Original Article 205

ANTI-INFLAMMATORY AND ANTIOXIDANT

ACTIVITIES OF THAI TRADITIONAL REMEDY CALLED
“YA-HA-RAK”
Thana Juckmeta, Arunporn Itharat*
Department of Applied Thai Traditional Medicine, Faculty of Medicine, Thammasat University,
Rangsit Campus, Klong Luang, Pathumtani 12120, Thailand

ABSTRACT: Benjalokawichien (BLW) or Ya-Ha-Rak remedy (HR) is the drug list in herbal
medicinal products A.D 2011 of Thailand and used as antipyretic drug and treat rash in Thai
Traditional Medicine. Its plant ingredients consists with five plant roots such as Ficus racemosa
Linn., Capparis micracantha DC., Clerodendrum petasites S. Moore., Harrisonia perforata
Merr., Tiliacora triandra Diels. This study are investigation anti-inflammatory and antioxidant,
activities of the extract of HR and its plant components by inhibitory activity on the release of
inhibitory activities against lipopolysaccharide (LPS) induced nitric oxide (NO) production in
RAW 264.7 cell lines and DPPH scavenging assays respectively. HR extract possessed the
highest NO inhibitory activity with an IC50 value of 40.4µg/ml. The ethanolic extract of Ficus
racemosa exhibited the highest antioxidant activity (IC50= 4.9 µg/ml) and HR extract also show
antioxidant activity (EC50=40.9µg/ml). This study can support the use of HR in Thai Traditional
Medicine for treatment inflammatory diseases.
Keywords: Benjalokawichian remedy, Ya-Ha-Rak remedy (HR), Ficus racemosa, anti-
inflammatory, antioxidant

INTRODUCTION The LPS-induced activation of macrophages results

Nitric oxide (NO) is one of the inflammatory in the production of bioactive lipids, reactive
mediators causing inflammation in many organs and oxygen species, and in particular, inflammatory
it is an inorganic free radical which has been cytokines to fight and clear the bacterial infection
implicated in physiological and pathological [6, 7]. Thus, the evaluation of anti-inflammation of
processes, such as vasodilation, non-specific host drug used the inhibitory nitric oxide production
defense and acute or chronic inflammation [1, 2]. NO assay as the one model of antiinflammation.
and its derivatives, including reactive nitrogen Benjalokawichian or Ya-Ha-Rak is a Thai
intermediates (nitrite and nitrate), produced by iNOS Traditional medicine preparation which is in the list
have been identified as important effector molecules of Herbal Medicinal Products A.D 2011 Thailand.
that restrict pathogen growth in infected hosts, and It consist of five plant roots such as Ficus
monocytes and macrophages, endothelial cells, racemosa Linn., Capparis micracantha DC.,
hepatocytes, and neutrophils can synthesize reactive Clerodendrum petasites S.Moore., Harrisonia
nitrogen intermediates by constitutive pathways, perforata Merr., Tiliacora triandra Diels. It has
inducible pathways, or both [3]. However, excessive been commonly used to reduce fever which is
production of this free radical is pathogenic to the relative with the inflammatory mechanism and also
host tissue itself, since NO can bind with other used to apply to inflammation for allergic skin in
superoxide radicals and acts as a reactive radical Thai traditional medicine long time ago
which directly damages the function of normal cells [Rehabilitation Foundation for the Promotion of
[4, 5]. LPS is one of the major constituents of the traditional Thai medicine. Ayurvedic College
outer membrane of Gram-negative bacteria, and the (Cheevakakomalapaj) [8]. Although BLW or Ya-
immune system is constantly exposed to low levels Ha-Rak (HR) remedy is widely used as an
of LPS through infections. LPS recognition and antipyretic by many traditional practitioners in
signal transmission are the key events aimed at Thailand, there have been only three scientific
eliminating an invading pathogen. studies that support its use. Konsue et al.
demonstrated that the root powder of BLW formula
*
Correspondence to: Arunporn Itharat showed the antipyretic efficacy by using a Baker’s
E-mail: [email protected] yeast-induced fever model in rats [9].
Tel. +66 (0) 2926 9749; Fax: +66 (0) 2926 9749 Jongchanapong et al. initially determined the

