全 文 ：Iamsaard et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2014 15(4):405-408

405




Phenolic contents and antioxidant capacities of Thai-Makham Pom
(Phyllanthus emblica L.) aqueous extracts*

Sitthichai IAMSAARD†1,2,3, Supatcharee ARUN1,2,
Jaturon BURAWAT1,2, Wannisa SUKHORUM1,2,
Jintanaporn WATTANATHORN3,
Somsak NUALKAEW4, Bungorn SRIPANIDKULCHAI2
(1Department of Anatomy, Faculty of Medicine, Khon Kaen University,
Khon Kaen 40002, Thailand)
(2Center for Research and Development of Herbal Health Products,
Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon
Kaen 40002, Thailand)
(3Integrative Complementary Alternative Medicine Research and
Development Group, Faculty of Medicine, Khon Kaen University,
Khon Kaen 40002, Thailand)
(4Department of Pharmaceutical Sciences, Faculty of Pharmacy,
Mahasarakham University, Mahasarakham 44150, Thailand)
†E-mail: iamsaard_sitt@yahoo.com; sittia@kku.ac.th
Received Nov. 10, 2013; Revision accepted Feb. 10, 2014
Crosschecked Mar. 10, 2014

doi:10.1631/jzus.B1300284
Document code: A CLC number: Q505; R931.71


This study was aimed at determining the amount
of the total phenolic contents (TPCs) and to evaluate
the antioxidant activities of different extracts (leaves,
branches, and barks) of Phyllanthus emblica (PE) L.,
a traditional medicinal plant that has been recently
used as an anti-inflammatory, wound healing, and
anti-aging agent. PE leaves, branches, or barks were
aqueous-extracted and the amount of the TPC was
determined using a Folin-Ciocalteu reagent method.
The antioxidant activity of each PE extract was as-
sessed using 1,1-diphenyl-2-picrylhydrazyl radical
scavenging and ferric reducing antioxidant power
(FRAP) methods. Among plant extracts, the PE bark
extract possessed the highest TPC. The TPCs of the
PE leaves, branches, and barks were (513.83±20.52),
(650.50±9.76), and (2196.33±11.02) mg gallic acid
equivalent/g dried material, respectively. Interest-
ingly, the IC50, which was calculated from 50% inhi-
bition against concentration (µg/ml) plot, of the PE
leaf ((7.72±0.25) µg/ml), branch ((6.92±0.22) µg/ml),
or bark ((6.54±0.27) µg/ml) extract was lower than
that of the ascorbic acid ((8.06±0.01) µg/ml). As
calibrated to the standard, the potent reducing power
abilities of the PE leaf, branch, and bark extracts were
(696.73±78.48), (729.33±36.9), and (966±64.73) mg/g
ascorbic acid equivalent, respectively.
The Phyllanthus emblica L. (also called “Ma-
kham Pom” in Thai) is a traditional medicinal plant
that has been used in many Asian countries including
Thailand. Focusing on the fruit, it has been demon-
strated that the fruits of PE are rich in phenolics (She
et al., 2013) and possess antioxidant activities (Chal-
ise et al., 2010; Sawant et al., 2010). In addition, PE
fruits have been proven to have many therapeutic
properties (Krishnaveni and Mirunalini, 2010; Chu-
larojmontri et al., 2013). PE fruits have been used for
anti-inflammation, anti-aging, and anti-melanogenesis.
Besides the fruits, in Thailand, the leaves, branches,
and barks of PE have also been used for many tradi-
tional treatments. PE leaves and branches (by boiling
them in a cup of water) are used for reducing fever
and edema. Moreover, the fresh extracts of PE leaves
and branches are used for anti-inflammatory and
external wound healing treatments. For additional
uses of the PE barks, they have been used as a strin-
gent, astringent drug, for wound healing in the oral
cavity, and in treating of dermatitis. Recently, it has
been demonstrated as a potential nanoemulsion
product, developed from PE branch extract, which
has a high entrapment efficiency (Chaiittianan and
Sripanidkulchai, 2013). Because the developed-PE
nanoemulsion particle is small, stable, and can release
phytochemical components, it is also promising to
apply it for skin treatments. These applications may
include such applications as cosmetic cream, muscle
pain relief balm, and herbal-compress ball. With its

Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology)
ISSN 1673-1581 (Print); ISSN 1862-1783 (Online)
www.zju.edu.cn/jzus; www.springerlink.com
E-mail: jzus@zju.edu.cn


* Project supported by the Center for Research and Development of
Herbal Health Products (RD-HHP), Faculty of Pharmaceutical Sci-
ences, Khon Kaen University, Thailand (No. RD-HHP 05)
© Zhejiang University and Springer-Verlag Berlin Heidelberg 2014

Letter:
Iamsaard et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2014 15(4):405-408

