″ position of G248-9 is determined based on the
method of calculating the quantum electron quantum chemistry (ECD - Electronic Circular
Dichroism) based on the Gaussian 09 software. The theory for 2S,2″S- and 2S,2″R- allows the


17
determination of 2S, 2″S configurations of G248-9 compound in accordance with the
calculation model (Figure 4. 66).
Table 4.3. NMR spectral data of compounds G248-9 and compounds refer to G246-1

C

C#,a

Ca

H (multi, J, Hz)

2

75,5

75,0

5,55 (dd, 2,5; 13,0)

3

48,2

45,4

2,82 (dd, 6,0; 17,0)
2,90 (dd, 13,0; 17,0)

4

193,9

198,8

5

164,9

164,7

6

109,6

109,6

7

161,9

161,9

8

93,4

93,6

9

164,9

165,2

10

105,7

105,6

1′

118,5

119,7

2′

156,6

160,0

3′

103,4

99,8

4′

159,5

159,0

5′

107,6

108,1

6,47 (dd, 2,0; 8,0)

6′

128,5

128,5

7,39 (d, 8,5)

1′′

28,2

28,2

2,61 (m)

2′′

48,2

48,2

2,50 (m)

3′′

32,4

32,4

2,03 m

4′′

124,8

124,8

5′′

132,0

132,0

6′′

17,8

17,8

1,48 (s)

7′′

25,9

25,8

1,58 (s)

8′′

149,8

149,8

9′′

111,2

111,2

10′′

19,2

19,1

1,65 (s)

OMe

55,9

55,9

3,83 (s)

55,9

3,82 (s)

OMe
a) measured in CD3OD;

δC#

data of G246-1 compound

6,13 (s)

6,49 (d, 2,0)

4,97 (t, 6,5)

4,53 (m)/ 4,58 (m)


18
From the analysis on HR-ESI-MS, IR, 1D and 2D NMR spectra, CD and ECD spectrum
has been established, the structure of G248-9 is (2S,2″S)-6-lavandulyl-7,4′ -dimethoxy-5.2′dihydroxylflavanone. Finding on the Scifinder database allows to conclude that this is a new
compound.
4.8.2. Compound G248-12: (2″S)-5′-lavandulyl-2′-methoxy-2,4,4′,6′-tetrahydroxylchalcone

Figure 4. 4. Chemical structure and interactions HMBC (H → C), COSY (H─H) of G248-12
compound and chemical reference structure.
Compound G248-12 was isolated as an amorphous solid, with negative optical rotation
[α]25D -1.8 (c 0.54, CH2Cl2) . It’s positive HR-ESI-MS showed the proton adduct ion [M+H]+ at

m/z 439.2117 (calcd for C26H31O6, 439.2121). Considering the

13

C-NMR data, a molecular

formula of C26H30O6 was suggested for G248-11. Comparison of the 1D NMR spectra with
those of G248-9 revealed the same substructures, l A- and B-ring systems, avandulyl group for
compound G248-12. The differences were noted for the signals of the CH=CH system instead
of the resonances of the coupling system CH-2/CH2-3. This observation suggested a chalcone
skeleton for G248-12 which was confirmed by cross-peaks of H- with C-2, C-6 and C-γ in the
HMBC spectrum of G248-12. The location of the lavandulyl at C-5’ was indicated by the
HMBC correlations of H-1’’ with C-5’ (δC 108.9) and C-6’ (δC 166.6), and H-2″ with C-6’ (δC
166.6) (Figure 2). Similarly, HMBC cross-peaks of C-2′ (δC 162.3) with the protons of the
methoxy at H 3.91 assigned the connection of C-2’ with the methoxy group. Complete
analyses of 2D-NMR spectra established the structure of G248-12 as 5’-lavandulyl-2′methoxy-2,4,2’,6’-tetrahydroxylchalcone. This compound has the same planar structure of
kuraridin, which was previously isolated from Albizzia julibrissin [6]. However, the two
compounds have different rotational degrees, so in terms of stereoscopic structure of the
compound G248-12 is different from kuraridin (with polarity [α]25D −25.5 (c 0.1, MeOH).
Absolutely at position C-2″ of G248-12 is determined based on the method of calculating
quantum electron quantum chemistry (ECD - Electronic Circular Dichroism) based on software
Gaussian 09. Theoretical calculation for 2 isomers 2″S- and 2″R- allow to determine the
configuration 2″S of the compound G248-12 in accordance with the calculation model (Figure


