doi : 10.5958/0975-6892.2018.00030.

8
Medicinal Plants
Vol. 10 (3), September 2018, 185-190
IndianJournals.com
A product of Diva Enterprises Pvt. Ltd.

Research Article

Anti-inflammatory effect of Black cincau (Mesona palustris BL) based

supplement in male carrageenan-induced mice

Tri Dewanti Widyaningsih*, Sudarma Dita Wijayanti and Nurfiany Tisnaningrum

Department of Food Science and Technology, Faculty of Agricultural Technology, Brawijaya University, Jl. Veteran 65145,
Malang, East Java, Indonesia

Received: October 03, 2017; Accepted: June 06, 2018

ABSTRACT

This study aims to investigate the potency and optimum dose of Mesona palustris supplement as an anti-inflammatory in
carrageenan-induced mice (Musmus culus). In this study, twenty male Balb/C mice were divided into five groups. The
treatment groups are including a negative control, positive control, drug control, and two different doses of black cincau
based herbal supplement (BCBHS) groups. In this experiment, edema was observed surrounding mice feet, and the relative
number of NF-α and CD68 were measured using flow cytometry analysis. The results revealed that both of BCBHS doses were
effective to inhibit edema incidence in carrageenan-induced mice. The expression level of TNF-α and CD68 in both of
BCBHS doses were lower than the positive control. Further, the tissue recovery was observed with M. palustris supplementation.
Our result suggests that both of BCBHS doses have an anti-inflammatory effect in carrageenan-induced mice.

Keywords: Anti-inflammatory, black Cincau, CD68, edema, Mesona palustris BL, TNF-α

INTRODUCTION saponin, terpenoid, and steroid (Maslukhah et al., 2016).

Inflammation is a response to tissue damage due to infection,
antibody activation, or physical injury (Brunton et al., 2005).
During the inflammation, several biological components
such as a leukocyte, chemical mediator, the other blood
derivate are infiltrated into the injured or infected area to
eliminate the harmful substances and recover the tissue. In
general, non-steroidal anti-inflammatory drugs (NSAID) are
consumed to recover inflammation by preventing the tissue
damage. However, high dose consumption of NSAID
contributes to an adverse side effect, such as tinnitus, loss of
hearing capacity and vertigo (Katzung and Payan, 2002).
Therefore, the herbals anti-inflammatory activity is needed
to reduce those side effects. Black Cincau (Mesona palustris
BL) has been considered as suitable herbs that contain
bioactive compounds, i.e., phenol, glycoside, flavonoid,
As one of the traditional foods, M. palustris has been
utilized as a black cincau based herbal supplement (BCBHS)
and has been practically packaged in a capsule (Maslukhah
et al., 2016). Also, red ginger was added as the flavoring due
to its role as antioxidant and anti-inflammation (Shimoda et
al., 2010). However, another study conducted by Huang et
al. (2012) failed to reveal the anti-inflammatory activity of
black cincau (M. procumbens Hemsl) in hyper cholesterol,
hypertension and diabetic-induced Rat (Wistar) model.
The macrophages are activated during inflammation
process since it involves in antigen phagocytes is reaction.
Within the blood, this cell presence is stimulated by microbes
and its products, antigen-antibody complex, inflammation,
and cytokine (Baratawidjaja, 2004). The CD68 antigen is
the standard marker used for macrophage identification,
whereas Tumor Necrosis Factor Alpha (TNF-α) is the primary