406
various medicinal properties, the PE trees are com-
monly planted in the agriculture farms as an economic
plant in many provinces in Thailand. Although many
parts of the PE trees have been believed to possess
various medicinal activities, the scientific demon-
stration of the amount of the TPCs and antioxidant
activities of particular PE leaf, branch, and bark
aqueous extracts is still limited. To gain basic me-
dicinal data of PE before treating in research animals
or humans for future clinical trials, this study at-
tempted to primarily demonstrate the total phenolics
and in vitro antioxidant activities of different part
aqueous extracts of Thai P. emblica.
In our study, the results showed that three dif-
ferent parts of the PE extracts possessed the total
phenolics (Fig. 1) as determined from the calibration
curves of the gallic acid (Y=5.4081x+0.093, R2=
0.9968, where Y is peal area, x is the concentration
(µg/ml) of the marker compound). In comparison,
however, it was significantly demonstrated that the
bark extract contained the highest TPC ((2196.33±
11.02) mg gallic acid equivalent/g dried material),
followed by the branch and leaf extracts ((650.50±
9.76) and (513.83±20.52) mg gallic acid equivalent/g
dried material, respectively).















For the 2-diphenyl-picrylhydrazyl (DPPH) scav-
enging assay, the results showed that each part of the
PE extracts possessed a concentration-response rela-
tionship in the DPPH scavenging activity which was
similar to the trend of positive control (ascorbic acid).
As compared to IC50 of ascorbic acid ((8.06±0.01) µg/ml),
the IC50 of PE leaf ((7.72±0.25) µg/ml), branch ((6.92±
0.22) µg/ml), or bark ((6.54±0.27) µg/ml) extract was
very close to and lower than that of the ascorbic acid
(Table 1).










In the reducing power capacity assay, the results
showed that each part extract of the PE displayed
concentration-dependent increases in the reducing
power as also shown in the calibrated standard
ascorbic acid (Y=0.007x+0.3769, R2=0.9802). Among
the PE extracts, the result (Fig. 2) showed that the PE
bark extract had the highest reduction levels of ferric
ion to ferrous sulfate ((966±64.73) mg/g ascorbic acid
equivalent) as compared to the PE branch and leaf
extracts ((729.33±36.9) and (696.73±78.48) mg/g
ascorbic acid equivalent, respectively).
















The PE fruit aqueous extract should be also as-
sayed in parallel to compare those phenolics and an-
tioxidant activities of its leaf, branch, and bark ex-
tracts. Unfortunately, the season for PE fruit produc-
tion in July was limited, but it is assumed to have
those capacities as previously demonstrated (Krish-
naveni and Mirunalini, 2012; She et al., 2013). In
traditional use, however, both fresh and dried samples
of PE leaves, branches, or barks have been used in all
seasons. According to recent results, it implies that
besides PE fruits (produced once a year), the rest of
the parts of the PE tree can be used at least in July for
traditional uses or for experimental research. In Thai
Table 1 Antioxidant activities of the leaf, branch, and
bark aqueous extracts of P. emblica (PE) by using the
DPPH radical method
Standard/PE extract IC50 (µg/ml)
Ascorbic acid (standard) 8.06±0.01
Leaves 7.72±0.25
Branches 6.92±0.22
Barks 6.54±0.27
Data are expressed as mean±SD (n=3)
0
200
400
600
800
1000
1200
F
R
A
P
(m
g/
g
as
co
rb
ic
a
ci
d
eq
ui
va
le
nt
)
*
Leaves Branches Barks
Fig. 2 Ferric reducing antioxidant powers (FRAPs) of
the leaf, branch, and bark aqueous extracts of P. em-
blica (PE)
Data are expressed as mean±SD (n=3). * P<0.05 vs. leaves
or branches
0
500
1000
1500
2000
2500
Leaves Branches Barks
**
Fig. 1 Amount of total phenolic contents (TPCs) of the
leaf, branch, and bark aqueous extracts of P. emblica
(PE) using the Folin-Ciocalteu reagent method
Data are expressed as mean±SD (n=3). ** P<0.01 vs. leaves
or branches
TP
C

(m
g
ga
lli
c
ac
id
e
qu
iv
al
en
t/g
d
rie
d
m
at
er
ia
l)
Iamsaard et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2014 15(4):405-408

407
traditional medicine, PE barks have been used as a
stringent, astringent drug, for wound healing in the
oral cavity, and also in the treating of dermatitis.
These properties of PE may be partially explained and
supported by the traditional uses since the PE branch
extract showed amounts of total phenolic compounds
and antioxidant activities. To our knowledge, PE bark
aqueous extract here is demonstrated to possess the
highest amount of total phenolics and antioxidant
activities as compared to the rest of the parts. In the
review of literature, α-tocopherol, ascorbic acid, and
reduced glutathione are three major molecules com-
monly present in all higher plants and have antioxi-
dant capacity (Shao et al., 2008). Since PE is classi-
fied as a higher plant, it may also contain such anti-
oxidant molecules, especially rich in the bark sample.
However, the profile of those molecules in PE parts
needs to be further investigated. If the antioxidant
molecule amount of PE bark has demonstrated to be
higher than those of the other parts, it can be used as a
good part for clinical trials in herbal drug develop-
ments and other medical applications.


