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B I O D I V E R S I T A S
ISSN: 1412-033X
Volume 26, Number 3, March 2025 E-ISSN: 2085-4722
Pages: 1258-1270 DOI: 10.13057/biodiv/d260325
DNA barcoding and High-Performance Liquid Chromatography
as golden standards of authentication in Blumea balsamifera and
Ehretia microphylla products
ALYSSA MARIE A. LOLA1,2,3,4,♥, SAM DOMINIC A. BINAG1,4,5, NIÑA KATHRYN G. ALFECHE1,4,5,
GRECEBIO JONATHAN D. ALEJANDRO1,4,5
1Graduate School, Thomas Aquinas Research Center, University of Santo Tomas. Manila, Metro Manila, 1008, Philippines. Tel.: +63-2-7315396,
Fax.: +63-2-7409732, email: aalola@ust.edu.ph
2Research Center for the Natural and Applied Sciences, Thomas Aquinas Research Center, University of Santo Tomas. Manila, Metro Manila, 1008,
Philippines
3Graduate School of Science, Tokyo Metropolitan University, Minami-Osawa, Hachioji-shi, Tokyo 192-0397, Japan
4Makino Herbarium, Tokyo Metropolitan University. Minami-Osawa, Hachioji-shi, Tokyo 192-0397, Japan
5Department of Biological Sciences, College of Science University of Santo Tomas. Manila, Metro Manila, 1008, Philippines
Manuscript received: 26 September 2024. Revision accepted: 11 March 2025.
Abstract. Lola AMA, Binag SDA, Alfeche NKG, Alejandro GJD. 2025. DNA barcoding and High-Performance Liquid Chromatography
as golden standards of authentication in Blumea balsamifera and Ehretia microphylla products. Biodiversitas 26: 1258-1270. In the
Philippines, the Department of Health (DOH) has included Blumea balsamifera (sambong) and Ehretia microphylla (tsaang-gubat)
among the top ten medicinal plants in the country. Sambong is known for treating urinary stones, while tsaang-gubat is used for helping
increase the motility of intestines. Despite their known healing effects, there remains a lack of strict implementation for the approval of
these products to be commercially available. This study aimed to employ DNA barcoding and High-Performance Liquid Chromatography
(HPLC) by creating a Standard Reference Material (SRM) for both methods. Eight blindly selected sambong herbal medicinal products
(HMPs), and six blindly selected tsaang-gubat HMPs are authenticated using the stated methods. DNA barcoding reveals that samples
BB4 and EMP1 are cases of substitution for the products containing Oryza glaberrima and Cymbopogon citratus, respectively. HPLC
analysis also reveals that all sambong derived HMPs contain quercetin, except for samples BB2, BB4, and BMP3, whereas for the
tsaang-gubat herbal products, only sample EMP1 does not contain rosmarinic acid as its active compound. Dissidence in the results
indicates that both protocols should be used in coordination as standard authentication tools to promote accuracy of the results and to
provide safety to all the consumers. Furthermore, DNA barcoding authenticates the actual plant material used while HPLC checks the
presence of active compounds.