*Corresponding author e-mail: tridewantiw@ub.ac.id

186 Tri Dewanti Widyaningsih et al.
factor in inducing inflammation (Coppack, 2001).
Furthermore, this study was aimed to investigate the anti-
inflammatory effect of black cincau based herbal supplement
by measuring the concentration of CD68 and TNF-α level in
mice blood serum after carrageenan induction.
MATERIAL AND METHODS
Bioactive compound contents and antioxidant activity of
BCBHS
Total phenol was measured according to George et al. (2005).
BCBHS absorbance was measured using spectrophotometer
(Lab Med. Inc) at λ=765 nm. The absorbance level then
calculated in linear regression using the caffeic acid standard
to obtain total phenol. Furthermore, the antioxidant activity
and IC 50 were measured according to 2,2-diphenyl-1-
picrylhydrazyl-hydrate (DPPH) method by Hatano et al.
(1989) at λ=517 nm.
Animal Model
The ethical clearance of this study licensed by The Research
Ethics Committee of Brawijaya University, Malang. There
were 20 male Balb/C mice aged 2-3 months and weighed
25-30 grams. Mice were divided into five experimental
groups including a negative control (healthy), positive
control (carrageenan-induced), drug control (carrageenan-
induced+diclofenac sodium), and two BCBHS treatment
groups. BCBHS dose I was composed of carrageenan-
induced+BCBHS 1.3 mg/20 g BW, whereas BCBHS dose II
was composed of carrageenan-induced+2.6 mg/20 g BW).
Mice were adapted to laboratory environment and fed with
milk pap pellet (PT. Japfa Comfeed) with ad libitum drink
for two weeks.
BCBHS Dose Adjustment
The doses of BCBHS aimed to experimental animals were
calculated according to Widyaningsih (2017) protocol as:
Human dose : 1 of BCBHS capsule = 500 mg
Mice dose : 500 mg x 0.0026 = 1.3 mg/20 g BW
Therefore, the appropriate doses for experimental animals
were 1.3 mg/20 g BW (Dose I) and 2.6 mg/20 g BW (Dose II)
Edema Observation
The anti-inflammatory activity could be observed by
carrageenan induction in mice feet (Winter et al., 1962).
About 1% carrageenan was injected sub planetary on the
right foot of experimental muce. Furthermore, diclofenac
sodium and BCBHS was intraperitoneally injected 1 hour
before carrageenan induction. Feet soles volumes were
measured 1-5 hours after this induction using 3 cc syringe
(Terumo). Edema volume was estimated from the volume
change of mercury by formula a-b, where a was mercury
level when mice foot was put on the syringe; and b was the
primary level of mercury. Edema percentage was measured
using this formula (1).
Vt - V0
x 100% ........... (1)
V0
Vt is edema volume at t certain time, and V0 is mice foot
original volume before carrageenan induction. Next, edema
inhibition was measured using this following formula (2)
% positive control Edema -
% sample of Edema % positive control Edema -
X % sample of Edema ........ .(2)
% positive control Edema
Flow cytometry
After the incubation period, the cells were harvested and
centrifuged at 2500 rpm at 10°C for 5 min. The pellet was
resuspended in 1 mL of PBS and divided into four microtubes
and centrifuged. The supernatant was discarded and pellets
were stained with conjugated antibodies. The combinations
of antibodies were: PE/Cy5-conjugated rat anti-mouse TNF-
a and FITC-conjugated rat anti-mouse CD68. Cells were
stained with extracellular antibodies and then incubated for
20 min in the ice box. A total of 50 µL of cytosis/cytoplasm
fixative solution was added and incubated for 20 min in the
ice box. Alternatively, 500 µL of washperm washing solution
was added and then centrifuged. The supernatant was
discarded, while pellets were stained with intracellular
antibodies and then incubated for 20 min in the ice box. A
total of 500 µL of PBS was added to both the cells that had
been incubated with either the extracellular or the intracellular
staining procedure. Each sample was transferred into a flow
cytometry cuvet and then the amount of binding analyzed
by flowcytometer - BD FACS CaliburTM (Hartati et al., 2017).
Data were analyzed using BD cell Quest PRO™ software
then tabulated and analyzed statistically.
Histopathology
For histological examination, biopsies of paws were taken at
5 h after Carr injection. The tissue slices were fixed in a
solution of 18.5 g L-1 formaldehyde and 10 g L-1 acetic
acid for 1 week at room temperature, dehydrated by graded
ethanol and embedded in paraffin. Sections (thickness 5 µm)
