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FITRIA NUGRAHA AINI / IRYASTI YUDISTIA / MERKURI PUSPITA S.

2018
INTRODUCTION
 Infammatory bowel diseases (IBD), such
as Crohn’s disease and Ucerative colitis, are
chronic relapsing disorders of the
gastrointestinal tract that are pathologically
characterized by intestinal infammation and
epithelial injury.
IRRITABLE
BOWEL
DISEASE
IBD Therapy
 Most of the current therapies for IBD involve treatment with
glucocorticosteroids, 5-aminosalicylic acid and immunosuppressive
drugs.
 Pharmacological effects :

Antibacterial

Antiulcerogenic

Anti-inflammatory

Portulaca Antioxidant

oleracea L. Woundhealing

Ameliorate acute intestinal


inflammation

Protective effect of ulcerative


colitis
MATERIALS AND
METHODS
Preparation of the
extract and purification
of compounds from
Portulaca oleracea
 Cell culture
 RAW264.7 cells, a mouse macrophage cell line were cultured in
DMEM supplemented with 10% heat-inactivated FBS and antibiotics
(100 U/mL penicillin, 100 μg/mL streptomycin) in a humidified, 5%
CO2 atmosphere at 37 °C.

 Cell viability assay


 Cell viability was determined using the MTT assay according to the
manufacturer's protocol.

 NO assay
 The culture supernatant (100 μL) was mixed with Griess reagent for
10 min, and the absorbance was measured at 550 nm.
 Experimental colitis was induced by providing mice drinking water containing 3%
dextran sulphate sodium (DSS) for 14 days ad libitum.

 For each experiment, the mice were divided into experimental groups:
Group 1  Control  administered PBS via the same route as P.oleracea
Group 2  Only d rinking water containing DSS throughout the experimental period.
Induction of Group 3-9  Comprised mice that received 3% DSS and were administered
sulfasalazine (50 mg/kg/day p.o.) or P. oleracea extracts (100, 300, and 500 mg/kg/day
Colitis p.o.) daily for 14 days according to the experimental design.
 Histopathology
 The resected large intestine  examined for mucosal defects,
haemorrhage, or ulcerative lesions  fixed with 4% neutral formalin
 stained with haematoxylin and eosin (H&E) and viewed under a
light microscope (10–1000×).

 Measurement of cytokine levels


 The Quantikine quantitative sandwich enzyme immunoassay
technique was used to analyse cytokine levels

 Statistical analysis
 p values were analysed using Student's t-test.
 Statistically significant at *p < 0.05, **p < 0.01, and ***p < 0.001.
RESULT
P. oleracea extracts inhibit LPS-
induced NO production and cell
viability
Effects of P. oleracea
extracts on LPS-
induced inflammatory
cytokine
production in
RAW264.7 cells

 in vitro ELISAs revealed significant increases in levels of the inflammatory cytokines TNF-α, IL-6 and
IL-1β in macrophages stimulated with LPS (1.0 μg/mL for 24h) compared to control macrophages
Effects of P. oleracea extracts on MAPK
activation in macrophages
P. oleracea ameliorated disease progression and pathological changes
associated with DSS-induced colitis in mice
Inhibitory effects of P. oleracea extracts
on inflammatory cytokines in
DSS-treated mouse serum
Purified active
compounds slow
disease progression
and ameliorate
pathological changes
in a mouse model of
DSS-induced IBD
Inhibitory effects of
bioactive compounds
on inflammatory
cytokine
production in DSS-
treated mice
 The innate immune response plays a crucial role in IBD, and
activated immune cells release inflammatory cytokines such as
TNF-α, IL-6 and IL-1β that regulate the differentiation of T cells and
the inflammatory response.
 Dysregulation of T cells or over-production of effector T cells is
involved in the progression and exacerbation of IBD.
DISCUSSION  Tissue analyses of patients with ulcerative colitis have reported
significant increases in the secretion of pro-inflammatory
cytokines, such as TNF-α, IL-6 and IL-1β  Thus, finding a
substance that modulates the production of
inflammatorycytokines is thought to be important for the
treatment of IBD.
 In this study, the effects of P. oleracea extracts and their active
compounds on IL-6 and TNF-α secretion in mice with DSS-induced
IBD.
 In the DSS-induced mouse model, the inhibitory effect of the P.
oleracea EtOAc fraction and its active compounds on TNF-α
DISCUSSION secretion was superior to SS, a commonly used therapeutic agent.
 IL-6 and IL-1β levels in animals treated with the EtOAc extract and
the active compounds were similar to the control group.
 Thus, P. oleracea inhibits IBD by controlling the levels of the
inflammatory cytokines TNF-α, IL-6 and IL-1β in mice with DSS-
induced IBD.
 In this study :
Evaluated cytotoxicity, the inhibition of NO production and
inflammatory cytokines by ELISA of P. oleracea extracts on
macrophage cells.
In addition, extracts were presumed to be involved in inhibiting the
expression of genes involved in various inflammationrelated
signalling pathways, such as ERK, JNK, and p38.

CONCLUSION Next, they isolated a potent active compounds from P. oleracea


extracts  found that this compounds has Portulacanone C, trans-n-
feruloyltyramine and cis-n-feruloyl-3′-methoxytyramine were
identified by HPLC, NMR and electrospray ionization-mass
spectrometry (ESI-MS).
Consequentially, extracts and compounds of P. oleracea display a
strong natural inhibition of IBD on in vitro and in vivo model.
Overall, this extracts and compounds has the potential to be
developed into newbiomaterials.
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