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Original article
Quality control standard values and stability study of ethanolic
Ya-Hom KAE-LOM-WING-WIEN remedy extract on nitric
oxide inhibition in LPS-stimulated RAW 264.7 macrophage
cells of its anti-inflammatory activity
* Student of Master of Sciences Program Applied Thai Traditional Medicine, Faculty of Medicine, Thammasat University
** Department of Applied Thai Traditional Medicine, Faculty of Medicine, Thammasat University
*** Center of Excellence on Applied Thai Traditional Medicine Research (CEATMR), Faculty of Medicine, Thammasat University
Corresponding author: Associate Professor Dr.Arunporn Itharat, Department of Applied Thai Traditional Medicine, Faculty of
Medicine, Thammasat University and Center of Excellence on Applied Thai Traditional Medicine Research (CEATMR), Faculty of
Medicine, Thammasat University E-mail: iarunporn@yahoo.com
Received: 18 June 2018 Accepted: 29 June 2018
Abstract
Introduction: Ya-Hom KAE-LOM-WING-WIEN remedy (KLWW) is included in the Thai National List of Essential
Medicines A.D. 2013 and used for treating dizziness, fatigue, and sleeplessness. Quality control
of plant ingredients and stability test of this remedy extract were ensured and controlled the
quality of herbal medicines. In addition, standard quality values of this remedy and its plant
ingredients have never been reported. The stability of this remedy extracts had also no report.
The aim of this study was to investigate the quality control and stability test of KLWW on
Method: Quality assessment method of remedy and its plant ingredients were determined by loss on
drying, extractive value, total ash and acid insoluble ash, following Thai Herbal Pharmacopeia
(THP) method. The stability testing of KLWW extract was stored over a six-month period, under
activity by nitric oxide (NO) inhibitory assay in LPS-stimulated RAW 264.7 macrophage cells.
Results: The KLWW showed loss on drying value of 6.40 ± 0.20%, total ash value of 6.77 ± 0.48%, acid
insoluble ash value of 0.91 ± 0.11%, and the ethanol and water-soluble extractive values of
7.64 ± 0.33 and 18.37 ± 0.74%, respectively. Stability test of the 95% ethanolic extract of KLWW
on NO inhibition at days 0, 15, 30, 60, 90, 120, 150 and 180 showed IC50 values of 19.38 ± 1.13,
28.43 ± 078, 29.06 ± 1.71, 34.02 ± 0.40, 33.82 ± 0.44, 34.66 ± 2.32, 34.13 ± 1.68 and 32.04 ±
2.32 µg/ml, respectively, compared with prednisolone (positive control) with IC50 value of 0.16
and the activity did not decreased any further up to 6 months.
Discussion and KLWW passed the standard value of THP standard in terms of loss on drying, extractive value,
Conclusion:
50 was in range 28 - 34 µg/ml.
Key words:
Stability
Nattanida Jantarach*, Pannawat Chaiyawatthanananthn**,
Pakakrong Thongdeeying**,***, Arunporn Itharat **, ***
511
Thammasat Medical Journal, Vol. 18 No. 4 October - December 2018
Introduction
Ya-Hom is a Thai traditional herbal remedy
and has long been used for treatment of dizziness,
fatigue, fainting, abdominal discomfort etc. in
Thailand. The remedy is a cardiotonic agent and is
usually used for adjustment of wind element affecting
healthy circulation1
Ya-Hom remedy are registered on the Thai National
List of Herbal Medicinal Products A.D. 2013 i.e.
Ya-Hom KAE-LOM-WING-WIEN remedy (KLWW)
has been used for treatment of dizziness, fatigue and
sleeplessness2. The KLWW is nice-smelling herbal
remedy and consists of various proportions of twenty-
three medicinal ingredients2. There are Glycyrrhiza
glabra L. (root), Myristica fragrans Houtt. (heartwood),
Syzygium aromaticum
Angelica sinensis (Oliv.) Diels (root), Ligusticum
sinense Oliv. cv.Chuanxiong Hort (rhizome), Vetiveria
zizanioides (L.) Nash ex Small (root), Nelumbo nucifera
Gaertn. (pollen), Cinnamomum bejolghota (Buch.-
Cinnamomum loureirii
Cinnamomum verumAquilaria crassna
Pierre ex Lecomte. (wood), Euphorbia antiquorum
L. (heartwood), Artemisia annua L. (aerial part),
Terminalia chebula Retz. var chebula (gall), Alyxia
reinwardtii
camphor, Mimusops elengi L. (wood), Mesua ferrea
Mimusops elengiMammea
siamensisEcdysanthera rosea
& Arn. (vine), sodium borate and Dracaena loureiri
Gagnep. (heartwood).