Medicinal Plants, 10(3) September 2018
 Anti-inflammatory effect of Black cincau (Mesona palustris BL) based supplement in male carrageenan-induced mice
were deparaff inised with xylene and stained with
haematoxylin and eosin (H&E) stain. Polymorpho nuclear
neutrophils were identif ied by positive staining and
morphology and counted using an Olympus BH-2
microscope (Hamburg, Germany). Only neutrophils present
within the dermal region of the tissue were counted. Every
three to five tissue slices were) groups. The number of
neutrophils was counted in each scope (400×) and there after
we obtained their average count from five scopes of every
tissue slice (Huang et al., 2012).
Statistical Analysis
Edema and the level CD68 and TNF-α was analyzed using
ANOVA two factors with four replications and ANOVA single
factor, respectively. Furthermore, data were analyzed using
DMR (Duncan’s Multiple Range) Test and LSD (Least
Significance Difference) Test (p=0.05).
RESULTS
Bioactive content and antioxidant activity of BCBHS
The total phenol content of BCBHS was 60.85% mg CAE/g,
thus there was 60.58 mg phenol as the same amount as the
caffeic acid in every gram of BCBHS. This result indicates
that total phenol in BCBHS is the highest among the other
herbal or amounted to 15.81% in Black Cincau extract and
12.12% in red ginger. Since the phenolic compound acts as
the antioxidant, the higher quantity of phenolic compound
indicates the higher antioxidant activity. Furthermore, the
Inhibition Concentration (IC50) is defined as the concen-
tration of sample that can reduce 50% of free radical activity.
This means that smaller IC50 related to the higher antioxidant
activity (Prakash, 2001; Molyneux, 2004) (Table 1).
Edema level
Edema percentage increases in the all experimental groups
after 1-3 hour of induction then decreases in the fourth and
fifth hours (Figure 1A). The edema inhibition was indicated
Table 1: BCBHS Comparison with other herbals
Parameter
Black Cincau Simplicia
Total Phenol (mg CAE/g) 15.81±0.36
Antioxidant Activity (%) 54.47±4.42
IC50 (ppm) 396.71±6.40
Water Concentration (%)* 10.17±0.22
Medicinal Plants, 10(3) September 2018
187
after 4-5 hour of induction, while in the positive control
there is no significant inhibition. On the other hand, mice
that treated with BCBHS (dose I and II) were found to
significantly inhibit the edema incidence. Both groups also
increased the inhibition level during1-5 hours, but there was
slight decreasing in three hours after induction (Figure 1B).
Specifically, BCBHSdose I inhibition was lower than dose
II, but diclofenac sodium inhibition was certainly as high as
dose II.
α concentration
CD68 dan TNF-α
It is noticeable that different treatments deliver different
results on CD68 and TNF-α levels (Figure 2C and 2D). The
total macrophage population in both BCBHS doses groups
were lower than a positive control. However, BCBHSII has
the similar concentration as a negative control. Also, SHBC
dose II group obtain lower TNF-α concentration than dose I,
and it was similar to negative control. Furthermore, anti-
inflammation drug group also has lower TNF-α level than
positive control.
Histopathology
Hyperplasia was not detectedin the negative control.
However, there were a considerable amount of inflammation
cells in dermis positive control tissue. This was considerably
lower in Na-diclofenac group. Furthermore, hyperplasia was
a presence inthe BCBHS dose I followed with a high amount
of inflamed cells. Contrary, hyperplasia was not detected,
and inflammatory cells were found at the lower amount in
BCBHS dose II.
DISCUSSION
Typically, acute inflammation disappears within hours until
1-2 days (Guyton, 1995; Underwood, 2000). Carrageenan
can apply as inflammation inducer due to its antigenicity
structure that consists of sulfated polysaccharides
(Underwood, 2000). Carrageenan induces a stronger response
to non-steroidal anti-inflammation drugs, which has been
Samples
Red Ginger Simplicia BCBHS
12.12±0.35 60.58±0.01
60.78±4.41 74.54±0.63
124.99±1.36 75.18±0.03
10.84±0.04 4.99±0.08
188 Tri Dewanti Widyaningsih et al.
Figure 1: BCBHS effect on edema percentage (A), inflammation inhibition percentage (B), CD68 expression (C) and TNF-α expression after
5 hours of carrageenan induction (D)
widely used in new drug discovery (Corsini et al., 2005).
The peak of edema volume after carrageenan induction can
