UNIVERSITY OF SCIENCE AND TECHNOLOGY OF HANOI

Pharmacological, Medical and Agronomical Biotechnology

FINAL REPORT

Project:
In vitro anti-inflammatory of several
compounds isolated from Fissistigma
pallens
Supervisors: Assoc. Prof. Do Thi Thao, PhD.
Dr. Nguyen Thi Hong Minh

Student: Le Nguyen Hai Giang - USTHBI8-052

ACKNOWLEDGEMENT
•I would like to express my gratitude to all those who aided me in my internship as well as all
experiences and encouragement that I received. First and foremost, I would like to give my sincere
to Associate Professor, Dr Do Thi Thao who provided me the opportunity to intern at her laboratory.
Her kindness and patience help me to overcome the hard situation. I am grateful to my internal
supervisor, Dr Nguyen Thi Hong Minh, too, who gave me helpful advices. I also want to thank to
Msc. Trieu Ha Phuong, who guided and taught me every lab technique during the internship and
aided me in writing this thesis. I also place on record, my thanks for the assitance of other member
of the laboratory of bioassays (IBT-VAST). Last but not least, I want to take this opportunity to
express my appreciation to my parents and my friends for their support and encouragement
throughout my study and internship.
 CONTENTS

 1) Introduction.
 2) Materials and Methods.
 3) Results.
 4) Discussion.
 5) Conclusion & Perspective.
 6) References.
 1. INTRODUCTION.

1
 1.1
INFLAMMATI
ON
 The natural process of body in fighting against
infections organisms, injuries, and toxins.

Two types:
o Acute: Immune cells recognize and destroy the
PAMPs (swelling, heat, redness and pain).
o Chronic: The resolution fail will lead to the
chronic stage.
• Example: asthma, stroke, even cancer.
2
 1.2 MACROPHAGES & ITS
PRODUCTION
 Macrophages: A major component of
mononuclear phagocytes system, formed via
monocytes differentiation.

 Functions:
 Innate: phagocytosis, produce NO &
cytokines.

 Adaptive: Expressing MHC to activate T-

cells, which kill the invaders directly and
precisely.

 Resolution of inflammation. Figure 1. Macrophage (Kumar, 2019)

3
 1.3 NITRIC OXIDE

 Endothelial-derived
relaxing factor.
Figure 2. Chemical formula of
 Colorless gas, short half- nitric oxide.
life, travel in limited
distances.

 Increased levels of NO
results in inflammation.

4 Figure 3. Synthesis of nitric oxide (Sharma and Parvathy, 2007)

  

Cytokines
 

   

Pro-inflammatory Anti-inflammatory
TNF- IL-6 IL-10
- Endogenous pyrogens
 
- Producing acute phase proteins in liver - IL10R
   
- Overproduction  diseases - Suppresion of macrophages and
their cytokines.
- TNFR1/TNFR2. - IL6R + gp130.  
    - Inhibiting MHC class II.
- NF-KB & MAPK. - JAK/STAT3.  
    - Reduced levels  diseases.
- Necrosis and apoptosis. - Development & infiltration of
  macrophages.
- Induction of other cytokines.  
- T cells activation.
 1.4 CURRENT ANTI-
INFLAMMATORY
Non-steroidal anti-inflammatory drugs
(NSAIDs) diverse of drugs that possess
anti-inflammatory, analgesic and
antipyretic properties.
Worldwide: Almost 70 million
people/day.
E.g.: Aspirin, piroxicam, celecoxib, etc.
6
 Figure 4. Mechanism and side effects of
7 NSAIDs (Cooper et al., 2019).
 1.5 FISSISTIGMA
PALLENS
 Fissistigma genus, Annonaceae family.
 Major compounds of Fissistigma genus:
aromatic alkaloids, flavonoids and
sesquiterpenes.
 Northern regions in Vietnam.
 Traditional uses: anti-malaria.
 Fissispallin A (FC-A, C30H42O7), fissispallin B
(FC-B, C30H44O8), fissispallin C (FC-C,
C31H46O8), fissispallin D (FC-D, C30H42O7)
showed significant cytotoxicity activity (IC50
0.4-7.8 μM) on three human cancer cell lines
Figure 5. F. pallens and 4 tested compound (Thinh et
8 (2019). al., 2019).
 Evaluating the anti-inflammatory
activity of these 4 compounds.
 2. MATERIALS

 2.1: Chemicals and cell lines.

