Industrial Crops & Products 122 (2018) 93–97

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Industrial Crops & Products

journal homepage: www.elsevier.com/locate/indcrop

A combination of simultaneous quantification of four triterpenes and T

fingerprint analysis using HPLC for rapid identification of Centella asiatica
from its related plants and classification based on cultivation ages
⁎
Mohamad Rafia,b, , Fitri Handayania, Latifah Kosim Darusmana,b, Eti Rohaetia,
Yudiwanti Wahyub,c, Sulistiyanib,d, Kohsuke Hondae, Sastia Prama Putrie
a
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Jalan Tanjung Kampus IPB Dramaga, Bogor, 16680, Indonesia
b
Tropical Biopharmaca Research Center-Institute of Research and Community Services, Bogor Agricultural University, Jalan Taman Kencana No. 3 Kampus IPB Taman
Kencana, Bogor, 16128, Indonesia
c
Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University, Jalan Meranti Kampus IPB Dramaga, Bogor, 16880, Indonesia
d
Department of Biochemistry, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Jalan Agatis Kampus IPB Dramaga, Bogor, 16680, Indonesia
e
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

A R T I C LE I N FO A B S T R A C T

Keywords: A simple and reliable method for the identification and classification based on cultivation ages of Centella asiatica
Centella asiatica (CA) was developed. The developed method used a combination of a simultaneous quantification of four tri-
Triterpene terpenes (madecassoside, asiaticoside, madecassic acid, and asiatic acid) and a fingerprint analysis of methanol
Simultaneous quantification extract of CA using a high-performance liquid chromatography (HPLC). The four triterpenes were well separated
Fingerprint analysis
under the optimized elution conditions. Fingerprint chromatogram of CA showed marked differences from those
HPLC
of closely related plants, namely Hydrocotyle sibthorpiodes and Hydrocotyle verticillata, so that it can be used for
discrimination of CA from those plants. Also the concentration of four triterpenes was different among CA
samples from different location and at 3, 4 and 5 months post-planting. Validity of the method was demonstrated
by evaluating the system suitability, linearity, precision, accuracy, limit of detection, and limit of quantification,
as well as by determining the stability of components in CA extract. All of the parameters met criteria for a
reliable and valid method according to the Association of Official Analytical Chemists (AOAC) guidelines. In
addition, the HPLC fingerprint with chemometrics analysis could be used for classification of CA according to
their cultivation ages.

1. Introduction
Centella asiatica (CA), which is known as Indian pennywort, is a
stoloniferous perennial herb which typically classified in tropical and
subtropical regions. In Indonesia, CA is called as pegagan and com-
monly used as a vegetable (salad), food supplement, and jamu (tradi-
tional Indonesia medicine). Some biological activities of CA have been
reported, such as peptic ulcers protection effect (Cheng et al., 2004),
acetylcholinesterase inhibitors (Mukherjee et al., 2007), skin damage
prevention (Sommerfeld, 2007), anti-inflammation (Li et al., 2009),
anticancer (Pittella et al., 2009), neuroprotective effect (Haleagrahara
and Ponnusamy, 2010), antioxidant (Singh et al., 2014), cytotoxic
(Alfarra and Omar, 2014), and antimutagenic (Akinboro et al., 2016).
The primary bioactive components in CA are madecassoside (MS),
asiaticoside (AS), madecassic acid (MA) and asiatic acid (AA), and all of
them belong to a class of triterpene (Fig. 1) (Hashim et al., 2011). These
four triterpenes have often been utilized as marker components for
quality evaluation of raw materials and herbal products of CA. In
general, however, quantity and quality of chemical components in
natural plants are markedly affected by environmental factors of
growth, such as climate (temperature, humidity, light, and wind) and
soil fertility. Harvest time and postharvest processing also have sig-
nificant effects on the chemical composition of plants. To ensure their
quality, efficacy, and safety, a reliable qualitative/quantitative method
is in high demand.
Marker components and fingerprint analysis are commonly used in
the quality analysis of medicinal plants and herbal products (Demirezer
et al., 2014). The marker component analysis focuses one or more