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206 Original Article

antipyretic activity of the root extract of BLW Macrophage Leukemia-like RAW264.7 cells was
remedy using lipopolysaccharide (LPS)-induced evaluated using a modified method Tewtrakul and
fever in rats compared to that of acetylsalicylic acid Subhadhirasakul [12]. RAW 264.7 cells line was
(ASA) [10]. Recently, Singharachai et al. diagnosed cultured in RPMI medium supplemented with 10%
and distinguished of five roots in Thai Traditional fetal bovine serum (FBS), penicillin (100 units/ml)
Medicine remedy: Ben-ja-lo-ka-wi-chian or Ya-Ha- and streptomycin (100 µg/ml). The cells were
Rak from the morphological and histological seeded in 96-well plate (cell concentration 1 × 105
characters [11]. Surprisingly, there is no previous cells/well) and incubated in CO2 incubator at 37oC
report for investigation on anti-inflammatory for 1 hour. 100 µl/well of RPMI medium
activity by inhibitory effect on NO production by containing 100µg/ml of LPS was added into control
Mouse Macrophage Leukemia-like RAW264.7 and sample wells, whereas only RPMI medium was
cells on these five root extract and HR. Thus, the added into a blank well. 100µl/well of different
results antiinflammatory testing of HR and its plant sample concentrations (1-100 µg/ml) were added
components should be support using this drug for into sample wells and their corresponding blank
development as the modern anti-inflammatory drug sample wells. Then cells were incubated at 37oC
from Thai traditional remedy in the future. for 48 hours. Supernatant (100µl) was added in
another 96-well plate and followed by the addition
MATERIALS AND METHODS of 100µl/well of Griess reagent. The color was
1. Reagents detected at a wavelength of 570 mm.
Animal cell lines, Chemicals and Reagents Cytotoxicity was also determined using the MTT
RAW 264.7 murine macrophage leukemia cell lines method. After 48 h incubation with the test samples,
were established and kindly provided by Assoc. MTT solution (10 µl, 5 mg/ml in PBS) was added
Prof. Dr. Supinya Tewtrakul, Department of to the wells and incubated at 37oC for 2 hours. The
Pharmacognosy and Pharmaceutical Botany, Faculty medium was removed, and isopropanol containing
of Pharmaceutical Sciences, Prince of Songkla 0.04 M HCl was then added to dissolve the
University, Thailand. RPMI Medium 1640 (RPMI formazan production in the cells. The formazan
1640) Medium powder with L-glutamine, Fetal solution was measured with a micro-plate reader at
Bovine Serum (FBS), Penicillin-Streptomycin 570 nm. The test sample was considered to be
(P/S), trypsin-EDTA and trypan blue were cytotoxic when the optical density of the sample-
purchased from Gibco, USA. Phosphate Buffer treated group was less than 70-80% of that in the
Saline (PBS) was from Amresco, USA. Sodium control (vehicle-treated) group. Indomethacin was
bicarbonate was from BDH, England. used as positive controls.
Lipopolysaccharide (LPS, from Escherichia coli), 4. DPPH radical scavenging activity
Dimethyl sulfoxide (DMSO) and 1,1-Diphenyl-2- The antioxidant activity of all extracts and fractions
picrylhydrazyl(DPPH) were from Fluka, Germany. was determined according to the modified method
2. Plant materials and extraction of Yamasaki et al. [13]. Samples for testing were
The parts of plants were collected from Dan-Chang, prepared by dissolution in absolute ethanol and
Suphanburi in Thailand. The voucher specimens prepare sample solution (4 concentration as two-
were deposited at the herbarium of Southern Center fold dilution) to be 100, 50, 10, 1 µg/ml. A portion
of Thai Medicinal Plants at Faculty of of sample solution (0.1 ml) was mixed with the
Pharmaceutical Science, Prince of Songkla same volume of 6 x 10-5 M DPPH in absolute
University, Songkhla, Thailand. Five plant materials ethanol. After the mixture had been allowed to
were dried at 50°C. One hundred gram of each protected from light for 30 minutes at room
plants was to provide homogeneous combined as temperature, its absorbance was measured at 520
Benjalokawichian or Ha-Rak preparation, each nm using a spectrophotometer.
roots was also ground and macerated with 95%
ethanol, then filtered and concentrated by rotary RESULTS
evaporator (under reduced pressure) to obtain the
1. The percent yield of extracts
ethanolic extracts. The percentage of yields of each
The percent yield of HR and each extract showed in
extract were calculated.
Figure 1. The HR extract showed the highest (4.26 %)
3. In vitro assay for Anti-inflammatory activity percentage of yields whereas FR showed the lowest
Inhibitory effect on NO production by Mouse (2.01%).