Materials and methods
Chemicals and reagents
Folin-Ciocalteu reagent, DPPH, acetate buffer,
2,4,6-tripyridyl-s-triazine (TPTZ), and ferric chloride
(FeCl3) were purchased from Sigma-Aldrich Co., Ltd.,
Thailand. Ascorbic acid, gallic acid, and other chemi-
cals were of analytical grade.
Plant collection and extraction
All parts (leaves, branches, and barks) of the PE
were collected from the Khundong 58 Punmai Farm,
Muang, Khon Kaen, Thailand, in July 2013. The plant
samples were further authenticated for their actual
species by Prof. Dr. Pranom CHANTARANOTHAI,
a plant taxonomist, Department of Biology, Faculty
of Science, Khon Kaen University (KKU), Thailand.
The voucher specimens of PE were deposited in the
KKU Herbarium (#Supatcharee Arun 01 [KKU]). For
extraction, the fresh leaves, branches, or barks of the
PE were washed with distilled water and dried in a hot
oven (60 °C) for 5 d, since PE has been commonly
prepared and used in the dried material form. Each
dried sample was crushed by a Hammer mill crusher
(AEG IP54 Lbi 07, Germany) for 30 min. Then, 2 kg
of each crushed sample was extracted with 10 L of
distilled water at 95–100 °C for 30 min and filtered
using nylon clothes. Sequentially, each filtrate was
dried using a spray dryer (Nitro A/S-Gladsaxeveg
305-DK-2860, Soeborg, Denmark) (Toyang et al.,
2012), as calculated from a formula of yield (%)=
(weight of plant powder extract after extraction)/(2 kg
of dried plant before extraction)×100%, and the ex-
traction yields of leaves, branches, and barks of the
PE aqueous extracts were 9.94%, 7.07%, and 12.68%,
respectively. Each sample was further prepared as
concentrated extract stock of 2 mg/ml (dissolved with
distilled water) before assessment.
Determination of TPC
The amount of TPC in PE extract (leaves, branches,
or barks) was measured using the Folin-Ciocalteu
reagent method as previously described (Singleton et
al., 1999). Briefly, each aqueous PE extract (0.2 ml,
0.01 mg/ml) was added into a test tube containing
1 ml of the Folin-Ciocalteu reagent (10%, v/v) and
incubated for 5 min before adding 3 ml of 0.2 g/ml
sodium carbonate. After mixing the solution thor-
oughly, the mixed sample was further incubated at
room temperature in a dark room for 2 h with inter-
mittent shaking. The absorbance was measured at
765 nm using an ultraviolet-visible spectrophotome-
ter (Jasco V530, Japan). All samples were tested in
triplicate. The standard sample used to gain a cali-
bration curve was at five concentrations of diluted
gallic acid solutions (0.026, 0.040, 0.064, 0.080, and
0.160 mg/ml). The results of the TPCs were expressed
as gallic acid equivalents in milligram per gram of dry
sample.
DPPH radical scavenging assay and determina-
tion of IC50
The radical scavenging activity of each part of
the PE extracts was determined by using a DPPH
assay as previously described (Brand-Williams et al.,
1995). Briefly, the distilled water-diluted solution of
the PE leaf, branch, or bark extract was prepared from
a concentrated extract stock into five concentrations
(1.60, 4.27, 6.10, 9.60, and 12.80 µg/ml) to perform a
concentration plot. Then, each concentration of ex-
tract (2 ml) was mixed with 2 ml DPPH in methanol
(0.004%) during 30 min at room temperature in a dark
room. The absorbance was recorded at 517 nm using
an ultraviolet-visible spectrophotometer. The ascor-
bic acid (1, 3, 5, 8, and 14 µg/ml) was used as the
positive standard. All samples and the standard were
Iamsaard et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2014 15(4):405-408

408
measured in triplicate. The scavenging activity of the
extracts or standard was calculated as the percent
inhibition of DPPH radical scavenged activity using
the formula of (absorbance of control–absorbance
of sample)/(absorbance of control)×100%. Data were
expressed as the IC50.
FRAP assay
Reducing power capacity was determined using
an FRAP assay as previously described (Benzie and
Strain, 1996). Briefly, 0.1 ml of each PE sample
(prepared from a stock solution) at six different
concentrations (0.80, 2.00, 5.12, 9.00, 12.80, and
32.00 µg/ml) was mixed with 3 ml of FREP reagent
(300 mmol/L acetate buffer, 10 mmol/L TPTZ,
20 mmol/L FeCl3 (10:1:1, v/v/v)). The mixture was
incubated in a dark room for 20 min and the absorb-
ance was recorded at 593 nm using an ultraviolet-
visible spectrophotometer. For standard preparation,
six concentrations of ascorbic acid (5.76, 9.60, 17.28,
24.00, 32.00, and 48.00 µg/ml) were plotted to de-
termine the reducing power.