Keywords: DNA barcoding, HMPs, HPLC, sambong, tsaang-gubat
INTRODUCTION
The Philippine Department of Health (DOH) has
recognized ten medicinal plants as effective and alternative
sources of medicine (Tupas and Gido 2021). Among these
herbal plants are Blumea balsamifera (L.) DC. (sambong)
and Ehretia microphylla Lam. (tsaang-gubat), which have
gained popularity due to their accessibility, availability,
and commonality. Traditionally, both plants are used as
decoctions in several regions of the country. They are also
commonly available in the market as tea, capsule, and
tablet. With their known curable properties, the Philippine
Institute of Traditional and Alternative Health Care
(PITAHC) has reported the processing of 10-15 million
tablets derived from sambong and tsaang-gubat yearly (de
Guzman 2013). The former or sambong is recommended
by the Philippine Kidney Transplant Institute (PKTI) as
diuretic treatment for kidney stones (Montealegre and De
Leon 2016; Rasonabe et al. 2023). Traditional local healers
also use sambong to treat different conditions such as
cough, fever, influenza, dysentery, sore eyes/throat, malaria,
boils, infected umbilical cords, and tuberculosis (Maramba-
Lazarte et al. 2020). Tsaang-gubat is traditionally utilized
to treat diarrhea, spasms, inflammation, gastrointestinal and
biliary colic (Mageswari and Karpagam 2015). Moreover,
its decoction tea is found to be effective in improving the
symptoms of allergic rhinitis and may be used as an
alternative to loratadine (Umali and Chua 2017). Legaspi
and Bagaoisan (2020) unveil the phytochemical constituents
of tsaang-gubat which include flavonoids, phenolics,
triterpenes, and alkaloids. Despite the increasing popularity
of these derived herbal medicinal products (HMPs) in the
Philippines, there remains a lack of strict regulation of the
production of HMPs in the country (Penecilla and Magno
2011). This poses a great threat to consumers as they are
not safeguarded from the ill effects of adulteration,
contamination, and substitution.
The authenticity of the source material ensures the
quality and effectiveness of the herbal products. HMPs
contain a mixture of active ingredients from plant parts or
plant materials (Tarmizi et al. 2021). Despite the World
Health Organization (WHO) emphasizing the importance
of using authentic raw plant material, studies have shown
that a great portion of products available in the market
LOLA et al. – Authentication of sambong and tsaang-gubat
1259
contain substitutes and unlabeled fillers (Buddhachat et al.
2015; Olivar et al. 2016; Pedales et al. 2016; Ichim et al.
2019). Industrial-scale protocols are available to test the
authenticity of these products. High-Performance Liquid
Chromatography (HPLC) has been the golden standard
used by the Food and Drug Administration (FDA) of the
United Kingdom and the United States to monitor the
quality of products. It utilizes well-characterized marker
compounds to set specifications for raw materials, standardize
botanical preparations during all aspects of manufacturing
processes, and obtain stability profiles (Upton et al. 2020).
Results from HPLC analysis provide “chromatographic
fingerprints” that allow the manufacturing of consistent
herbal products. However, HPLC only authenticates at the
level of the active compound. Mishra et al. (2016) have
provided instances indicating the ease of product adulteration
through replicated marker compounds and through external
factors that affect the specific compound. This suggests the
need for species level authentication of HMPs.
Hebert et al. (2003) have proposed a species authentication
system that utilizes a standard region of the genome. This
system, termed DNA barcoding, is a reliable tool since
genetic information is not affected by external factors
(Mishra et al. 2016). However, DNA Barcoding has been
less successful in the authentication of plant material due to
two factors: (i) limited agreement of gene regions used for
barcoding, and (ii) low DNA yield from powdered plant
material (Hollingsworth et al. 2011). Nevertheless, several
studies (Newmaster et al. 2013; Buddhachat et al. 2015;
Vassou et al. 2015; Olivar et al. 2016; Pedales et al. 2016;
Alfeche et al. 2019) have shown its use in the identification
of plant material from HMPs. Results of these studies
consistently show that there is a rampant adulteration in
herbal products, warranting the need to include DNA
Barcoding in industrial-scale authentication protocols. This
study, therefore, aimed to utilize DNA barcoding and HPLC
to authenticate B. balsamifera and E. microphylla derived
HMPs sold in Philippine markets. Both DNA barcoding
and HPLC are utilized in this study to test incongruences
regarding the authenticity of the sampled products. The
results of the study promote strict adherence to standard
authentication protocols that safeguard consumer health.
MATERIALS AND METHODS
Wild sample collection of Blumea balsamifera and Ehretia
microphylla
Five B. balsamifera and seven E. microphylla samples
were collected from various localities in the Philippines
(Table 1). These collections served as the wild samples that
were used in the construction of the SRM. Figure 1
illustrates the collected B. balsamifera and E. microphylla
from the wild. Most of these were obtained from private
properties and the Bureau of Plant and Industry, Manila.