19
4. 76). 1D, 2D-NMR spectrum CD and ECD we identified G248-12 as (2″S)-5′-lavandulyl-2′methoxy-2,4,4′,6′-tetrahydroxylchalcone. Whether Scifinder allows to conclude this is a new
compound.
Table 4.4. NMR spectral data of compound G248-12 and reference compound

Ca

*Ca

(G246-2)

(Kuraridin)

1

116,5

125,0

124,4

2

156,9

160,3

160,3

3

106,2

103,7

103,7

4

159,1

162,4

162,4

5

109,1

108,9

109,0

6,36 (m)

6

131,0

131,6

131,6

7,42 (d, 8,0)

1′

106,2

108,9

108,8

2′

161,0

164,0

164,0

3′

90,9

91,6

91,6

4′

161,0

162,4

162,3

5′

107,4

116,3

116,2

6′

165,8

166,6

166,6

1′′

29,7

28,2

28,2

2,64 (m)

2′′

46,5

48,0

48,0

2,57 (m)

3′′

31,9

32,4

32,4

2,10 (m)

4′′

122,8

125,0

125,0

5,06 (m)

5′′

132,9

131,8

131,8

6′′

18,0

17,9

17,9

1,58 (s)

7′′

25,8

25,9

25,9

1,65 (s)

8′′

150,5

149,9

149,9

9′′

110,7

111,1

111,1

C

#

Ca

H (multi, J, Hz)

6,37 (d, 2)

6,02 (s)

4,55 (d, 2,5)
4,61 (dd, 2,5; 1)

10′′

20,7

19,0

19,0

1,72 (s)

OMe

55,7

56,1

56,0

3,91 (s)

α

126,0

125,5

125,4

7,96 (d, 16,0)

β

137,5

139,8

139,8

8,02 (d, 16,0)

γ

193,2

194,8

194,7

#

*

a) measured in CD3OD; δC, δC data of reference compound G246-2 and kuraridin [115]


20
General remarks on secondary compounds extracted from 3 studied bacteriophage
strains: From 3 actinomyces strains showing antimicrobial resistance and cytotoxic activity
including G246, G261, G248 were isolated and determined chemical structure 36 compounds.
However, there are many similar compounds, so only 26 different compounds are actually
collected. There are 6 compounds of flavonoids, 9 compounds of the class of dicyclopeptide
and 3 compounds of indole and other compounds. New compounds are G246-1, G246-2,
G248-9, G248-11, G248-12 are all of flavonoids.