⁎
Corresponding author at: Department of Chemistry, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Jalan Tanjung Kampus IPB Dramaga, Bogor, 16680,
Indonesia.
E-mail address: mra@ipb.ac.id (M. Rafi).

https://doi.org/10.1016/j.indcrop.2018.05.062
Received 17 November 2017; Received in revised form 21 May 2018; Accepted 24 May 2018
0926-6690/ © 2018 Elsevier B.V. All rights reserved.
M. Rafi et al.
Fig. 1. Chemical structures of (a) asiaticoside, (b) asiatic acid, (c) madecassoside, (d) madecassic acid.
specific chemical components in the plants, and their quantitative in-
formation is used for quality evaluation. Meanwhile, the fingerprint
analysis, which allows us to monitor an overall profile of chemical
components in the medicinal plant (Zeng et al., 2008), are mostly used
for qualitative purposes, such as identification and/or authentication of
plant species to prevent contamination of a closely-related medicinal
plant (WHO, 1991). Owing to the complementary properties between
these approaches, combination of the multicomponent quantification
and fingerprint analysis is often employed in a quality control method
of some medicinal herbs (Alaerts et al., 2014; Kumar et al., 2015; Rafi
et al., 2015; Padilla-Gonzalez et al., 2016; Cui et al., 2016).
Two closely-related species from the same family are known for CA,
namely Hydrocotyle sibthorpiodes (HS) and Hydrocotyle verticillata (HV).
In Indonesia, HS and HV are called in their local names, semanggi
gunung and antanan air, respectively. These two plants could be po-
tential counterfeits for CA because they have similar morphology and
biological activities to CA, such as antioxidant, antitumor, and cyto-
toxic (Yu et al., 2006; Huang et al., 2008). Although their leaves have
different appearance, discrimination of these three plants is impaired
once they are pulverized into powder. An HPLC-based chemical fin-
gerprint has been developed for investigating the variance of chemical
components among CA from 14 locations in China (Zhang et al., 2009).
Simultaneous quantification of MS, AS, MA and AA in CA has also been
achieved using HPLC analysis (Rafamantanana et al., 2009). Moreover,
Schaneberg et al. (2002) developed an improved method for quantita-
tive determination of six triterpenes (MS, AS, MA, AA, terminolic acid
and asiaticoside-B) in CA extracts. An LC–MS/MS was also used for the
quantification of the four analytes (Gajbhiye et al., 2016). However, the
combination of fingerprint analysis and simultaneous quantification of
the bioactive triterpenes in CA has not been reported so far. In this
study, we developed an HPLC-based approach for the quality control of
CA by the combination of their fingerprint analysis and simultaneous
quantification of MS, AS, MA and AA. The method developed in this
study was demonstrated to be applicable to the discrimination of CA
from its closely-related species as well as to the determination of its
cultivation ages.
2. Materials and methods
2.1. Chemicals and reagents
Madecassoside (87.5%), asiaticoside (88.8%), madecassic acid
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Industrial Crops & Products 122 (2018) 93–97
(88.1%), and asiatic acid (92.6%) were obtained from ChromaDex Inc.
(Santa Ana, CA, USA). Solvents used in HPLC analysis were of analytical
or HPLC grade (Merck, Darmstadt, Germany). Whatman membrane
filters (0.22 μm pore size; PTFE; P/N E252, Buckinghamshire, England)
were used for filtration of sample solutions.
2.2. Plant materials
Four samples of CA from Bogor (West Java), Kuningan (West Java),
Sleman (Special Region of Yogyakarta) and Boyolali (Central Java), two
herbal products containing CA from a local market, one sample of HV
from Sukabumi (West Java) and one sample of HS from Bogor (West
Java) were used in this study. Identification of all samples was con-
ducted in Herbarium Bogoriense, Research Center for Biology,
Indonesian Institute of Sciences. Voucher specimens (BMK 201603001-
BMK 201603006) were deposited at Tropical Biopharmaca Research
Center, Bogor Agricultural University, Indonesia. For the classification
of CA according to their cultivation ages, we used 3 (BMK 201604001),
4 (BMK 201605001) and 5 (BMK 201606007) month-old samples after
planting with 7 replicates provided by Conservation and Cultivation
Unit of Tropical Biopharmaca Research Center, Bogor Agricultural
University, Indonesia. Before use, all samples were sieved, dried, and
pulverized.
2.3. Apparatus and chromatographic conditions
An HPLC system (LC-20A series, Shimadzu, Kyoto, Japan) equipped
with a diode-array UV–vis detector and shim-pack VP-ODS C18 column
(150 mm x 4.6 mm i.d, 4.6-μm particle size) (Shimadzu, Kyoto, Japan)
was used. The mobile phase consists of acetonitrile (A) and water (B)
using a gradient elution program for 40 min with a flow rate of 1 mL/
min. Column temperature was kept at 40 °C and a detection wavelength
at 206 nm was used for analysis.
2.4. Preparation of samples and standard solutions
About 100 mg powdered samples were suspended in 5 mL methanol
and ultrasonicated for one hour at room temperature using an ultra-
sonication device (Branson 1510E-MT 42 kHz, Danbury, USA) was used
for sample extraction. The sample solutions were filtrated through a
0.22 μm membrane filter and diluted to 10 mL with methanol before
being subjected to HPLC analysis. Standard solutions of MS, AS, MA and
M. Rafi et al. Industrial Crops & Products 122 (2018) 93–97