be observed after 3 hours (Winter, 1962). The result of this
study revealed that BCBHS significantly inhibits edema
(p<0.05) (Figure 1A). The volume of edema became the
highest at early 3 hours after induction then decreased at the
4-5 hours. Carrageenan releases inflammation mediator after
the induction, one of which is prostaglandin (Corsini et al.,
2005). As a result, there will be an interaction between these
mediators and lead the blood vessels to become more
permeable (Huang et al., 2012). There are two phases of edema
induction. In the early phase, histamine and serotonin are
released after 2 hours of induction. After that, in the late
phase, the bradykinin and prostaglandin are released between
3-5 hours after induction (Kumar and Kuttan, 2009).
Tissue lesion leads to arachidonic acid induces
inflammation mediators, such as prostaglandin and leuco-
nutrient, which produced in cyclooxygenase (COX) and
lipoxygenase pathway. Also, tissue trauma may recruit the
other pro-inflammation factors such as IL-1, IL-6, and TNF-
α (Ricciotti and Fitz Gerald, 2011). Pro-inflammatory
cytokines increase blood capillary permeability that allows
blood fluid move out to interstitial tissue and increases extra
vascular fluid called as edema (Scallan et al., 2010).
There are two main ingredients in BCBHS, i.e., black
cincau and red ginger. Further, black cincau contains a
phenolic compound that effectively prevents inflammation.
This compound has the ability to bounds to amino acid as
the enzyme composition (Contran and Mitchell, 2007).
Cycloxygenase enzyme consists of amino acids such as
tyrosine, valine, leucine, and the others. The longer polymer
of polypeptide induces the stronger anti-inflammation
activity. Beside of that, phenolic compound scavenges the
free radicals that can cause tissue damage. Furthermore, red
Medicinal Plants, 10(3) September 2018
 Anti-inflammatory effect of Black cincau (Mesona palustris BL) based supplement in male carrageenan-induced mice
Figure 2: Mice feet
histopathology (100X
magnification). (a)
negative control; (b)
positive control; (c) Na-
diclofenac; (d) supplement
dose I; (e) supplement
dose II
ginger contains gingerolas anti-inflammatory activity by
inhibition of TNF-α (Sutyarso and Yap, 2016).
The macrophage is the central mononuclear phagocytic
cell in the blood. It can be stimulated by antigen-antibody
complex, inflammation, cytokines, and trauma
(Baratawidjaja, 2004). This cell is the differentiated from
monocyte; it expresses CD68 as macrophage marker. There
was a significant effect on CD68 expression after carrageenan
induction (p<0.05). The levels of CD68 calculated from the
isolated PBMC (Peripheral Blood Mononuclear Cell).
Furthermore, TNF-α is the primary cytokine involved in
acute inflammation. It produced by various cells including
macrophage due to the infection that is called as pro-
inflammation cytokines (Khan, 2008). Its expression
neutrophil and monocyte infiltrate into infection site to
results in eliminate pathogenic microbes (Baratawidjaja,
2006). Additionally, there were significant differences in
TNF-α expression between treatment groups (p<0.05).
Phenolic compound prevents the release of arachidonic
acid and lysozyme by neutrophil and endothelial cells. It
Medicinal Plants, 10(3) September 2018
189
also inhibits the proliferation phase and exudate phase in
the inflammation process. This hydrophilic compound
hampers the bacterial adhesion onto phagocytic cells that
occurdue to the hydrophobicity of bacteria and phagocytic
cell membrane (Hoffmann, 2003).
Neutrophil and hyperplasia are the main indicators of
histopathology after carrageenan induction in mice feet.
Hyperplasia or cell enlargement is caused by the excess of
fluid accumulation (Dumbre et al., 2014). According to
Adnyana et al. (2012), the presence of neutrophil in
inflammation tissue is related to phagocytic activity due to
carrageenan induction. Ma et al. (2013) also observed that
there was a huge amount of neutrophil found after 5 hours of
incubation in positive control mice group with hyperplasia
(Figure 2).
In summary, BCBHS inhibited inflammation activity in
carrageenan-induced mice. Both doses of BCBHS and Na-
diclofenac reduced the inflammation. Specifically, BCBHS
dose I induction level was lower than Na-diclofenac and
dose II. Therefore, the optimal recommended dose of BCBHS
190 Tri Dewanti Widyaningsih et al.

