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The 6 International Conference on Natural Products for Health and Beauty (NATPRO6)
January 21-23, 2016
Organized by Khon Kaen University

Effect of Cissus quadrangularis, Pluchea indica and Clerodendrum

serratum extracts on expression of cyclooxygenase proteins
Lawan Siangjong*, Auayporn Apirakaramwong, Penpun Wetwitayaklung
Faculty of Pharmacy, Silpakorn University, Nakorn Pathom 73000, Thailand
*SIANGJONG_L@su.ac.th

phone +66-34255800

fax +66-34255801

ABSTRACT
Several Thai herbal medicines have been safely and effectively used for treatment of hemorrhoid, a disease of
enlarged anal veins associated with inflammation. Cissus quadrangularis L. (CQ), Pluchea indica (L.) Less (PI) and
Clerodendrum serratum Moon var. wallichii Clarke (CS) are commonly used for hemorrhoid treatment. To investigate the
anti-inflammatory effect of CL, PI and CS crude extract, we examined cyclooxygenase (COX)-1 and COX-2 protein
expression using western immunoblotting technique. MTT cell viability test indicated that the ethanoic extract of CQ, PI
and CS showed minimum toxicity in HeLa cells at a concentration of 3.13, 1.56 and 3.13 g/ml, respectively. In LPSactivated HeLa cells, COX-2 protein expression did not change with the treatment of CL, PI and CS extract. Similar findings
were obtained with COX-1 protein. Our results suggest that these medicinal plant extracts may mediate antiinflammatory effect through different pathways. Alternatively, the inhibition does not change protein expression but
could involve enzyme activity, which should be further investigated.
Keywords: Cissus quadrangularis L., Pluchea indica (L.) Less, Clerodendrum serratum Moon, anti-inflammation,
cyclooxygenases

2. INTRODUCTION
Hemorrhoid is a common disease characterized by enlarging of veins in the anal canal, in which can cause
bleeding and pain if ruptures. The lack of exercise, low-fiber diets and modern-day lifestyle, are ones of the major factors
contributing to the increasing prevalence of hemorrhoid. Hemorrhoid can be manifested by chronic inflammation of
tissues in the rectal area causing pain, discomfort and embarrassment to patients. Treatments for hemorrhoid depend on
the disease stage. Many Thai herbal medications have been commonly used for treatment of hemorrhoid due to their
safety and low cost. Cissus quadrangularis L. (CQ), in Vitaceae family, is the only herbal remedy for hemorrhoid that is
listed in the National List of Essential Medicine (NLEM). While CQ has been intenstively studied for its mechanism of
action, other herbs such as Pluchea indica (L.) Less (PI) and Clerodendrum serratum Moon var. wallichii Clarke (CS) are
also widely and effectively used. It was reported that CQ and PI elicited its anti-inflammatory effect through inhibition of
nitric oxide (NO), lipoxygenase (LO) and cyclooxygenase (COX) pathways [1-4]. They inhibited prostaglandin E2
production, EPP-induced ear edema and carrageenan-induced paw edema in animal models. In contrast, CS has only few
studies on its anti-inflammatory action. COX is an important enzyme that produces many inflammatory mediators from
arachidonic acids, particularyly PGE2. It is also a direct target of non-steroidal anti-inflammatory drugs (NSAIDs), which
are effectively used for combating inflammation and pain. Thus, in the present study, we investigate the effect of PI and
CS, compared with CQ, on the expression of COX-1 and COX-2 proteins in order to explore the mechanism of action of
three commonly used herbal medicines for hemorrhoid treatment.

2. MATERIALS AND METHODS

Ethanol extraction of crude extract from plant samples
Pluchea indica (L.) Less (PI), Clerodendrum serratum Moon var. wallichii Clarke (CS) and Cissus quadrangularis L.
(CQ) were cut into small pieces followed by drying in a hot air oven at 50C for 3-5 days. 5 g of dried plants was extracted
with 95% ethanol above boiling water for 15 min and the filtrate was consequently collected. Then, the residue was