3. Results and discussions

3.1. Optimization of HPLC conditions

In the initial step of the method development for quantitative ana-

Fig. 2. HPLC chromatogram of CA (a), HV (b), HS (c). Peak assignment: ma-
decassoside (2), asiaticoside (5), madecassic acid (8) and asiatic acid (10).
AA were prepared in methanol at concentrations of 1000 μg/mL. An
appropriate volume of each standard solution was mixed and diluted
with methanol to obtain 6 concentrations ranging from 1 to 200 μg/mL
for MS, 1–250 μg/mL for AS, and 1–150 μg/mL for MA and AA to obtain
calibration curves.
lysis and the fingerprint analysis, we optimized HPLC conditions to
obtain a sufficient separation of the four analytes (MS, AS, MA and AA).
We have tried several mobile phase consisting acetonitrile-water with
different composition in linear gradient elution mode. Total number of
peaks, resolution of the analytes, and total analysis time were used as
variables to determine the optimum conditions for analysis. We main-
tained the flow rate, detection wavelength and column temperature at
1 mL/min, 206 nm and 40 °C, respectively.
The use of linear gradient of acetonitrile in water (20–45% in the
initial 20 min followed by 45–65% in 20–40 min) resulted in a good
separation of the four analytes and an excellent fingerprint chromato-
gram with a total analysis time below 30 min for separation of the four
triterpenes (Fig. 2a), which was shorter than those reported in previous
reports (Schaneberg et al., 2002; Rafamantanana et al., 2009). In these
conditions, the resolutions of MS, AS, MA and AA were higher than 1.5.
There was no extra peak overlapping with those of the analytes in
contrast to a previous report in which MS could not be separated from
asiaticoside B (Rafamantanana et al., 2009).