Standard values of quality control on all Thai
remedy and its plant ingredients were determined
for quality controlling of the raw materials by fol-
lowing Thai Herbal Pharmacopoeia standard (THP)3.
assay had been studied on Glycyrrhiza glabra L.4,
Myristica fragrans Houtt.5, Angelica sinensis (Oliv.)
Diels6, Ligusticum sinense Oliv. cv.Chuanxiong Hort7,
Vetiveria zizanioides (L.) Nash ex Small8 , Nelumbo
nucifera Gaertn.9, Cinnamomum verum J. Presl.10,
Mesua ferrea Linn.11, and Mammea siamensis Kosterm6.
The stability test of the remedy extract implied that
the extract had effective assurance activity. Therefore,
this study was to determine standard values of quality
control methods of this remedy and its plants ingredi-
ents and also investigated its stability test of KLWW on
accelerated condition for assessing quality and effective
assurance activity of this remedy.
Method
Chemicals
RPMI medium 1640 (RPMI 1640), Penicillin-
Streptomycin (P/S), trypsin-ethylene diamine tetra
acetic acid (EDTA) and trypan blue were purchased
from Gibco, USA. Fetal Bovine Serum (FBS) was
purchased from Biochem, Germany. Phosphate
buffered saline (PBS) was purchased from Ameresco,
USA. Sodium bicarbonate (NaHCO3) was purchased
from BHD, England. Lipopolysaccharide (LPS, from
Escherichia coli) and 3-[4, 5-Dimethyl-2-thiazolyl]-2,
5-dipheyl-2H-tetrazalium bromide (MTT) were
purchased from Sigma-Aldrich Chemical Company,
USA. Absolute ethanol was purchased from QREC,
New Zealand. Others chemicals were purchased at
analytical grade.
Medicinal ingredients in Ya-Hom KAE-LOM-WING-
WIEN
Ya-Hom KAE-LOM-WING-WIEN remedy
consists of 23 medicinal ingredients which were
collected from various parts of Thailand and India,
China, Vietnam, Indonesia, Australia, and America in
Southern Center of Thai Medicinal Plants at Faculty of
plant and voucher specimens are shown in Table 1.
512
Species Places for Voucher specimen Thai name Part of plant used
specimen collection number
A. annua India SKP051010101 Kot chula lampa aerial part
A. sinensis China SKP199011901 Kot chiang root
A. crassna
A. reinwardtii
Borneo camphor China - Phim sen -
C. bejolghota
C. loureirii
C. verum
D. loureiri
E. antiquorum
E. rosea
G. glabra China SKP072070701 Cha aim thet root
L. sinense China SKP199121901 Kot hua bua rhizome
M. ferrea
M. elengi
M. elengi*
M. fragrans Australia SKP121130601 Chan thet stem
M. siamensis
N. nucifera
S. aromaticum
Sodium borate America - Nam phra santoeng -
T. chebula India SKP0459200301 Kot phung pla gall
V. zizanioides
Table 1 List of ingredients in Ya-Hom KAE-LOM-WING-WIEN remedy
Standard values of quality control of KLWW and
its plant ingredients12
Standard values of quality control of the
KLWW and its ingredients were investigated following
THP standard12 including loss on drying, extractive
value, total ash, and acid insoluble ash. All of plant
ingredients were investigated quality control methods
except borneo camphor and sodium borate.
Moisture contents
Moisture content or loss on drying was
analyzed by electronic moisture analyzer (Scaltec,
Model: SMO 01). 5 g of each sample was put into the
moisture analyzer at 105 ºC. The weight of the dried
sample was displayed and moisture content was
calculated by the formula.
Extractive value
The study includes ethanol and water-soluble
extractive values. Dried plant powder (5 g) were
macerated in 100 ml of 95% ethanol in Erlenmeyer
hours and then allowing to stand for 18 hours. After
that, the extract was dried at 105°C until constant
weight. The procedure for determination of water-
soluble extractive value is similarly to the method
513
Thammasat Medical Journal, Vol. 18 No. 4 October - December 2018
for ethanol-soluble extractive value but using 0.25%
chloroform in water instead of ethanol. The percentage
of ethanol and water-soluble extractive values were
calculated.