as anti-inflammatory herbal should be 2.6 mg/20 g BW (dose Huang GJ, Liao JC, Chiu CS, Huang SS, lin TH and Deng JS
II = 130 mg/kg BW). On the other hand, histopathology (2012). Anti-inflammatory activities of aqueous extract of
Mesona procumbens in experimental mice. J. Sci. Food. Agr.,
observation revealed that BCBHS treatment induces wound
92: 1186-1193.
healing in damage tissue of mice. Katzung BG and Payan DG (2002). Non-steroid antiinflammation
drug; non-opioid analgesics; drug used on gout. In Katzung
ACKNOWLEDGMENT BG (eds). Basic and clinical pharmacology. 6th ed. EGC,
Jakarta, pp. 558-582.
The author would like to thank Faculty of Agricultural Khan MM (2008). Role of cytokines. In Khan MM (eds).
Technology, Brawijaya University, Malang Indonesia for Immunopharmacology. Elsevier, Amsterdam, pp. 33-59.
providing lab facility. Kumar PP and Kuttan G. Vernonia cinerea L (2009). Scavenges free
radicals and regulates nitric oxide and proinflammatory
cytokines profile in carrageenan-induced paw oedema model.
REFERENCES
Immunopharmacol. Immunotoxicol., 31: 94-102.
Ma Y, Li Y, Li X and Wu Y (2013). Anti-Inflammatory effects of 4-
Adnyana IK, Sukandar EY and Indriasari W (2012). Anti-rheumatoid
methylcyclopentadecanone on edema models in mice. Int. J.
arthritis effect of water fraction of siwalan fruit (Borassus
Mol. Sci., 14: 23980-23992.
flabellifer L.) to mice. J. Medika. Planta., 2: 53-61.
Maslukhah YL, Widyaningsih TD, Waziiroh E, Wijayanti N and
Baratawidjaja KG (2004). Basic Immunology. 6th ed. Jakarta: Faculty
Sriherfyna FH (2016). Influence factor of black Cincau
of Medicine, University of Indonesia, pp. 153-157.
(Mesona palustris BL) extraction in pilot plant scale: a review.
Baratawidjaja KG (2006). Basic Immunology. 7th ed. Jakarta: Faculty
Jurnal Pangan dan Agroindustry, 4: 245-252.
of Medicine, University of Indonesia, pp. 124-126.
Molyneux P (2004). The use of the stable free radical
Brunton LL, Lazo JS and Parker K (2005). The Pharmacological
diphenylpicrylhydrazyl (DPPH) for estimating antioxidant
Basis of Therapeutics Eleventh Edition. 11th ed. The McGraw-
activity. Songklanakarin J. Sci. Technol., 26: 211-219.
Hill Companies, New York, pp. 237-295.
Prakash A (2001). Antioxidant Activity. Medallion Laboratories
Contran RS and Mitchell RN (2007). Acute and Chronic
Analytical Progress, 19: 1-4.
Inflammation. In Kumar V et al. (eds) Textbook of Pathology.
Ricciotti E and Fitz Gerald GA (2011). Prostaglandins and
7th ed. EGC, Jakarta, pp. 33-60.
inflammation. Arterioscler. Thromb. Vasc. Biol. Rev. A., 31:
Coppack SW (2001). Pro-Inflammatory cytokines and adipose tissue.
986-1000.
Proc. Nutr. Soc., 60: 349-356