Dulbecco’s Modified Eagle Medium (DMEM/F-12), fetal bovine serum antibiotics,

glutamine, HEPES (Gibco, Thermo Fisher Scientific - US), trypan blue solution (Gibco,
Thermo Fisher Scientific), trypsin (Gibco, Thermo Fisher Scientific), pure compound
extracted from Fissistigma pallens, Dimethyl sulfoxide (DMSO) (Invitrogen, Thermo
Fisher Scientific), Lipopolysaccharides (LPS) ( Invitrogen, Thermo Fisher Scientific), 3-
(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) (Invitrogen,
Thermo Fisher Scientific), NaCl (Invitrogen, Thermo Fisher Scientific), NaH 2PO4
(Tribioscience, US), Na2HPO4 (Tribioscience, US), the RAW 264.7 cell lines
(Laboratory of Bioassay, VAST), Mouse IL-6 DuoSet ELISA kit (R&D System, USA),
Mouse IL-10 (R&D System, USA), 4 tested compounds (VAST).
 2.2: Equipments
Micropipette (Eppendorf, Germany), plastic pipette (Corning Stripette Serological
Pipettes, US), pipette aid, Eppendorf 15ml tubes (Eppendorf, Germany), 50 ml tubes
(Eppendorf, Germany), 96-well plate (SPL Life Science, Korea), T25 flask (Thermo
Figure 6. RAW 264.7
Fisher Scientific-US), haemocytometer (Hirschmann Techcolor, Germany)
Microscope (Olympus Tokyo, Japan), CO2 incubator (Sanyo, Japan), autoclaves machine
cells (Nga et al., 2020).
(Japan), Microflow Advanced Bio Class 2 Safety Cabinet (Bioquell, UK), pH meter (pH
211, Hanna Instrument, Romania), microplate reader (ELX 800, BioTek Instrument Inc.,
Vermont, US), centrifuge machine (Z323K, HERMLE Labortechnik Gmbh, Germany).
 2. METHODS

24h 24h

11
 3. RESULTS (MTT ASSAY)
 Table 1: The cytotoxicity of four extracts of F. pallens on RAW 264.7 cells in vitro

Concentration Percentage of alive cells (%)

of samples
(M) FC-A FC-B FC-C FC-D

1.25 101.17  1.55 100.70  1.60 93.22  5.19 100  1.20

2.5 95.27  0.00 109.07  0.55 86.51  0.50 90.71  0.45
5 74.40  1.85 90.43  0.05 74.51  0.50 76.34  5.59
10 68.86  3.04 85.03  1.30 68.43  1.70 72.63 1.35

 The cells viability at all concentration was higher than 68%.

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 3. RESULTS (NO
INHIBITORY ASSAY)
 Table 2: Inhibitory effect of four compounds extracted from F. pallens on NO at different concentration

Percentage of NO inhibition (%)

Concentration
(M) FC-A FC-B FC-C FC-D

1.25 31.81  3.40 34.21  1.70 2.40  2.55 22.21  5.09

2.5 42.02  2.55 39.62  0.85 56.42  9.34 54.02  2.55

5 51.62  5.94 53.42  3.40 61.22  4.24 63.63  4.24

10 67.23  9.34 71.43  5.09 72.63  0.00 70.83  2.55

IC50 3.91  0.10 3.68  0.35 3.66  0.49 3.04  0.02

 All compounds had effective inhibitory activity (IC 50: 3.04 – 3.91 M).
13
 3. RESULTS (TNF- PRODUCTION)

Table 3. Effect of four compounds extracted from F. pallens on TNF- production at 4 different concentrations

 Concentration of  Percentage of TNF- production (%)

samples (M) FC-A FC-B FC-C FC-D

1.25 72.37*  2.49 100.02  2.34 93.69  0.73 92.65  2.49

2.5 59.85**  0.59 99.38  3.22 64.71**  3.07 62.96**  1.17
5 57.68**  0.15 56.33**  1.17 49.50**  3.22 60.47**  0.88
10 50.95**  1.46 51.06**  0.44 40.91**  5.71 53.02**  0.59

 All compounds highly affected TNF- at 5-10 M

 Strongest: FC-C (inhibited 59.09% at 10 M).
14  FC-A inhibited significantly at all concentration.
3. RESULTS (IL-6 PRODUCTION)

Table 4. Effect of four compounds extracted from F. pallens on IL-6 production at 4 different concentrations