Total ash contents
Weight of sample (2 g) in crucible was recorded
The crucible was cooled in a dessicator and then put
in the dessicator until cool down. This procedure was
weight, % total ash was calculated compared with
the weight of before burning.
Acid insoluble ash contents
This method was continued from the total
ash content method. The total ash was added to
9 hours. After burning, weight of the ash was mea-
sured until the weight was stable. Percentage of acid
insoluble ash was calculated.
The stability test of KLWW extract13, 14
The 95% ethanolic extract of KLWW was
stored for 180 days under 40 ± 2 °C with 75 ± 5% RH as
(control sample), 15, 30, 60, 90, 120, 150 and 180 and
activity by nitric oxide (NO) inhibitory assay.
Assay of nitric oxide production inhibitory effect
15, 16
Inhibitory activity on NO production in RAW
264.7 murine macrophage cells was used to evaluate
(ATTC TIB-71) were cultured in RPMI 1640 medium
supplemented with 10% FBS and 1% P/S stored at
37 °C in 5% CO2 atmosphere with 95% humidity.
The cells were washed with PBS according to 0.25%
trypsin-EDTA and then added fresh medium. After
that it was centrifuged at 1,500 rpm for 5 min, the
supernatant was removed and replaced with 10 ml
of fresh medium. Viable cells were counted by using
-
tion of 1 x 105 cells/well in 96 well plates. After incu-
bation for 24 hours, the medium was removed and
added fresh medium with 100 µl containing 2 ng/ml
of lipopolysaccharide (LPS) together with sample
solution at various concentrations (1-100 µg/ml). The
extracts were dissolved with DMSO and diluted in
medium according to the desired concentration, and
then added 100 µl/well. After incubation for 24 hours,
NO production was determined by measuring the
accumulation of nitrite in the supernatant by adding
100 µl/well of the Griess reagent consist of 1% sulfa-
nilamide in 5% H2SO4 and 0.1% naphthalene diamine
dihydrochloride). Cytotoxicity testing was also tested
to ensure that NO production was not produced
by destroying cell membrane (% cytotoxicity less
than 30%) by using MTT colorimetric method. The
absorbance was measured at 570 nm by a microplate
inhibitory concentration (IC50) value were determined
by Prism software.
Statistical analysis
All data are mean ± standard error of mean
(n=3). The data were analyzed with SPSS (License
Authorization Wizard) statistical software using repeated
test for data with non-normality and Paired t-test for
data with normality. A p-value of less than 0.05 was
Results
The plant ingredients data of KLWW included
voucher specimen number, Thai name and part used
were shown in Table 1. The results of standard values
of quality control of KLWW and its plant ingredients
were shown in Table 2. The stability of the 95%
activity through inhibitory activity of NO release were
shown in Table 3.
514
Sample %Loss on drying %Extractive value %Ash content
Ethanol-soluble Water-soluble Total ash Acid insoluble ash
A. annua 8.08 ± 0.23 5.10 ± 0.25 19.09 ± 0.24 13.18 ± 0.15 0.43 ± 0.01
A. sinensis 3.47 ± 0.29* 6.36 ± 0.62* 48.56 ± 4.47* 8.65 ± 0.37* 2.44 ± 0.34*
(not more (not less (not less (not more (not more
than 14%) than 12%) than 52%) than 7%) than 2%)
A. crassna 6.92 ± 0.10 3.62 ± 0.14 2.74 ± 0.02 0.74 ± 0.01 0.08 ± 0.01
A. reinwardtii 6.30 ± 0.34 1.28 ± 0.09 15.99 ± 0.23 8.20 ± 0.17 0.08 ± 0.02
