Indonesian Journal of Cancer Chemoprevention, October 2015

ISSN: 2088–0197
e-ISSN: 2355-8989

Anti-Inflammatory Effect of Ethanolic Extract of Curcuma

aeruginosa Roxb Rhizome, Morinda Citrifolia Fruit and
Apium graveolens Leaf on Lipopplysaccharide-induce
RAW 264.7 Cell Lines

Siska Andrina*, Churiyah, Nuralih

Center of Pharmaceutical and Medical Technology,

Agency for the Assessment and Application of Technology, Indonesia

Abstract
Curcuma aeruginosa Roxb., Morinda citrifolia and Apium graveolens are Indonesian plants
which have been used in traditional medicine as an antihelmintic, antimicrobial,
antiinflammation and antioxidant. In this study, the antiinflammatory activity of Curcuma
aeruginosa Roxb. rhizome, Morinda citrifolia fruit and Apium graveolens leaf extract was
investigated. All of extract was prepared by maceration with ethanol 70% and treated for
antiinflammatory effect by using inducible-nitric oxide secretion measurement on
lipopolysaccharide (LPS) - induce macrophage RAW 264.7 cells. We used three concentration
of Curcuma aeruginosa Roxb. rhizome and Apium graveolens leaf extract at 25 ppm; 50 ppm; 100
ppm while for Morinda citrifolia fruit extract with 50 ppm; 100 ppm; 200 ppm. This study
revealed that rhizome of Curcuma aeruginosa Roxb. and fruit of Morinda citrifolia extract
inhibited inducible-nitric oxide synthesis with highest value of 84.2% at 25 ppm and 85.71% at
100 ppm in cells. However, leaf of Apium graveolens extract showed low inhibition with highest
values of 18.85% at 100 ppm. This study demonstrates the potential of Curcuma aeruginosa
Roxb. rhizome and Morinda citrifolia fruit as antiinflammatory agents.

Keywords: Curcuma aeruginosa Roxb., Apium graveolens, Morinda citrifolia, antiinflammation,

inducible-nitric oxide, lipopolysaccharide-induce macrophage RAW 264.7 cells

INTRODUCTION NADPH and molecular oxygen, is a short-lived free

radical and an intercellular messenger produced by a
Inflammation is one of self-defense variety of mammalian cells, which include
mechanisms in body which is characterized by macrophages, neutrophils, platelets, fibroblasts,
redness, pain, swelling and a sensation of heat. The endothelium, neuronal, and smooth muscle cells. NO
inflammatory responses play an important role in mediates a variety of biological actions ranging from
host survival although it can also lead to chronic vasodilatation, neurotransmission, inhibition of
inflammatory diseases such as asthma (Cragg and platelet adherence and aggregation, as well as the
Newman, 2008), cancer (Hendra, et al., 2011), macrophage- and neutrophil-mediated killing of
rheumatoid arthritis (Hsieh, et al., 2007), Crohn’s pathogens (Moncada, et al., 1991, MacMicking, et
disease (Moncada, et al., 1991) and ulcerative colitis al., 1997; Oh, et al., 2008). Chronic inflammation
(UC) (MacMicking, et al., 1997). The production of and infections lead to the up-regulation of a series of
inflammatory cytokines such as interleukin-6 (IL6), enzymes and signaling proteins in affected tissues
tumor necrosis factor-alpha (TNF-alpha), and other and cells. The inducible forms of NOS are the most
inflammatory mediators including nitric oxide (NO) important.
and prostaglandin E2 (PGE) will arise immediately.
Nitric oxide (NO), which is synthesized by nitric
oxide synthase (NOS) from L-arginine using
*Corresponding author e-mail: [email protected]

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Indones. J. Cancer Chemoprevent., 6(3), 84-88