Scallan J, Huxley VH and Korthuis RJ (2010). Capillary fluid
Corsini E, Paola RD, Viviani B, Genovese T, Mazzon E, Lucchi L,
exchange: regulation, function, and pathology. Morgan &
Marinovich M, Galli CL and Cuzzocrea S (2005). Increased
Claypoll Life Sciences, San Rafael.
carrageenan-induced acute lung inflammation in old rats.
Shimoda H, Shan SJ, Tanaka J, Seki A, Seo JW, Kasajima N, Tamura
Immunology, 115: 253-261.
S, Ke Y and Murakami N (2010). Anti-inflammatory properties
Dumbre RK, Kamble MB and Patil VR (2014). Inhibitory effects by
of red ginger (Zingiber officinale var. Rubra) extract and
ayurvedic plants on prostate enlargement induced in rats.
suppression of nitric oxide production by its constituents. J.
Pharmacogn. Res., 6: 127-132.
Med. Food., 13: 156-162.
George S, Brat P, Alter P and Amiot MJ (2005). Rapid determination
Sutyarso TS and Yap S (2016). The effect of red ginger ethanol
of polyphenols and vitamin C in plant-derived products.
extract (Zingiber officinale Roxb var Rubrum) to airway goblet
J.Agric. Food.Chem., 53: 1370-1373.
cells count and cilliary length on cigarette smoke-induced white
Guyton CA (1995). Textbook of Medical Physiology. 7th Ed. EGC,
male rats sprague dawley strains. Jurnal Kedokteran Unila.,
Jakarta, p. 307.
181-189.
Hartati FK, Widjanarko SB, Widyaningsih TD and Rifa’i M (2017).
Underwood JCE (2000). General and Systemic Pathology. 3th Ed.
Anti-Inflammatory evaluation of black rice extract inhibits TNF-
Churchill Livingstone, London, pp. 1-839.
α, IFN-γ and IL-6 cytokines produced by immunocompetent
Widyaningsih TD (2010). Potential black Cincau (Mesona palustris
cells. Food Agric. Immunol., 28 (6): 1116-1125.
BL) as functional food ingredients is immunomodulator.
Hatano T, Edamatsu R, Hiramatsu M, Mori A, Fujita Y, Yasuhara T,
Seminar of Proceeding of the Local Food, LIPI, Yogyakarta.
Yoshida T and Okuda T (1989). Effect of the interaction of
Widyaningsih TD, Mar tati E and Lukitasari DM (2017).
tannins with co-existing substances. VI. : effect of tannin and
Immunomodulatory effects of black Cincau (Mesona palustris
related polyphenols on superoxide anion radical and on 1,1-
BL) supplement on Escherichia coli Strain O157-Infected
diphenyl-2-picrylhidrazil radical. Chem. Pham. Bull., 37: 2016-
Mice. Asian J. Pharm. Clin. Res., 10: 326-330.
2021.
Winter CA, Risley EA and Nuss GW (1962). Carrageenin-Induced
Hoffmann D (2003). Medical Herbalism: The Science and Practice
Udem in Hind Paw of the Rat as an Assay for Anti-
of Herbal Medicine. Healing Arts Press, Vermont, pp. 1-672.
inflammatory Drugs. Proc. Soc. Exp. Biol. Med. 111: 544-
554.

Medicinal Plants, 10(3) September 2018