2.5. Method validation 3.2. Validation of the developed method

Validity of the developed method was evaluated in accordance with
the guidelines of Association of Official Analytical Chemists (AOAC,
2013). System suitability, the linearity of calibration curves, limit of
detection (LOD), limit of quantification (LOQ), precision, accuracy, and
stability of extracted samples were evaluated. In HPLC fingerprint
analysis, the repeatability of the analysis method and the stability of
samples were determined by evaluating relative standard deviations
(RSD) of relative retention times (RRT) and relative peak areas (RPA) of
characteristic peaks. The elution peak of asiaticoside (peak number 5 in
Fig. 2) was used to calculate RRT and RPA. CA from Bogor was used for
the validation test.
2.6. Data analysis
Discriminant analysis was used for classification of CA according to
its cultivation ages. The analysis was performed in XLSTAT software
version 2012.2.02 (Addinsoft, New York, USA).
Validation of the developed method was evaluated in terms of
system suitability, linearity of the calibration curves of the four ana-
lytes, LOD, LOQ, precision, and accuracy. We also evaluated the sta-
bility of the analytes to assess the effect of sample preservation time on
elution profiles. The results were summarized in Table 1. System suit-
ability was assessed to determine whether the system performance has
met the required standards. System suitability test was performed by
injecting five replicates of the standard mixture of MS, AS, MA and AA
(10 μg each/mL). Relative standard deviation of the retention time,
peak area, capacity factor, tailing factor, and theoretical plate number
of the four analytes were below 0.15, 3.50, 1.50, 2.70 and 2.40, re-
spectively. Low variation was obtained in the separation of MS, AS, MA
and AA, indicating system performance have met the required stan-
dards.
Linearity was checked by generating calibration curves of each
analyte in the concentration range between 1 and 200 μg/mL for MS,
1–250 μg/mL for AS, 1–150 μg/mL for MA and AA. Correlation coeffi-
cient (r) was used as the value to evaluate the linearity of the

Table 1
Analytical data of calibration curves, LOD, LOQ, precision, recovery, and stability for the quantitative analysis of madecassoside, asiaticoside, madecassic acid, and
asiatic acid.
Analyte Regression equation (y = a + bx)a(correlation LOD LOQ Precision (RSD, %) Recoverya Stabilityc (n = 6)
coefficient, r)
Intra-day Inter-day Average recovery RSD (%)
(n = 6) (n = 3) (%)b (n = 3)

MS 5140.6x − 8656.4 0.0004 0.0013 Day 1 = 2.01 1.17 98.36 2.51 0.64
(0.9996) Day 2 = 2.25
Day 3 = 4.03
AS 5228 x − 4801.4 0.0004 0.0013 Day 1 = 2.01 1.09 98.28 2.74 0.37
(0.9996) Day 2 = 1.12
Day 3 = 2.72
MA 13058x − 18756 0.0003 0.0010 Day 1 = 1.09 3.82 114.81 3.40 0.58
(0.9984) Day 2 = 2.65
Day 3 = 3.43
AA 14672x + 29944 0.0002 0.0007 Day 1 = 0.39 3.88 103.05 3.99 3.90
(0.9981) Day 2 = 4.02
Day 3 = 3.33

a
Concentration range 1–200 μg/mL for madecassoside, 1–250 μg/mL for asiaticoside, and 1–150 μg/mL for madecassic acid and asiatic acid.
b
Three levels of added standard compounds (25, 50, and 100 μg/mL) to the sample solution with each level measured in triplicat.
c
Six measurements at 0, 4, 8, 12, 24, and 48 h after extraction of the sample.

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M. Rafi et al. Industrial Crops & Products 122 (2018) 93–97