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Original Article 207

5
4.26%
3.9%
4 3.72%

3.12%
2.83%
3

2.01%
2

0
HR TT HP CP CM FR

Figure 1 The percent yield of Ya-Ha-Rak remedy and each component extracts by maceration with 95% ethanol

IC50(µg/ml)
120
>100
100

80
61.35 54.65 53.16
60 46.55
40.36
40
20.32
20

0
FR CM TT HP CP HR Indomethacin*

Figure 2 Anti-inflammatory Activity of Benjalokawichian Remedy (Ya-Ha-Rak) and its plant ingredients (n=3),
*Positive control

Table 1 The results of Anti-Inflammatory Activity of Benjalokawichian Remedy (Ya-Ha-Rak) and its ingredients (n=3)

Sample %Inhibition (µg/ml)/ (percentage of viable cells) at various concentration

(Ethanolic IC50SEM
extracts) 100 50 30 10 1
Ficus racemosa 47.946.90 14.914.47 2.505.09 -0.575.21 -5.634.31 >100
Linn. Z (FR) (85.375.14) (105.742.57) (112.846.29) (120.473.98) (128.341.72)
Capparis 79.682.05 40.873.12 25.761.88 15.252.53 2.971.07 61.354.26
micracantha DC. (116.9720.22) (127.9316.61) (142.6921.64) (106.189.75) (127.7920.48)
(CM)
Tiliacora 79.634.00 48.055.43 28.192.20 15.801.45 0.302.17 54.655.34
triandra Diels. (109.225.84) (90.91.19) (99.734.11) (106.8710.98) (112.567.74)
(TT)
Harrisonia 83.451.78 46.556.96 19.967.86 6.148.52 -1.605.20 53.165.21
perforata Merr. (57.518.01)* (83.627.09) (106.795.61) (100.442.50) (109.359.87)
(HP)
Clerodendrum 88.001.46 55.181.79 26.353.20 8.605.15 -2.124.51 46.550.92
petasites S.Moore (137.4627.28) (150.2324.65) (159.3218.69) (141.0021.93) (155.2512.64)
(CP)
Benjalokawichian 86.502.76 62.771.30 36.421.81 17.5113.60 4.635.78 40.361.99
(BJW, Ya-Ha- (95.150.64) (92.534.97) (119.096.79) (126.1012.10) (122.4210.03)
Rak, HR)
Indomethacin 63.701.50a 38.603.80 a -9.153.40 a - -32.9515.70 a 20.323.23
(91.261.15) (82.464.87) (79.661.76) (84.2711.96)
* Cytotoxic effect was observed, a Value in μM

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208 Original Article

EC50
(mcg/ml) Ethanolic extracts

120
>100
100

80
61.37
60
40.93
40

15.38 16.91
20 12.75
4.87
0
FR BHT* TT HP HR CM CP

Figure 3 The results of DPPH radical scavenging assay of Benjalokawichian Remedy (Ya-Ha-Rak) and its ingredients
(n=3), *Positive control