Multiple specimens of each species were considered to
represent the extent of genetic variation. Leaf samples of
the individual plants were placed in zip-lock bags with
silica-gel beads for DNA Barcoding (Semagn 2014).
Herbarium specimens were prepared for accessioning at the
University of Santo Tomas Herbarium (USTH) and for
authentication at the Philippine National Herbarium (PNH
Control No. 16-07-870). Table 1 illustrated the collection
information. A kilogram of leaf samples each of CB17-070
(B. balsamifera) and CB17-067 (E. microphylla) were
collected and air-dried for HPLC analysis.
Figure 1. Wild samples of the collected. A. B. balsamifera (Photo
taken by NKG Alfeche); B. E. microphylla (Photo taken by SDA
Binag)
Table 1. Collection information of B. balsamifera and E. microphylla samples
Species
name
Sample code
Collection locality
Usth accession number
Genbank accession numbers
ITS
trnH-psbA
Blumea
balsamifera
16-605
Davao del Sur
USTH013666
OP602345
OP584405
16-606
Polillo Island, Quezon
USTH013667
OP602346
OP584406
16-607
Bureau of Plant
Industry, Malate, Manila
USTH013668
OP602347
OP584407
CB17-070
Davao Oriental
USTH014281
OP602348
OP584408
SB17-002
Tumauini, Isabela
USTH014182
OP602349
OP584409
Ehretia
microphylla
16-600
Orani, Bataan
USTH013525
OP604158
OP584399
16-601
Bureau of Plant
Industry, Malate, Manila
USTH013541
OP604159
OP584400
16-602
Valenzuela City
USTH013523
OP604160
OP584401
16-603
Pantok, Rizal
USTH013663
OP604161
OP584402
CB17-067
Davao Oriental
USTH014279
OP604162
OP584404
UG17-900
Aklan
USTH014280
OP604163
OP584403
17-019
Alcoy, Cebu
USTH014144
OP604164
Not sequenced
A
A
B
B
B I O D I V E R S I T A S
26 (3): 1258-1270, March 2025
1260
Herbal product sampling
Eight and six blindly selected B. balsamifera and E.
microphylla derived HMPs, respectively, were selected
from different markets in the Philippines. The identities of
these products were concealed to show anonymity, and
were labeled randomly as BB1, BB2, BB3, BB4, BMP1,
BMP2, BMP3, BMP4, EM1, EM2, EM3, EM4, EMP1, and
EMP2.
DNA extraction, amplification, purification, and
sequencing of wild samples and HMPs of B. balsamifera
and E. microphylla
The total genomic DNA from silica-gel dried leaf
samples of the wild samples and from powdered HMPs was
extracted using DNeasy Plant Mini kit (Qiagen, Hilden,
Germany), following the manufacturer’s protocol. The ITS,
matK, rbcL, and trnH-psbA barcodes were generated to
test their efficiencies in discriminating B. balsamifera and
E. microphylla from their closest relatives. Amplification
was accomplished in 25 µL PCR aliquots of 15.3 µL dH2O,
2.5 µL 10X PCR buffer, 2.0 µL 25 mM MgCl2, 1.5 µL 2
mM dNTPs, 1.0 µL of 10 mM forward and reverse primers,
respectively, 0.2 µL KAPA Taq DNA polymerase, and 1.0
µL DNA. Reactions were performed in Biometra TGradient
Thermocycler. Primer information and amplification protocols
were shown in Table 2. PCR amplicons were checked on
1% TBE agarose gel and were purified using Qia-Quick
PCR purification kit (Qiagen, Hilden, Germany), following
the manufacturer’s protocol. Purified products were sent to
MACROGEN South Korea for bidirectional sequencing.
Sequences were then assembled and edited using Codon
Code Aligner v5.1.5.