 Concentration  Percentage of IL-6 production (%)

of samples (M) FC-A FC-B FC-C FC-D
 

1.25 93.35  3.34 88.47  2.37 100.01  7.25 99.44  3.16

2.5 90.13  4.86 85.27  1.00 99.35  5.01 87.15  4.74
5 86.27  4.25 82.12  5.53 93.30  8.69 78.21  1.58
10 85.19  5.16 54.19*  2.37 61.45*  9.48 40.22*  3.16
 IL-6 decreased with increasing concentrations of compounds.
 FC-B, FC-C, FC-D (10 M): significant inhibition (P < 0.05).
15
 3. RESULTS (IL-10 PRODUCTION)

Table 5. Effect of four compounds extracted from F. pallens on IL-10 production at 4 different
concentrations

 Concentration of  Percentage of IL-10 production (%)

samples (M)
FC-A FC-B FC-C FC-D

1.25 102.73  5.41 94.81  9.66 102.73  4.64 87.70  15.07

2.5 101.91  3.48 95.22  1.16 104.64  8.89 96.72  0.77
5 104.10  3.48 97.27  2.32 104.73  0.77 101.37  1.93
10 106.01  4.64 99.18  3.48 109.02  6.57 103.55  2.70

 IL-10 were not much stimulated.

16
 4. DISCUSSION

 The inhibitory effect of four compounds on NO and pro-inflammatory was highly

effective.
 Previous reports:
o Z23 (Fissistigma oldhamii) (6.25, 25, 100 M) inhibited NO, TNF-, IL-6
(RAW264.7).
o Ethanol CHCl3–soluble extract and alkaloids-2 (10 M) (Fissistigma oldhamii)
decreased TNF- (38.25 g/mL) and IL-6 (18.98 g/mL) (RAW264.7).
o Terpennoids 1-5 (Fissistigma polyanthoides) (IC50 : 13.7 μM - 49.0 μM)
had the dose-dependent inhibitory impacts on NF-κB/AP-1 produced by LPS-
stimulated THP1-Blue-CD14 cells.
o 2′,4,4′-trihydroxy-6′-methoxy-3′(3″-hydroxybenzyl) dihydrochalcone
(Melodorum siamensis) inhibited the nuclear factor-κB significantly in
pancreatic β cell line (MIN-6 cells) with IC50 value of 9 M.
17
 5. CONCLUSION &
PERSPECTIVE
 Conclusion:
 Four compounds (FC-A, FC-B, FC-C, FC-D) might presented
the potential anti-inflammatory activity and slightly toxic effects.

 Perspective: further study

 Testing again 3 cytokines at different time point.
 Testing inhibitory activity on:
• Other pro-inflammatory cytokines: IL-1, IL-4, IL-8, etc.
• Other inflammatory factors: COX-2, PGE2, NF-KB/AP-1,
etc.
18  Preclinical levels test.
 6.
REFERENC
ES
 Ge, Y. W. et al. (2013) ‘Aristololactams and aporphines from the stems of Fissistigma
oldhamii (Annonaceae)’, Phytochemistry, 86(6), pp. 201–207. doi:
10.1016/j.phytochem.2012.09.011.
 Hu, X. et al. (2008) ‘Anti-inflammatory effects of Z23 on LPS-induced inflammatory
responses in’, 120, pp. 447–451. doi: 10.1016/j.jep.2008.09.026.
 Jaidee, W. et al. (2019) ‘Amides and flavonoids from the fruit and leaf extracts of melodorum
siamensis’, Journal of Natural Products, 82(2), pp. 283–292. doi:
10.1021/acs.jnatprod.8b00696.
Kumar, V. (2019) ‘Macrophages : The Potent Immunoregulatory Innate Immune Cells’.

 Nguyen Ngoc, H. et al. (2019) ‘Terpenoids from the Stems of Fissistigma polyanthoides and
Their Anti-Inflammatory Activity’, Journal of Natural Products, 82(11), pp. 2941–2952. doi:
10.1021/acs.jnatprod.9b00208.
 Sharma, J. N. and Parvathy, S. S. (2007) ‘Review Role of nitric oxide in infl ammatory
diseases’, 15, pp. 252–259. doi: 10.1007/s10787-007-0013-x.
 Thinh, N. S. et al. (2020) ‘Cytotoxic sesquiterpene glucosides from Fissistigma pallens’,
Phytochemistry, 172(December 2019), p. 112255. doi: 10.1016/j.phytochem.2019.112255.
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