2. Anti-inflammatory activity by NO inhibitory previous study of anti-inflammatory activities,

effect and cytotoxicity test in RAW264.7 cells Indomethacin (10 mg/kg) decreased the writhing
The ethanolic extract of Ya-Ha-Rak (HR) remedy response by 89% whereas HR doses of 200 and 400
exhibited the most potent inhibitory activity in this mg/kg decreased the number of writhes induced by
test (IC50= 40.36µg/ml) but it exhibit NO acetic acid by 63%) [10]. The Clerodendrum
production inhibitory effect lower than petasites extract possessed moderate inhibitory
Indomethacin (IC50= 20.32µg/ml). Moreover, the activity on acute phase of inflammation in a dose-
ethanolic extract of Clerodendrum petasites, related manner as seen in ethyl phenylpropiolate-
Harrisonia perforata, Tiliacora triandra, Capparis induced ear edema (ED50 = 2.34 mg/ear) as well as
micracantha showed moderate inhibition activity carrageenin-induced hind paw edema (ED30 =
(IC50<60µg/ml). The Ficus racemosa extracts 420.41 mg/kg) in rats [14]. Its related with CP
showed no inhibition activity (IC50> 100µg/ml) extracts showed the second exhibited NO inhibitory
(Figure 2). The cytotoxicity test, all of extracts effect (IC50= 46.55µg/ml). The ethanolic extract of
showed anti-inflammatory and no toxicity in RAW FR showed no inhibitory activity by NO effect, but
264.7 cells. There are showed survival more than on the other hand, the study of Ficus racemosa
80% in Table1. leaves reported that the petroleum ether extract (400
3. DPPH radical scavenging activity mg / kg) exhibited maximum anti-inflammatory
The ethanolic extract of Ficus racemosa exhibited effect, that is 30.4, 32.2, 33.9 and 32.0% at the end
higher DPPH radical scavenging activity (EC50= of 3 h with carrageenin, serotonin, histamine,
4.87µg/ml) than BHT which is the standard (EC50= dextran-induced rat paw edema, respectively [15].
12.75 µg/ml). The Tiliacora triandra and Furthermore, extracts of the bark from Ficus
Harrisonia perforata showed strong antioxidant racemosa inhibited COX-1 [16] and 5-LOX
activity(EC50= 15.38, 16.91µg/ml, respectively). enzymatic activities at 90 and 18µM [17].
The results of Benchalokawichian remedy and However, this result showed synergistic
Capparis micracantha showed antioxidant activity antiinflammatory effect of the plant ingredients of
(EC50=40.93, 61.37µg/ml, respectively) whereas HR, so HR exhibited the highest anti-inflammatory
Clerodendrum petasites showed no antioxidant activity. The antiinflammatory compound occurred
activity (EC50>100 µg/ml) (Figure 3). after macerating the mixture of plant ingredients.
Thus, it is interesting for isolation anti-
DISCUSSION inflammatory compound from Ya-Ha-Rak remedy
The results of anti-inflammatory activity showed extract.
that HR exhibited maximum NO inhibitory effect The FR, TT, HP showed high antioxidant activities
but less than indomethacin (positive control). In with EC50= 4.87, 15.38, 16.91 µg/ml, respectively.
this study showed results in the same way with This results related with the previous report that the

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Original Article 209

Table 2 Comparison of Percent yield, anti-inflammatory activity by NO inhibitory effect, EC50 values of DPPH radical
scavenging activity from Benjalokawichian remedy and it plant ingredients (n=3)

EC50 values of DPPH

IC50 of NO inhibitory
Part radical scavenging
Sample %Yield activity
of use activity
( IC50±SEM, µg/ml)
( EC50±SEM, µg/ml)
Ficus racemosa Linn. (FR) root 2.01 % >100 4.87  0.24
Capparis micracantha DC. (CM) root 2.83 % 61.35  4.26 61.37  0.34
Tiliacora triandra Diels. (TT) root 3.90 % 54.65  5.34 15.38  0.25
Harrisonia perforata Merr. (HP) root 3.72 % 53.16  5.21 16.91  0.73
Clerodendrum petasites S.Moore (CP) root 3.12 % 46.55  0.92 >100
Benjalokawichian( Ya-Ha-Rak, HR) root 4.26 % 40.36  1.99 40.93  1.25
Indomethacina - - 20.32  3.68 -
BHTb - - - 12.75  0.46
a ,b
positive control

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