C. bejolghota 8.01 ± 0.35 20.37 ± 2.37 13.48 ± 0.22 1.30 ± 0.02 0.06 ± 0.00
C. loureirii 6.27 ± 0.50 2.35 ± 0.25 5.89 ± 0.09 2.98 ± 0.02 0.09 ± 0.01
C. verum 4.47 ± 0.32 2.47 ± 0.34 9.65 ± 0.20 4.10 ± 0.07 0.06 ± 0.00
D. loureiri 4.40 ± 0.22** 18.73 ± 0.18** 2.61 ± 1.04** 5.62 ± 0.23** 0.37 ± 0.08
(not more (not less (not less (not more -
than 8%) than 12%) than 1%) than 1%)
E. antiquorum 4.53 ± 0.18 24.02 ± 1.25 2.05 ± 0.21 4.20 ± 0.10 0.12 ± 0.01
E. rosea 5.81 ± 0.28 2.43 ± 0.30 10.14 ± 0.80 4.14 ± 0.38 0.14 ± 0.03
G. glabra 6.73 ± 0.62 6.99 ± 0.41 16.33 ± 0.50 8.83 ± 0.39 1.32 ± 0.18
L. sinense 8.68 ± 0.43 9.71 ± 0.57 21.61 ± 2.66 4.20 ± 0.12 0.36 ± 0.01
M. ferrea 7.13 ± 0.22** 15.77 ± 0.53** 9.39 ± 1.21** 4.59 ± 0.35** 1.54 ± 0.12**
(not more (not less (not less (not more (not more
than 11%) than 4.5%) than 2.5%) than 5%) than 1.5%)
M. elengi 8.72 ± 0.38** 8.06 ± 0.17** 11.96 ± 0.05 5.67 ± 0.23** 1.12 ± 0.18**
(not more (not less - (not more (not more
than 16%) than 8%) than 7%) than 3%)
M. elengi* 5.68 ± 0.21 5.02 ± 0.04 5.09 ± 0.02 1.02 ± 0.03 0.26 ± 0.01
M. fragrans 6.48 ± 0.44 6.61 ± 0.34 1.52 ± 0.04 7.78 ± 0.02 1.68 ± 1.08
M. siamensis 8.01 ± 0.20 15.08 ± 0.20 25.43 ± 0.31 8.12 ± 0.05 0.39 ± 0.01
N. nucifera 7.82 ± 0.29** 6.05 ± 0.07 7.62 ± 0.55** 7.89 ± 0.18** 1.77 ± 0.02**
(not more - (not less (not more (not more
than 12%) than 10.5%) than 6%) than 1%)
S. aromaticum 5.78 ± 0.49 6.43 ± 0.16 20.74 ± 0.44 5.71 ± 0.13 0.19 ± 0.01
T. chebula 5.23 ± 0.37 21.56 ± 2.05 45.14 ± 0.82 3.72 ± 0.10 0.08 ± 0.03
V. zizanioides 7.91 ± 0.44 10.21 ± 0.51 40.10 ± 0.24 9.54 ± 0.07 1.85 ± 0.07
KLWW 6.40 ± 0.12 7.64 ± 0.33 18.37 ± 0.74 6.77 ± 0.48 0.91 ± 0.11
Table 2 The results of quality control of KLWW and its plant ingredients
Note: * indicated the standard value of THP 2016,
** indicated the standard value of Vol.4 2014,
- indicated not reported
515
Thammasat Medical Journal, Vol. 18 No. 4 October - December 2018
The standard values of quality control of
KLWW was found that the percentage loss on drying
of KLWW was 6.40% ± 0.12%.The highest percentage
of loss on drying was Mimusops elengi Linn. (8.72 ±
0.37%) and the lowest percentage of loss on drying
was Angelica sinensis (Oliv.) Diels (3.47 ± 0.29%). The
percentages of ethanol and water-soluble extractive
values of KLWW were 7.64 ± 0.33% and 18.37 ± 0.74%,
respectively. The highest percentage of ethanol-
soluble extractive value was Euphorbia antiquorum
L. (24.02 ± 1.25%) and the lowest percentage of
ethanol-soluble extractive value was Alyxia reinwardtii
percentage of water-soluble extractive value was
Angelica sinensis (Oliv.) Diels (48.56 ± 4.47%) and the
lowest percentage of water-soluble extractive value
was Myristica fragrans Houtt. (1.52 ± 0.04%). The
percentage total ash of KLWW was 6.77 ± 0.48%. The
highest percentage of total ash was Artemisia annua
L. (13.18 ± 0.15 %) and the lowest percentage of total
ash was Aquilaria crassna Pierre ex Lecomte. (0.74 ±
0.01%). The percentage acid insoluble ash of KLWW
was 0.91 ± 0.11%. The highest percentage of acid
insoluble ash was Angelica sinensis (Oliv.) Diels (2.44
± 0.34 %) and the lowest percentage of acid insoluble
ash was Cinnamomum bejolghota (Buch.-Ham.) Sweet
and Cinnamomum verum J. Presl. (0.06 ± 0.00% and
0.06 ± 0.00%, respectively). The KLWW passed the
standard criteria of THP12.