calibration curve. The correlation coefficient of each calibration curve Table 2

was higher than 0.9900. Signal to noise ratio (S/N) was used to de- Madecassoside, asiaticoside, madecassic acid, and asiatic acid content in CA
termine the LOD and LOQ of the four analytes and estimated by mea- samples.
suring S/N equal to 3 and 10, respectively. We found LODs and LOQs of
the four analytes were in the range of 2.0 × 10−4–4.0 × 10−4 and
7.0 × 10−4–13.0 × 10−4 μg/mL, respectively. The low LOD and LOQ
value indicated that the developed method has an excellent sensitivity.
The precision of the method was determined by analyzing six
samples for three consecutive days (intra-day and inter-day) and ex-
pressed as RSD. Intra- and inter-day precision were found to be less
than 4.1%. The accuracy of the developed method was determined by
standard addition method and expressed as a percentage of recovery.
The addition of each standard analyte solutions to the test sample was
performed at three different concentration levels which followed by
measurements of three replicates at each level of concentration. The
average percentages of recovery for MS, AS, MA and AA were 98.4%,
98.3%, 114.0%, and 103.0%, respectively with RSD below 4% for each
analyte. These results demonstrated that the developed method has a
sufficient accuracy. Stability of analytes in the sample solution was
evaluated by analyzing the sample solutions after 0, 4, 8, 12, 24, and
48 h storing at room temperature. RSD ranged between 0.37–3.90%,
demonstrating all of the analytes are stable after their extraction from
CA at least for 48 h.
In the HPLC fingerprint analysis of CA, repeatability of the method
was tested by injecting 5 different samples which were prepared daily
for three consecutive days (intra-day and inter-day). The RSD values of
RRT and RPA were less than 0.5% and 4.3%, respectively. Method
stability was determined based on the analysis of sample solution for
two days which resulted in the RSD values of RRT and RPA below 2.6%
and 3.5%. From the result of method validation indicated that the
proposed method was reliable and valid.

3.3. Simultaneous quantification of the four triterpenes in C. asiatica

The HPLC analysis was used for simultaneous determination of MS,

AS, MA and AA. CA from distinct areas in Java Island (Bogor, Kuningan,
Sleman, and Boyolali) and commercial herbal medicine containing CA
were subjected to the analysis. Triplicate measurements were per-
formed to determine the mean amount of MS, AS, MA and AA in each
sample. Table 2 showed the level of the four analytes in the samples. CA
from Bogor had higher levels of MS, AS, MA and AA compared to the
other samples.
We also used the developed method for quantification of the four
triterpenes in CA samples with 3, 4 and 5 months of post-planting
(MPP). Samples were measured in duplicate, and the amounts of the
four triterpenes were showed in Table 2. The contents of MS, AS, MA,
and AA were ranging from 2.59 to 15.1, 2.45 to 13.6, 0.65 to 6.39, and
0.04 to 4.76 mg/g, respectively; demonstrating that contents of the four
components were significantly affected by the post-planting period. MS
and AS were found to be the most dominant component while levels of
MA and AA were considerably low in the majority of the samples in-
vestigated. Comparison of the amount of the four triterpenes in CA with
different MPP showed that the amount of MS, AS, MA, and AA in CA
samples of 4 MPP was higher than those of 3 MPP and 5 MPP, except
one sample of 4 MPP. MA and AA were found in a slight concentration
in 5 MPP of CA. These results clearly showed that the levels of chemical
components in CA could be markedly affected by difference in their
environmental growth conditions (climate and soil fertility), harvest,
and post-harvest processing.
3.4. Identification of C. asiatica by HPLC fingerprint analysis
Fingerprint analysis is often employed for the identification and
discrimination of some closely-related medicinal plants. In this study,
we aimed to identify and discriminate CA from HV and HS. By com-
paring their fingerprint chromatograms (Fig. 2), we found that only
a
CA sample from some area (Bogor, Kuningan, Sleman, and Boyolali); com-
mercial herbal medicine contain of CA (CA-1 and CA-2); CA samples of 3 MPP
(CA-3A1–CA-3A7), 4 MPP (CA-4A1–CA-4A7), 5 MPP (CA-5A1–CA-5A7).
peak 12 commonly appeared in CA, HV, and HS. Peak 3 appeared in CA
and HS but not in HV whereas peak 11 was found only in HV and HS.
The other five remaining peaks (peak 2, 4, 5, 8, and 10) were specifi-
cally found only in CA. Therefore, based on the RRT of the five char-
acteristic peaks (peak 2, 4, 5, 8, and 10) including those of MS, AS, MA,
and AA (peak 2, 5, 8, and 10, respectively), we can identify an unknown
sample to be CA.
These peaks are also available for the discrimination of CA from HV
and HS since they appear only in the chromatogram profile of CA.
Furthermore, to detect the contamination of HV in CA, we can use peak
7, 9, and 13 which are specific to HV. Similarly, we can use peak 1 and
6, which appear only in HS, to detect HS contamination in CA.
The proposed method (HPLC fingerprint analysis) was then applied
to 21 samples of CA consisting of 7 samples of 3 MPP, 7 samples of 4
MPP, and 7 samples of 5 MPP. Seven peaks (peak 2, 3, 4, 5, 8, 10 and
12) which were commonly found in all the samples with sharp signals
were selected as the common peaks, and the peak of asiaticoside (peak
5) was employed as a reference to calculate RRT and RPA of the
common peaks. RRT of the common peak was relatively consistent,