Establishment of a Standard Reference Material (SRM)
of B. balsamifera and E. microphylla wild samples
A multi-locus/tiered dataset was prepared using 78 gene
accessions of 13 closely related Blumea species, including
accessions for Ligularia sp. as the outgroup, and 26 gene
accessions of closely related Ehretia species, including
accession for Bourreria sp. as the outgroup. GenBank
sequences of species closely related to B. balsamifera and
E. microphylla and the collected wild samples for each
species were used in the establishment of an SRM. GenBank
accession numbers of the newly generated sequences of the
wild B. balsamifera and E. microphylla are shown in Table
1. Variable and parsimony- informative sites and resolutions
of species were computed to provide data regarding the
strengths of the utilized barcoding loci. Inter- and intraspecific
Kimura-2-Parameter (K2P) distances were computed using
MEGA 7.0 to measure the extent of genetic variation
(Kumar et al. 2016). The SRMs were established by
constructing Maximum Likelihood (ML) trees using the
K2P distances in MEGA 7.0.
Authentication of B. balsamifera and E. microphylla
derived HMPs using DNA barcoding
Sufficient DNA quality and quantity were extracted
from the HMPs following the standard DNA extraction
protocols (Table 3). Amplification of the ITS, trnH-psbA,
matK, and rbcL loci was then conducted. Authentication
through DNA Barcoding was followed using BLASTn and
ML tree reconstruction criteria. The generated sequences
were queried using the BLASTn algorithm (https://
blast.ncbi.nlm.nih.gov). An herbal product was considered
to pass the BLASTn criterion if the BLAST search resulted
in B. balsamifera or E. microphylla as its top hit with an e-
value cut-off <0.01. The generated sequences were also
incorporated within the established SRM tree. An herbal
product was considered to pass the ML tree criterion if the
sequences nested within a strongly supported (BS>70%)
monophyletic clade with the authentic B. balsamifera and
E. microphylla samples. If both criteria were met, an herbal
product was considered authentic in terms of the presence
of the actual plant material.
Table 3. Spectrophotometric results* of the B. balsamifera and E.
microphylla derived HMPs showing its DNA concentration and
purity
Product code
Concentration (μg/μL)
A260/A280
BB1
0.045
2.004
BB2
0.099
2.011
BB3
0.017
1.983
BB4
0.024
2.019
BMP1
0.021
2.016
BMP2
0.018
1.856
BMP3
0.016
1.878
BMP4
0.019
1.589
EM1
0.011
1.426
EM2
0.049
1.906
EM3
0.015
1.747
EM4
0.060
1.692
EMP1
0.021
1.848
EMP2
0.019
1.730
Note: *Average of three trials per sample
Table 2. PCR primers and amplification protocols
Gene region
Primer name
Primer sequence (5’ → 3’)
References
trnH-psbA
trnH
CGCGCATGGTGGATTCACAATCC
Lv et al. 2020
psbA
GTTATGCATGAACGTAATGCTC
ITS
5
GGAAGTAAAAGTCGTAACAAGG
Alfeche et al. 2019
4
TCCTCCGCTTATTGATATGC
matK
1RKIM-f
ACCCAGTCCATCTGGAAATCTTGGTTC
Kuzmina et al. 2012
3FKIM-r
CGTACAGTACTTTTGTGTTTACGAG
rbcL
1F
ATGTCACCACAAACAGAAAC
Fay et al. 1997
724R
TCGCATGATCCTGCAGTAGC
LOLA et al. – Authentication of sambong and tsaang-gubat
1261
Authentication of B. balsamifera and E. microphylla
derived HMPs using HPLC analysis
Standard HPLC profiles were created following
protocols of Nessa et al. (2005) and Toralba et al. (2015)
for B. balsamifera, and Makino et al. (2003) and Wang et
al. (2003) for E. microphylla. The chromatographic profiles
of the targets, quercetin (QA) and rosmarinic acid (RA),
were obtained using the Shimadzu LC-20AT, a Photodiode
Array (PDA) detector, CBM-20A, and a C-18 Shimdazu
column. These profiles were created to specifically detect
the presence of QN for B. balsamifera and RA for E.
microphylla. Approximately 200 mg of powdered samples
from the wild samples and herbal products were used. B.
balsamifera derived samples were extracted for 6 hours
with 20 mL methanol in glass-stopped vessels on IKA®C-
MAG HS7 hot plate with magnetic stirring at 40°C
followed by filtration using Whatman® Filter Paper grade
602 h ½ and further extraction with 10 mL methanol for
three times. The extract was evaporated to dryness in an
Eyela®N-1200B rotary evaporator at 40°C. For E. microphylla
collected wild and HMPs, these were dissolved in 100 mL
ethanol/water (30:70, v/v) with sonication using Daikan®
Labtech Power Ltd. Sonic410 for 40 minutes followed by
filtration and further extraction using 80 mL ethanol-water
with sonication for 20 minutes.