Plants have formed the basis of sophisticated containing 2 mM glutamine, 10 mM 4-[2

traditional medicine systems that have been in hydroxyethyl]1-piperazineethanesulfonic acid
existence for thousands of years. These plant based (HEPES), penicillin (Gibco) (100 units/mL),
medicinal systems continue to play an essential role streptomycin (Gibco) (100 µg/mL) and 10% Fetal
In health care today, and it has been estimated by the Bovine Serum (Gibco). Cells were cultured at 37°C
World Health Organization that Approximately 80% in a humidified incubator with 5% CO2.
of the world’s inhabitants rely mainly on traditional
medicines for primary health care (Cragg and MTT Assay
Newman, 2008; Hsieh, et al., 2007). Indonesia is one Cells were cultured in 96-well plates for 24
of rich country that have more a thousand plants hr. After 24 hours, plant extracts at concentrations of
growing in distributed areas of Indonesia. Most 200; 100; 50; 25; 12.5; 6.25 µg/mL was added into
plants have been used by local people as a traditional the well in triplicate then incubation overnight. The
medicine or jamu by history, as example Curcuma next day, 3-(4,5dimethylthiazol-2-yl)-2,5-
aeruginosa Roxb. which originated from South East diphenyltetrazoliu bromide or MTT (Sigma) was
Asia including Indonesia. Curcuma aeruginosa added to the medium for 4 hours followed by SDS
Roxb grows throughout the year in tropical areas with HCl 0.01 N to dissolve the formazan crystals.
reaching a height of around 1.5 m which has dark Incubate for 24 hours and then measured with
rhizome with unique odor. Rhizome of Curcuma microplate reader with absorbance at 540 nm. The
aeruginosa Roxb. have been used as a traditional percentage of dead cells was determined related to
medicine or jamu by Indonesian people to treat many control group.
disease such as inflammation, fungi and infection of
bacteria. Morinda citrofolia is one of famous plant Nitric Oxide Assay
from Indonesia. Leaf of Morinda citrofolia have The nitric oxide assay was performed as
been used as a medicine in many years ago as an described previously with slight modification (Yoon,
analgetic, immunostimulant, antiinflammation and et al., 2009). After pre-incubation of RAW 264.7
also antibacterial. Leaf of Morinda citrofolia has cells (5×105 cells/mL) with LPS (Sigma) 2 µg/mL
been proven being as anticancer agent because its for 24 h, the quantity of nitrite in the culture medium
potential to ihibit some regulatory gene of cancer was measured as an indicator of NO production.
cells. Apium graveolens is originated from South of Amounts of nitrite, a stable metabolite of NO, were
Europe and has distributed all of the world. Apium measured by using Griess reagent (1% sulfanilamide
graveolens or celery herbs is used by local people as and 0.1% naphthylethylenediamine dihydrochloride
one of food ingredients and also as a medicine. As a in 2.5% phosphoric acid). Briefly, 100 µL of cell
medicine, Apium graveolens is used to reduce the culture medium was mixed with 100 µL of Griess
blood pressure, paint killer and inducer of appetite. reagent. Subsequently, the mixture was incubated at
This study aims to investigate Indonesian plants for room temperature for 10 min and the absorbance at
the inhibitory activity toward Nitric Oxide 540 nm was measured in a microplate reader. Fresh
production by measuring its production in LPS- culture medium was used as a blank in every
stimulated macrophage RAW 264.7 cell line. experiment. The quantity of nitrite was determined
from a sodium nitrite standard curve.
MATERIALS AND METHODS
RESULT AND DISCUSSION
Plant and Extract Preparation
The plants were collected from Serpong, Effect of extracts on cells viability in Raw
Banten, Indonesia. Plant materials were air-dried, 264.7 macrophages
grounded and extracted three times with 70% In this study, we prepared crude extracts of
ethanol at room temperature. The filtrates were three plants from Java Island, Indonesia: Curcuma
concentrated under reduced pressure, and stored in a aeruginosa Roxb, Morinda citrifolia and Apium
closed container until treatment. graveolens to test their potential for anti-
inflammation activity. All the plant samples were
Cell Culture macerated in 70% ethanol and evaporated to
Murine macrophage RAW 264.7 cells were obtained viscous extracts. The cytotoxic effect of the
obtained from KRIBB Korea. They were cultured in extract was evaluated on macrophages RAW 264.7
Roswell Park Memorial Institute medium (RPMI) cell lines using MTT method to ensure that the anti-