Compliance with ethics guidelines
Sitthichai IAMSAARD, Supatcharee ARUN, Jaturon
BURAWAT, Wannisa SUKHORUM, Jintanaporn WAT-
TANATHORN, Somsak NUALKAEW, and Bungorn
SRIPANIDKULCHAI declare that they have no conflict of
interest.
This article does not contain any studies with human or
animal subjects performed by any of the authors.

References
Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of
plasma (FRAP) as a measure of “antioxidant power”: the
FRAP assay. Anal. Biochem., 239(1):70-76. [doi:10.
1006/abio.1996.0292]
Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a
free-radical method to evaluate antioxidant activity.















Lebensm.-Wiss. Technol., 28(1):25-30. [doi:10.1016/
S0023-6438(95)80008-5]
Chaiittianan, R., Sripanidkulchai, B., 2013. Development of a
nanoemulsion of Phyllanthus emblica L. branch extract.
Drug Dev. Ind. Pharm., online. [doi:10.3109/03639045.
2013.838580]
Chalise, J.P., Acharya, K., Gurung, N., et al., 2010. Antioxi-
dant activity and polyphenol content in edible wild fruits
from Nepal. Int. J. Food Sci. Nutr., 61(4):425-432. [doi:
10.3109/09637481003591590]
Chularojmontri, L., Suwatronnakorn, M., Wattanapitayakul,
S.K., 2013. Phyllanthus emblica L. enhances human
umbilical vein endothelial wound healing and sprouting.
Evid. Based Complement. Alternat. Med., 2013:720728.
[doi:10.1155/2013/720728]
Krishnaveni, M., Mirunalini, S., 2010. Therapeutic potential of
Phyllanthus emblica (amla): the ayurvedic wonder. J.
Basic Clin. Physiol. Pharmacol., 21(1):93-105.
Krishnaveni, M., Mirunalini, S., 2012. Chemopreventive
efficacy of Phyllanthus emblica L. (amla) fruit extract on
7,12-dimethylbenz(a)anthracene induced oral carcino-
genesis—a dose-response study. Environ. Toxicol. Phar-
macol., 34(3):801-810. [doi:10.1016/j.etap.2012.09.006]
Sawant, L., Pandita, N., Prabhakar, B., 2010. Determination of
gallic acid in Phyllanthus emblica Linn. dried fruit pow-
der by HPTLC. J. Pharm. Bioallied Sci., 2(2):105-108.
[doi:10.4103/0975-7406.67012]
Shao, H.B., Chu, L.Y., Lu, Z.H., et al., 2008. Primary antiox-
idant free radical scavenging and redox signaling path-
ways in higher plant cells. Int. J. Biol. Sci., 4(1):8-14.
[doi:10.7150/ijbs.4.8]
She, G., Cheng, R., Sha, L., et al., 2013. A novel phenolic
compound from Phyllanthus emblica. Nat. Prod. Com-
mun., 8(4):461-462.
Singleton, V.L., Orthofer, R., Lamuela-Raventos, R.M., 1999.
Analysis of total phenols and other oxidation substrates
and antioxidants by means of Folin-Ciocalteu reagent. In:
Packer, L. (Ed.), Methods in Enzymology. Academic
Press Inc., San Diego, Vol. 299, p.152-178.
Toyang, N.J., Ateh, E.N., Keiser, J., et al., 2012. Toxicity,
antimicrobial and anthelmintic activities of Vernonia
guineensis Benth. (Asteraceae) crude extracts. J. Eth-
nopharmacol., 144(3):700-704. [doi:10.1016/j.jep.2012.
10.016]
中文概要：

本文题目：泰国余甘子水提物中总酚含量及抗氧化活性
Phenolic contents and antioxidant capacities of Thai-Makham Pom (Phyllanthus emblica L.)
aqueous extracts
本文概要：通过对余甘子不同部位（叶、枝和树皮）水提物的总酚含量的测定及体外抗氧化活性的研究，
为其体内及临床试验研究奠定了基础。余甘子叶、枝和树皮的水提物皆具有抗氧化活性，其中
树皮中的总酚含量最高，抗氧化活性最强。
关键词组：余甘子；总酚含量；抗氧化能力；传统药用植物