All extracts from wild and HMP samples of sambong
and tsaang-gubat were evaporated to dryness in an Eyela®
N-1200B rotary evaporator at 40°C. Five mg of the solid
residue was then weighed and mixed with 5 mL methanol
for the wild sambong and derived HMPs, and 5 mL
ethanol/water (30:70, v/v) for wild tsaang-gubat and derived
HMPs to produce a 1000 ppm mixture. The mixtures were
then filtered using an Agilent® 0.45 μm nylon syringe
filter and 20 μL of the resulting mixtures were injected into
the HPLC for analysis. Three trials for each sample were
done for the consistency of data.
For QN, the mobile phase consisted of methanol-0.5%
phosphoric acid in water (50:50, v/v). The flow rate was
held constant at 0.9 mL/min. For RA, solvents used for
separation were 0.1% orthophosphoric acid in water (v/v)
(eluent A) and 0.1% orthophosphoric acid in methanol
(v/v) (eluent B). The gradient used was: 0-10 min, linear
gradient from 40% to 50% B; 10-15 min, linear gradient
from 50% to 60% B, maintained at 60% B until 25 min. The
flow rate was 1.0 mL/min and the detection wavelength at
330 nm.
The chromatographic peaks of QN in both wild B.
balsamifera and RA in wild E. microphylla, together with
their respective HMPs, were confirmed by comparing their
retention times (tR) with that of their respective reference
standards. The herbal product was deemed authentic if it
showed similar HPLC profiles with the standards for QN
and RA. The standard chromatographic profile of QN
shows a distinct peak at a retention time of 20-30 minutes
(Toralba et al. 2015; Jirakitticharoen et al. 2022) while RA
exhibits a distinct peak at a retention time of 16-18
minutes, following the protocols of Makino et al. (2003).
To further validate chromatographic peaks, standards of
QN or RA were used to spike wild samples and herbal
products of B. balsamifera or E. microphylla, respectively.
From the 100 ppm standards of QN and RA, 50 μL was
taken. This was thoroughly mixed with 50 μL of the 1000
ppm samples of either wild samples or HMPs. A 20 μL of
the mixture was injected into the HPLC instrument. The
chromatographic profiles were then assessed to confirm the
presence of the active compounds in the tested HMPs.
RESULTS AND DISCUSSION
Establishment of the Standard Reference Material
(SRM) herbal barcode library of B. balsamifera and E.
microphylla wild samples
An ideal barcode must be easily amplified, possess high
variable sites, high parsimony-informative sites, high
interspecific K2P distances, low intraspecific K2P distances,
and high resolution of species. Table 4 shows the values for
the amplification and sequencing success of the four loci
which are used to describe the versatility of the primer
amplified over a wide range of species. All the primers
were successfully amplified (100%) in both B. balsamifera
and E. microphylla, with ITS having the longest aligned
length of 553 bp for B. balsamifera and matK with the
longest aligned length of 719 bp for E. microphylla. ITS
provided the best values for variable sites in both B.
balsamifera (29.66%) and E. microphylla (37.76%). It also
gave the most parsimony-informative characters, 27.49%
for B. balsamifera and 31.82% for E. microphylla. The
trnH-psbA marker had the second-highest parsimony-
informative values with 7.89% and 7.08% for both species,
respectively. Both matK and rbcL had the lowest variable
and parsimony-informative sites for both species. This
suggests that ITS and trnH-psbA can be readily amplified
for both species and both loci provide universality while
matK and rbcL, though successfully sequenced, are not
enough to provide species discrimination.