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Indones. J. Cancer Chemoprevent., 6(3), 84-88

inflammatory activity was not due to cytotoxicity all extract at concentration lower than 200 µg/mL
effect from the extract. During inflammation, the did not give cytotoxicity effect in RAW 264.7 cell.
ultimate phase of a series of signaling events, Therefore, we used non-toxic at concentration 25; 50
macrophages induce the expression of pro- and 100 µg/mL to deterime its anti-inflammatory
inflammatory genes such as inducible nitric oxide activity.
synthase (iNOS). This enzyme is up-regulated
secretion of pro-inflammatory cytokines, and Effect of Extracts on Nitrit Oxide Production
produces NO from L-arginine. The regulation of NO in LPS-induced Macrophage
production is therefore an important target for Antiinflammation activity assay using LPS
inflammatory disease. LPS stimulation alone has stimulated-RAW 264.7 cell was determined to
been demonstrated to induce iNOS transcription and quantify nitric oxide production after LPS induced.
its protein synthesis in murine macrophage RAW Extracts of plants were diluted with DMSO and
264.7 cells, with a corresponding increase in NO Phosphate Buffer Saline (PBS) to obtain
production. Furthermore, LPS stimulation has also concentration at 25; 50 and 100 µg/mL. The Griess
been shown to induce IκB proteolysis and NF-κB reaction, a spectrophotometric determination for
nuclear translocation (Xie, et al., 1994; Henkel, et nitrite, was carried out to quantify the nitrite levels in
al., 1993). Thus, this cell system is an excellent the conditioned medium of RAW 264.7 cells treated
model for drug screening and the subsequent with LPS 2 ug/mL. The final concentration of
Evaluation of potential inhibitors against iNos and DMSO in the culture media was 0.2% and this
NO production. concentration of DMSO did not show any effect on
We tested cytotoxicity assay of plants at the cells. Sodium nitrate as a standard solution was
concentration of 200; 100; 50; 25; 12.5; 6.25 µg/mL. tested separately to obtain standard curve of
The result showed that all of extract have percentage nitrate.
of proliferation inhibition less than 50% at the Table 1 shows the inhibitory activity by plant
highest concentration except for Curcuma extracts towards NO production by LPS-activated
aeruginosa Roxb. rhizome which showed 97% macrophages.
inhibition at 200 µg/mL (Fig.1). It is indicated that

Figure 1. Effects of Curcuma aeruginosa Roxb. rhizome, Morinda citrofolia fruit and Apium graveolens Leaf
extracts on cell viability in Raw 264.7 mouse macrophage cells. Cell viability was tested by MTT reduction
assays. Data were mean of three replications x ±SD (p<0,05)

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Indones. J. Cancer Chemoprevent., 6(3), 84-88

Table 1.Nitrit oxide inhibition of Curcuma aeruginosa Roxb. rhizome, Morinda citrofolia Fruit,
and Apium graveolens leaf extract

No Name of Extract Concentration (ug/mL) % Inhibition

1 Curcuma Aeruginosa 25 84.426
Roxb Rhizome 50 83.606
100 78.278
2 Morinda citrofolia Fruit 50 53.968
100 85.714
200 85.714
3 Apium Graveolens Leaf 50 0
100 -146.72
200 18.852

Based on this study, we can see that among of CONCLUSION

three extracts at each concentration, Morinda
citrofolia fruit extracts showed the highest inhibition We summarized that Curcuma aeruginosa
at 100 µg/mL and 200 µg/mL, however Curcuma Roxb. rhizome and Morinda citrifolia fruit show
aeruginosa Roxb rhizome extract showed inhibition potency as antiinflammatory agents based on NO
above 80% at lower concentration compared to inhibition in LPS-induced RAW 264.7 cells.
Morinda citrofolia fruit extracts. In the other hand,
Apium graveolens leaf extracts appeared to did not REFERENCES
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