4.9. Interpretation of the results of testing the biological activity of compounds isolated
from actinomycete strains.
4.9.1. The results of activity tests for test microorganisms of some compounds
Twenty-six isolated compounds were tested to be active for seven tested microorganism
strains, including three Gram-negative strains: E. coli ATCC25922, P. aeruginosa ATCC27853,
S. enterica ATCC13076; 3 strains of Gram-positive bacteria: E. faecalis ATCC29212, S. aureus
ATCC25923, B. cereus ATCC 13245 and 1 strain of C. albicans ATCC10231. The results
showed that 13 antimicrobial compounds tested, including highly active compounds such as
compounds G246-2, G246-6, G248-12, G248-9 isolated from 2 strains of Streptomyces sp.
(G246, G248) in Da Nang and Quang Nam sea. Especially 4 compounds G246-2, G248-9,
G248-11, G248-12 show strong activity for all 6 tested microorganism strains, 5 new
compounds are G246-1, G246-2, G248-11, G248-12, G248-9 both show strong anti-microbial
antimicrobial activity, as compound G248-11 has very interesting antimicrobial activity with
MIC value of 1 (µg/ ml) with 5 strains of tested microorganisms. Particularly compound G24812 shows strong activity for 7 strains of tested microorganisms. There are 3 substances that can
inhibit Gram (-) E. coli bacteria, among them the compound G248-12 inhibits Gram (-) E. coli
bacteria quite strongly with MIC value of 4 (µg/ ml). In addition, compounds G246-2, G246-6,
G248-11, G248-12, G248-9 show good fungal inhibitory activity with MIC values of 8, 2, 1,
16, 16 µl/ ml. (See Table 3.5)
4.9.2. Results of anti-tuberculosis activity test of some compounds
Several isolated compounds have also been investigated for anti-tuberculosis activity
against Mycobacterium tuberculosis H37Rv. However, most of the compounds did not show
activity, except for compound G246-1 which showed the strongest activity with MIC value =
6.00 µg/ ml, compound G248-9 had weak activity with the price MIC value = 48.02 µg/ ml is
due to the structure of the two compounds G246-1 and G248-9 differing in position 4'of round
B in the flavanone frame, while the compound G248-10 exhibits activity good with MIC value


21
of 11.17 µg / ml. These compounds are isolated from G246 and G248 strains in Da Nang
waters of Vietnam (see table 3.7).

4.9.3. Test results of cytotoxic activity of some isolated compounds
14 compounds with antimicrobial activity and tuberculosis resistance were isolated from
3 strains G246, G261, G248 tested cytotoxic activity for 4 cancer cell lines KB, Lu-1, Hep- G2
and MCF7. The results have 12 compounds with cytotoxic activity, including 9 active
substances with KB cell line, 10 active compounds with 10 liver cancer cell lines Hep-G2, 8
substances with lines Lu-1 cells, 9 substances with MCF7 cell line. Compound G246-1, G24810 have quite strong activity for KB epithelial cancer cell lines with IC50 values of 11.05, 5.65
µM respectively. Compound G246-1, G246-2, G246-10 have strong activity against liver
cancer cells Hep-G2 with IC50 values of 5.18; 6.51; 5.65 µM. The compounds G246-1, G24810 also exhibited strong cytotoxic activity for 3 cancer: KB, Hep-G2, Lu-1 with IC50 values
between 5.18 and 11, 05 µM (see details in table 3.6)

CONCLUSIONS AND RECOMMENDATIONS
* CONCLUSIONS
The main results of the thesis are:
1. 32 actinomycetes strains were isolated from 25 samples collected in 5 seas (Thanh
Hoa, Quang Binh, Quang Tri, Da Nang, Quang Nam) and screened biological activity of 32
isolated actinomycetes strains. 3 actinomycetes strains with the best activity were selected to
carry out the next studies, including strains G246, G248 collected in Son Tra peninsula, Da
Nang, G261 strains collected at Cu Lao Cham, Quang Nam . Three strains have been identified
(G246, G261, G248) by 16S rARN sequencing and they belong to the genus Streptomyces.
2. The chemical structure of 36 secondary compounds from 3 actinomycetes strains
G246, G261 and G248 were collected, respectively 10, 13 and 13 clean compounds. In which
there are 5 new flavonoid compounds are (2S,2″S)-6-lavandulyl-7-methoxy-5,2′,4′trihydroxylflavanone (G246-1), (2″S)-5′-lavandulyl-4′-methoxy-2,4,2′,6′-tetrahydroxylchalcone
(G246-2), (2S,2″S)-6-lavandulyl-7,4′-dimethoxy-5,2′-dihydroxylflavanone ( G248-9), (2S, 2 ″
S)-6-lavandulyl-5,7,2′,4′-tetrahydroxylflavanone (G248-11), (2 ″S)-5′-lavandulyl-2′-methoxy2,4,4′,6′-tetrahydroxylchalcone (G248-12) and 1 first compound isolated from nature are 2oxo-2,3-dihydrobenzo [d] oxazole-4-carboxylic acid (G261- 4).
3. The isolated compounds were investigated for biological activity. In which, 4
compounds (G246-1, G246-2, G246-4, G246-6) isolated from Streptomyces sp. G246, 4
compounds (G261-1, G261-4, G261-10, G261-13) isolated from Streptomyces sp. G261, 5