The pairwise sequence divergences indicate the primer’s
discriminatory power. Wilcoxon signed-rank and two-sample
tests performed in SPSS (IBM) confirmed the significant
differences between inter- and intraspecific Kimura-2-
parameter (K2P) distances of the investigated primers. The
K2P interspecific distances depict how close different species
are to each other, while the K2P intraspecific distances
show the closeness of individuals within the same species.
For the barcode to be ideal, it should have a significantly
higher interspecific divergence to distinguish one species
from another, and not the individuals of the same species
(Galimberti et al. 2019). As illustrated in Table 5, each of
the candidate barcodes has shown a higher interspecific
distance compared to its intraspecific distance. Among the
barcodes tested, the mean interspecific divergences were
the highest in ITS (0.1048 and 0.2397).
B I O D I V E R S I T A S
26 (3): 1258-1270, March 2025
1262
Table 4. Properties of the four candidate DNA barcoding loci for A. B. balsamifera and B. E. microphylla
Properties
ITS
matK
rbcL
trnH-psbA
B. balsamifera
Number of sequences
33
6
6
33
Aligned length (bp)
553
251
560
393
Variable sites (%)
29.66
1.99
2.68
9.67
Parsimony-informative characters (%)
27.49
1.99
0
7.89
Mean interspecific K2P distance
0.1048 ± 0.0062
0.0193 ± 0.0018
0.0083 ± 0.0007
0.0838 ± 0.0111
Mean intraspecific K2P distance
0.0026 ± 0.0011
0.0026 ± 0.0018
0
0
Resolution of species (%)
100.00
66.67
100.00
84.62
E. microphylla
Number of sequences
7
6
7
6
Aligned length (bp)
286
719
443
339
Variable sites (%)
37.76
4.03
1.13
8.55
Parsimony-informative characters (%)
31.82
4.03
1.13
7.08
Mean interspecific K2P distance
0.2397 ± 0.0109
0.0418 ± 0
0.0083 ± 0.0007
0.0838 ± 0.0111
Mean intraspecific K2P distance
0.0035 ± 0.0028
0
0
0
Resolution of species (%)
100.00
100.00
100.00
100.00
Note: aEntries shown in bold are considered the best performing for a particular parameter
Table 5. Wilcoxon two-sample test for inter- and intra-specific divergences of four barcodes
Barcode
Number of
inter specific
Mean inter
specific
Number of
intra specific
Mean intra
specific
Wilcoxon W
P value
B. balsamifera
ITS
669
0.1048
72
0.0026
2712.50
0.000
matK
21
0.0192
45
0.0000
1057.50
0.000
rbcL
7
0.0273
21
0.0000
231.00
0.000
trnH-psbA
673
0.0235
71
0.0000
2905.00
0.000
E. microphylla
ITS
46
0.2397
32
0.0028
528.00
0.000
matK
14
0.0418
22
0.0000
253.00
0.000
rbcL
47
0.0083
31
0.0000
511.50
0.000
trnH-psbA
46
0.0838
32
0.0000
528.00
0.000
For Blumea, ITS had the highest interspecific divergence,
followed by rbcL, psbA-trnH, and matK (p<0.05) (Table
6), while for Ehretia, ITS had the highest interspecific
divergence, followed by trnH-psbA, matK, and rbcL
(p<0.05). For the intraspecific divergence of Blumea, the
lowest was for trnH-psbA, matK, and rbcL (0), followed
by ITS (0.0026) (Table 6), while for Ehretia, all markers
obtained a value of zero. Thus, ITS and trnH-psbA provided
the best discriminatory power over the other candidate
barcodes.