The stability results of the 95% ethanolic
KLWW extract on inhibited NO production at days 0,
15, 30, 60, 90, 120, 150 and 180 presented IC50 values
of 19.38 ± 1.13, 28.43 ± 078, 29.06 ± 1.71, 34.02 ± 0.40,
33.82 ± 0.44, 34.66 ± 2.32, 34.13 ± 1.68, and 32.04 ±
2.32 µg/ml, respectively.
Discussion and Conclusion
The KLWW remedy is mostly used for
adjustment of wind element for healthy circulation.
The study of KLWW based on the Thai National List
of Herbal Medicinal Products and control of quality
ingredients in relation to the use of herbal medicines.
Quality control methods and stability test of KLWW
that presented the stability of ethanolic KLWW extract
on NO inhibition in LPS-induced macrophage cells.
Stability of 95% ethanolic Comparison of the
Storage time of extract extract of Ya-Hom remedy by p-value with Day 0
inhibition nitric oxide
production IC50 ± SEM (µg/ml)
Day 0 19.38 ± 1.13 -
Day 15 28.43 ± 0.78 0.04
Day 30 29.06 ± 1.71 0.02
Day 60 34.02 ± 0.40 0.00
Day 90 33.82 ± 0.44 0.00
Day 120 34.66 ± 2.32 0.00
Day 150 34.13 ± 1.68 0.00
Day 180 32.04 ± 2.32 0.00
Table 3
production (IC50 µg/ml ± SEM), (n=3)
Note:
516
reduce NO release although it was different from the
fresh extract (day 0). For stability study, KLWW extract
50 were
in range 28 - 34 µg/ml after 15 days until 180 days
compared with day 0 (IC50 as 19.38 µg/ml). Thus, it
was concluded that KLWW extract is nearly stable
container protected from light and stored at room
temperature for at least two years13, 14. Although, the
results were reduction of activity, KLWW extract was
The standard quality value of THP repre-
sented by loss on drying is not more than 10%. KLWW
and its plant ingredients were within the standard
value. The highest percentage of loss on drying was
Mimusops elengi
so it should be the highest moisture content because
-
ture. The lowest percentage of loss on drying was
Angelica sinensis (Oliv.) Diels because this plant part is
rhizome and export from China, so it should be made
dryness for protection of microbial contamination. The
highest percentage of total ash was Artemisia annua
L. (13.18 ± 0.15 %) which is high value and did not
pass of criteria of THP which ash content of the plant
is not more than 10%. Aerial part of Artemisia annua
L. which bought from India may contaminate from
sand and small gravel from preparing plant. Its acid
this value is less than 0.5% (0.43%). If this value also
showed higher than 2%, it is described that this plant
was contaminated from heavy metal12. Thus, these
results showed that the source of raw material for
preparing traditional drug is necessary for standard
values of plants. Although the percentage of total
ash of Artemisia annua L., Angelica sinensis (Oliv.),
Dracaena loureiri Gagnep., and Nelumbo nucifera
Gaertn. did not pass of criteria, KLWW was within
12. The lowest
percentage of total ash was Aquilaria crassna Pierre
ex Lecomte. (0.74 ± 0.01%). This plant is wood part
so it can be burned and no contaminated from sand.
Acid insoluble ash of this plant also related with total
ash content because it showed small value amount
(0.08 ± 0.01%). The percentage of acid insoluble
ash of KLWW was within the standard value of THP
Angelica sinensis (Oliv.) Diels and
Nelumbo nucifera Gaertn. did not pass the standard
showed the percentage of water-soluble extractive
value more than ethanol-soluble extractive value.
Angelica sinensis (Oliv.) Diels and Nelumbo nucifera
Gaertn. showed the percentage of ethanol and water-
soluble extractive values less than the standard value
of THP. These plants related with high total ash and
acid insoluble ash contents.
In conclusion, the results indicated that the
inhibition and stable within 2 years. The standard
for quality control of raw materials of plant ingredients
and be guided for preparing KLWW. However, KLWW
compounds and testing in animal model.
Acknowledgements
Excellence on Applied Thai Traditional Medicine
Research (CEATMR), Faculty of medicine, Thammasat
University for grant and laboratory facilities.
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