22

compounds (G248-2, G248-7, G248-9, G248-11, G248-12) isolated from Streptomyces sp.
G248 exhibits antimicrobial activity. Special compounds G246-2, G248-11 have strong
antimicrobial activity. For anti-tuberculosis activity, only compound G246-1 exhibited good
activity against Mycobacterium tuberculosis H37Rv with MIC value of 6.00 μg/ ml.
4. In addition, 14 compounds have been evaluated to inhibit the growth of four cancer
cell lines (KB, Lu-1, Hep-G2 and MCF7). Compound G246-2, G248-10 exhibited cytotoxic
activity for KB epithelial cell line with IC50 values of 11.05, 5.65 µM, compound G246-1,
G248-10 exhibited good cytotoxic activity for all 3 cancer cell lines: KB, Hep-G2, Lu-1 with
IC50 values ranging from 5.65 to 11.05 µM.
* RECOMMENDATIONS
- Need to expand research in other sea areas of our country to detect marine microbial strains
capable of producing compounds with high biological activity, in order to research
applications. At the same time, it is necessary to continue to study the isolated bacteriophage
strains, as well as the fermentation conditions in order to find new substances with
antimicrobial, tuberculosis and cytotoxic activity in the theoretical framework. This project has
no conditions for research.
- It is necessary to have follow-up studies for compounds with anti-microbial antibiotic activity,
tuberculosis resistance and strong cytotoxic activity as compounds G246-1, G246-2, G248-11
with next generation further testing in vitro activity, studying the mechanism of action.


23
NEW FINDINGS OF THE THESIS
Content of new findings of the thesis
1. 32 bacteria strains were isolated from 25 samples collected in 5 seas (Thanh Hoa,
Quang Binh, Quang Tri, Da Nang, Quang Nam) and tested biological activity of 32 isolated
actinomycetes strains, the results: 29/32 tested antimicrobial strains, 21/32 TB resistant strains,
20/32 strains with KB cytotoxic activity. Three antimicrobial strains have been selected (G246,
G261, G248) with good biological activity, they all belong to Streptomyces genus and ferment
in large quantities (50 liters) to carry out further studies.

2. There are 36 compounds isolated from 3 bacteriophage Streptomyces G246, G261,
G248,

including

5

new

compounds

(2S,2″S)-6-lavandulyl-7-methoxy-5,2′,4′-

trihydroxylflavanone (G246-1), (2″S)-5′-lavandulyl-4′-methoxy-2,4,2′,6′-tetrahydroxylchalcone
(G246-2), (2S,2″S)-6-lavandulyl-7,4′-dimethoxy-5,2′-dihydroxylflavanone (G248-9), (2S,2″S)6-lavandulyl-5,7,2′,4′-tetrahydroxylflavanone

(G248-11),

(2″S)-5′-lavandulyl-2′-methoxy-

2,4,4′,6′-tetrahydroxylchalcone (G248-12) and have 1 substance first isolated from nature 2oxo-2,3-dihydrobenzo[d]oxazole-4-carboxylic acid (G261-4)
3. Newly isolated compounds have good biological activity. Especially compounds
G246-2, G248-11 have very strong antiviral activity. The compound G246-1 exhibits very
good activity for Mycobacterium tuberculosis H37Rv with MIC value of 6.00 μg/ ml. The
compound G246-1 exhibits good cytotoxic activity for 3 KB cancer cell lines, Hep-G2, Lu-1
with an IC50 value between 5.18 and 11.05 µM.