The resolution of the species was tested using BLASTn
and the tree construction criterion which established the
Standard Reference Material (SRM) herbal barcode library
for B. balsamifera and E. microphylla. For a barcode to be
efficient, it should be able to identify samples down to the
species level. Single locus analysis following BLASTn
criteria was able to resolve the species identity to a certain
degree. Both ITS and trnH-psbA were able to identify all
wild Blumea samples (100%) while ITS, matK, and trnH-
psbA were able to resolve all the wild Ehretia samples
(100%) down to genus level only. Tree construction criterion
utilizing single locus also resolved the species to a certain
degree. ITS and matK were able to resolve the individuals
of B. balsamifera to a monophyletic clade with BS=100%,
while trnH-psbA BS=99%, and rbcL BS=92%. ITS and
trnH-psbA resolved the individuals of E. microphylla to a
monophyletic clade with BS=100%, while matK and rbcL
had BS=99%. The multi-locus/tiered approach utilizing the
four loci successfully provided distinctness of B. balsamifera
and E. microphylla clades with both forming strongly
supported (BS=100%) monophyletic groups (Figure 2).
Evident in both SRMs is the distinctness of B. balsamifera
and E. microphylla from their close allies as conspecific
samples form strongly supported (BS=100%) monophyletic
groups (highlighted in yellow). The concatenated dataset
also supported stronger bootstrap values. This indicates that
the established SRM can be used to associate unknown
samples to B. balsamifera and E. microphylla when DNA
barcoding protocols are followed.
LOLA et al. – Authentication of sambong and tsaang-gubat
1263
Table 6. BLASTn results for the authentication of plant derived herbal products
Product
code
ITS
trnH-psbA
BLASTn ID
e-value
% identity
BLASTn ID
e-value
% identity
BB1
Blumea balsamifera L.
0.0
99
Blumea balsamifera L.
0.0
98
BB2
Blumea balsamifera L.
0.0
99
Blumea balsamifera L.
0.0
99
BB3
Blumea balsamifera L.
0.0
99
Blumea balsamifera L.
0.0
99
BB4
Oryza glaberrima Steud.
1e-131
97
Oryza glaberrima Steud.
1e-131
97
BMP1
Blumea balsamifera L.
0.0
99
Blumea balsamifera L.
0.0
99
BMP2
Blumea balsamifera L.
0.0
99
Blumea balsamifera L.
0.0
99
BMP3
Blumea balsamifera L.
0.0
99
Blumea balsamifera L.
0.0
96
BMP4
Blumea balsamifera L.
0.0
97
Blumea balsamifera L.
0.0
99
EM1
Ehretia microphylla Lam.
0.0
95
Ehretia laevis Roxb.
1e-135
93
EM2
Ehretia microphylla Lam.
0.0
97
Ehretia laevis Roxb.
2e-153
96
EM3
Ehretia microphylla Lam.
0.0
97
Ehretia laevis Roxb.
3e-136
94
EM4
Ehretia microphylla Lam.
0.0
97
Ehretia laevis Roxb.
3e-143
95
EMP1
Cymbopogon citratus Stapf.
0.0
98
Cymbopogon citratus Stapf.
0.0
99
EMP2
Ehretia microphylla Lam.
0.0
99
Ehretia laevis Roxb.
3e-140
94
Note: aEntries shown in yellow are considered substitutions observed for the selected medicinal products
Figure 2. Standard reference material (SRM) herbal barcode library consensus tree (ML analysis) for combined ITS, matK, rbcL, and
trnH-psbA dataset of A. 94 gene accessions of Blumea species and B. 38 gene accessions of Ehretia species. Highlighted are the A. B.
balsamifera and the B. E. microphylla clades. Numbers on nodes are bootstrap (BS) support values
Authentication of B. balsamifera and E. microphylla
derived HMPs following DNA barcoding
Authentication of 14 blindly selected B. balsamifera
and E. microphylla derived HMPs followed the BLASTn
and Maximum-Likelihood (ML) Tree reconstruction criteria.
In terms of primer universality, both sambong and tsaang-
gubat derived HMPs provided 28 good-quality sequences
for both ITS and trnH-psbA. However, problems were
experienced in the PCR amplification of matK and rbcL for
the HMPs. This does not necessarily point to matK and
rbcL as unsuitable loci, but unsuccessful amplifications
may have been due to primer mismatch, degradation of the
DNA material, or due to inhibition of polymerase. As
Newmaster et al. (2013) stated, difficulties are often
encountered based on the primers used. Table 6 reveals the
BLASTn results, where seven out of eight sambong HMPs
were identified to be B. balsamifera. BB4 was substituted
with Oryza glaberrima. Substitution occurs when barcodes
of other species, other than the target species, are sequenced
in the analysis, following the definition of Newmaster et al.
