efek pluchea indica pd cox

7/25/2019 Efek Pluchea Indica Pd Cox

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The 6th

International Conference on Natural Products for Health and Beauty (NATPRO6)

January 21-23, 2016

Organized by Khon Kaen University

249

Effect of Cissus quadrangularis, Pluchea indicaand Clerodendrum

serratumextracts on expression of cyclooxygenase proteins

Lawan Siangjong*, Auayporn Apirakaramwong, Penpun Wetwitayaklung

Faculty of Pharmacy, Silpakorn University, Nakorn Pathom 73000, Thailand

*[email protected] 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 serratumMoon 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 LPS-

activated 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 anti-

inflammatory effect through different pathways. Alternatively, the inhibition does not change protein expression but

could involve enzyme activity, which should be further investigated.

Keywords: Cissus quadrangularisL., Pluchea indica (L.) Less, Clerodendrum serratumMoon, 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 quadrangularisL. (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 serratumMoon 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 E 2production, 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 serratumMoon var. wallichii Clarke

(CS) and Cissus quadrangularisL.

(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 extractedwith 95% ethanol above boiling water for 15 min and the filtrate was consequently collected. Then, the residue was


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International Conference on Natural Products for Health and Beauty (NATPRO6)January 21-23, 2016


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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% fetalbovine 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,

HeLa cells (1.5x104cells) 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

To measure the expression of COX proteins, HeLa cells (3x105

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, crudeextracts 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 (ThermoScientific).

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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The 6t

Figure 1. Effect of PI, CS and CQ

concentrations of PI, CS and CQ ext

compared with cells treated with DM

Figure 2.Effect of PI, CS and CQ extra

g/ml), CS (1.56 g/ml) or CQ (3.13 using specific monoclonal antibody a

hInternational Conference on Natural Products for H

Organi

extracts on viability of HeLa cells. HeLa cells w

racts for 24 h and assessed with MTT cell viability

SO as a control.

cts of COX-1 protein expression. HeLa cells were tre

g/ml) extracts for 24 h. Total lysate were analyzed wainst COX-1 protein (1:1,000, Cell Signaling Technolo

alth and Beauty (NATPRO6)

January 21-23, 2016

zed by Khon Kaen University

251

ere treated with different

assay. % Cell viability was

ted with DMSO (C), PI (3.13

ith western immunoblottinggy).


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International Conference on Natural Products for Health and Beauty (NATPRO6)January 21-23, 2016


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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 PGE2production [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).

REFERENCES

1.

Bhujade AM, Talmale S, Kumar N, Gupta G, Reddanna P, Das SK, Patil MB. 2012. Evaluation of Cissus quadrangularis

extracts as an inhibitor of COX, 5-LOX, and proinflammatory mediators. J. Ethnopharmacol. 141, 989-996.

2. Panthong A, Supraditaporn W, Kanjanapothi D, Taesotikul T, Reutrakul V. 2007. Analgesic, anti-inflammatory and

venotonic effects of Cissus quadrangularisLinn. J. Ethnopharmacol. 110, 264-270.

3.

Buapool D, Mongkol N, Chantimal J, Roytrakul S, Srisook E, Srisook K. 2013. Molecular mechanism of anti-

inflammatory activity of Pluchea indica leaves in macrophage RAW 264.7 and its action in animal models of

inflammation. J. Ethnopharmacol. 146, 495-504.

4. Sen T, Ghosh TK, Chaudhuri AK. 1993. Studies on the mechanism of anti-inflammatory and anti-ulcer activity of

Pluchea indica-probable involvement of 5-lipoxygenase pathway. Life Sci. 52, 737-743.

efek pluchea indica pd cox

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