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replenished with ethanol and the extraction was repeated. All filtrates were combined and the ethanol was evaporated.
The dried crude extract was then stored at 4C and protected from light.
Cell culture and MTT cell viability assay
HeLa cells were cultured in D
ulbeccos Modified Eagles Medium (DMEM) supplemented with 10% fetal
bovine serum (FBS), 4.5 g/l D-glucose, 100 units/ml penicillin, 100 g/ml streptomycin, 2 mM L-glutamine and 1%
nonessential amino acids. The cells were maintained in a humidified chamber at 37C, 5% CO2. To perform MTT assay,
4
HeLa cells (1.5x10 cells) were seeded in a 96-well plate at 100 l. Next day, cells were treated with crude extract of PI,
CS and CQ dissolved in DMSO for 24 h. Subsequently, 10 l of MTT solution (2 mg/ml, Sigma) was added to each well and
incubated in the humidified incubator for 2 h. After removing the media, DMSO was added to dissolve the formazan
product, which is generated by mitochondria of viable cells, and the absorbance was measured at 550 nm after 2 h of
incubation at room temperature. % Cell viability was calculated as percentage of the absorbance of the treatments
subtracted with that of cell-free control compared with DMSO.
Western immunoblotting
5
To measure the expression of COX proteins, HeLa cells (3x10 cells) were cultured in a 6-well plate until 70%
confluency. Cells were washed with phosphate buffer saline (PBS) and replaced with serum-free media. After 24 h, crude
extracts of PI, CS and CQ at the minimum toxicity concentrations were used to treat cells for another 24 h. When
appropriate, lipopolysaccharide (LPS, Sigma) activation of inflammation was induced at 30 min upon treatments.
Subsequently, the cells were lysed in lysis buffer containing 60 mM Tris-HCl buffer, pH 6.8, 2% SDS and protease
inhibitors, with sonication at 130 watts, 40% amplitude for 5 sec with 5-sec pause (5 times). The samples were then
centrifuged at 12,000 xg for 10 min at 4C (Haraeus Biofuge 15R). Thereafter, the supernatant was collected and used for
BCA protein concentration assay (Thermo Scientific). 30 g of total protein lysates were then subjected to SDS-PAGE
using 10% acrylamide gel, and, afterwards, transferred to PVDF membrane. The protein blot was subsequently blocked
for non-specific binding with 5% skimmed milk in Tris buffer saline (TBS) containing 0.1% tween 20 (TBS-T), at room
temperature for 1 h. Next, the blot was probed with rabbit anti-COX-1 and COX-2 antibodies (1:1000, Cell Signaling
Technology) in 5% BSA/TBS-T, overnight with gentle shaking at 4C. The secondary antibody was goat anti-rabbit IgG
conjugated with horseradish peroxidase (HRP) (1:10,000, KPL), which was probed for 1 h at room temperature. The
protein bands were detected with enhanced chemiluminescence (ECL, Thermo Scientific) and X-ray film (Thermo
Scientific).

3. RESULTS
To study the effect of our interested plant extracts on the expression of inflammatory COX proteins, we first
investigated the toxicity of the ethanoic extracts on HeLa cells. Upon treatments of different concentrations of the
extracts for 24 h, the cells were tested with MTT cell viability assay. The results showed that these ethanol extracts
affected HeLa cell survival in a concentration-dependent manner compared to the vehicle control DMSO (Figure 1).
However PI, CS and CQ at the concentration of 3.13, 1.56 and 3.13 g/ml, respectively, elicited the least toxicity effect on
the cells. Furthermore, at the high dose 400 g/ml, PI, CS and CQ caused 85, 60, 59% cell survival, respectively, suggesting
that PI is possibly less toxic to the cells than the others. When PI concentration was doubly increased to 800 g/ml, the
comparable survival effect (approximately 60 %) was then obtained. The minimum toxicity concentration was
subsequently used for examining the expression of COX-1 and COX-2 proteins using western immunoblotting technique.
However, we found that the 65 kDa protein bands of COX-1 in HeLa cells did not change with the treatments at the
concentration tested (Figure 2). Likewise, in HeLa cells activated with LPS, these extracts had no effect on the expression
of COX-2 protein, which is well known for its principal role in inflammation (Figure 3). Moreover, these finding is
consistent with our quantitative reverse transcriptase (RT) PCR (data not shown).

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Figure 1. Effect of PI, CS and CQ extracts on viability of HeLa cells. HeLa cells were treated with different
concentrations of PI, CS and CQ extracts for 24 h and assessed with MTT cell viability assay. % Cell viability was
compared with cells treated with DMSO as a control.

Figure 2. Effect of PI, CS and CQ extracts of COX-1

COX 1 protein expression. HeLa cells were treated with DMSO (C), PI (3.13
g/ml), CS (1.56 g/ml) or CQ (3.13 g/ml) extracts for 24 h. Total lysate were analyzed with western immunoblotting
using
ng specific monoclonal antibody against COX-1
COX 1 protein (1:1,000, Cell Signaling Technology).

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Figure 3. Effect of LPS and PI, CS and CQ extracts on COX-2 protein expression. HeLa cells were treated with DMSO as
a control or extracts of PI (3.13 g/ml), CS (1.56 g/ml), CQ (3.13 g/ml). Upon the treatments, cells were activated
with LPS to promote inflammation. The symbols + and ++ indicate the concentration of LPS; 1 and 5 g/ml,
respectively. The equally loaded cell lysates were subjected to western immunoblotting and detected for COX-2
protein expression using anti-COX-2 antibody (1:1,000, Cell Signaling Technology).

4. CONCLUSIONS
Our study showed that ethanoic extracts of PI, CS and CQ did not change the protein expression of COX-1 and
COX-2 in HeLa cells. Interestingly, previous studies showed that PI and CQ inhibited PGE2 production [1, 3], an important
inflammatory mediator produced by COXs. Therefore, our findings suggest that the mechanism of anti-inflammation of
these plants might not take place at the protein expression level but could involve the enzyme activity. Moreover, these
ethanol plant extracts might mediate anti-inflammatory effect through different pathways. Further studies are required
to investigate the exact mechanism and of these herbal medicines commonly used for treatment of hemorrhoids.

ACKNOWLEDGEMENTS
This work was supported by the Faculty of Pharmacy, Silpakorn Univeristy, and a New Researcher Scholarship of
CSTS, MOST, funded by Coordinating Center for Thai Government Science and Technology Scholarship Students (CSTS),
National Science and Technology Development Agency (NSTDA).

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