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M. Rafi et al.
Fig. 3. Plot of DA of CA samples according to its cultivation ages.
demonstrating RRT can be a reliable parameter for sample identifica-
tion. By contrast, large fluctuations were found in RPA values, in-
dicating cultivation ages of CA lead to a significant difference in the
levels of its chemical components.
3.5. Classification of C. asiatica acccording to its cultivation ages
As we know, the level of bioactive component in a medicinal plant
depends on its cultivation age, and it will relate to the level of their
biological activity (Ghasemzadeh et al., 2016). So, it is crucial for us to
determine in which cultivation ages the medicinal plant can be har-
vested to have a consistent and high level of biological activity in their
product. Chromatographic fingerprint analysis can be applied for the
classification of CA according to their different cultivation ages as well
as those for other plants. However, the common peaks in CA with dif-
ferent cultivation ages differed only in their intensities and peak area.
To solve this problem, the fingerprint profiles of HPLC analysis were
further processed by a multivariate analysis.
We employed discriminant analysis (DA), which is one of multi-
variate-analysis methods. DA is mostly used for discrimination and
classification of objects and belongs to supervised pattern recognition.
Areas of 7 common peaks (peak 2, 3, 4, 5, 8, 10 and 11) in the fin-
gerprint chromatograms of CA were used as variable. Before being
subjected to DA, we performed pre-processing of our data by dividing
each variable by its standard deviation. Discriminant function (DF) for
each group was obtained from DA by searching a linear combination of
data that will give separation of 2 or more observation groups. DF plot
gave total variance as much 100% (DF1 = 79.8% and DF2 = 20.3%).
According to DA plot (Fig. 3), all of the samples could be classified into
each group showing that estimated DF could differentiate CA based on
its cultivation ages, i.e. 3, 4, and 5 MPP.
Prediction ability of this model was evaluated using leave-one-out
cross-validation (LOOCV). LOOCV is the most general approach em-
ployed for the validation of discriminant model. In LOOCV, a sample is
tested in a model as an unknown sample to predict sample membership.
The procedure is repeated until all the samples have been tested in the
rotation. In this study, LOOCV result obtained was 97.6% showing that
model could give good classification prediction for the tested samples.
4. Conclusion
HPLC equipped with a diode array detector was used for fingerprint
analysis of methanol extracts of CA and its closely related plants (HV
and HS) as well as for simultaneous quantification of madecassoside,
asiaticoside, madecassic acid and asiatic acid in CA. Validation of the
method demonstrated that all the parameters met the acceptance cri-
teria. Discrimination of CA from HV and HS was achieved by using
fingerprint profiles obtained using the optimized HPLC method.
Further, CA samples could be classified according to their cultivation
ages using a combination of HPLC fingerprint and discriminant
97
Industrial Crops & Products 122 (2018) 93–97
analysis.
Acknowledgement
This work was partly support by the Penelitian Unggulan Perguruan
Tinggi Research Grant 2016 (No: 079/SP2H/LT/DRPM/Il/2016) from
the Directorate of Research and Community Services, Ministry of
Research, Technology and Higher Education, Republic of Indonesia.
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