(2013). For tsaang-gubat HMPs, five out of six derived
products (EM1, EM2, EM3, EM4, and EMP2) were
identified to be authentic, containing E. microphylla using
ITS gene region with an average e-value of 0.0, and E.
laevis identification using trnH-psbA with an average e-
value of 2.6e-136. The discrepancy in the BLASTn
identification could be attributed to the amounts of published
sequences in the NCBI database. Tsaang-gubat HMP
A
A
B
B
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26 (3): 1258-1270, March 2025
1264
EMP1 was also identified as a form of substitution, with
the plant component identified as Cymbopogon citratus.
All 16 and 12 newly generated sequences from sambong
and tsaang-gubat -derived HMPs, respectively, were
incorporated into the established SRM herbal barcode
libraries presented in Figure 3. Authentic sambong and
tsaang-gubat-derived products should nest within their
respective monophyletic clades. All codes in Figures 2.A
and 2.B, in red, are the products tested in this study. As
seen in the Figure 2A, product codes BB1, BB2, BB3,
BMP1, BMP2, BMP3, and BMP4 were well nested within
the B. balsamifera clade (BS=89%). Product code BB4 was
placed outside the outgroup Ligularia sp. indicating its
exclusion from family Asteraceae. While in figure 2B,
product codes EM1, EM2, EM3, EM4, and EMP2 reside in
the E. microphylla clade (BS=100%). Same with the
product code BB4, product code EMP1 was placed outside
the outgroup Bourreria sp., which indicates its exclusion
from the family Boraginaceae.
Following both BLASTn and SRM Herbal Library
criteria, it was concluded that out of the eight sambong and
six tsaang-gubat-derived HMPs, one product code of each
target HMP did not correspond to B. balsamifera and E.
microphylla, respectively. The identification of different
source plant materials in the two HMPs is evidence of
product substitution. BB4 was identified to contain Oryza
glaberrima as evidence of product fillers. African rice (O.
glaberrima) has been classified as product filler in several
commercial teas and capsules (Newmaster et al. 2013). On
the other hand, the identification of C. citratus is an example
of substitution wherein herbal plants may have been
replaced with another raw material with similar benefits but
cheaper production costs. Even though C. citratus has its
own ethno-pharmaceutical properties, the use of this plant
in the guise of another species is still a case of product
fraud which could pose health risks to unassuming consumers.
The results of the study show the success and effectiveness
of DNA Barcoding as a standard protocol for authenticating
medicinal products.
Several studies showed that DNA barcoding is useful
for HMPs’ authentication. Olivar et al. (2016) revealed
cases of adulterations in Vitex negundo (lagundi) HMPs by
using BLAST and ML tree criterion of combined ITS-trnH-
psbA datasets. BLAST unveiled only one of five samples
was authentic while other samples contained a fungus and
an uncultured eukaryote. Similarly, the study by Pedales et
al. (2016) indicated that the BLAST results showed 4 of 19
ITS2-barcoded HMPs contained possible substitution or
contamination. They further demonstrated that ITS2 can
effectively discriminate among species but recommended
an additional barcode to strengthen the resolution. Meanwhile,
the work of Michel et al. (2016) proved that ITS is an
effective marker in terms of species discrimination for the
HMPs sold in New York City. ITS is regarded as a core
DNA barcode in the identification of medicinal plants
primarily due to its length, easy expansion, and high success
rate (Yang et al. 2018). More importantly, the authentication
in Apocynaceae HMPs proven the effectiveness of using
both ITS and trnH-psbA (Lv et al. 2020). The strength of
ITS and trnH-psbA as a marker for sambong and tsaang-
gubat derived products’ authentication was evidently seen as
well in this study. Frigerio et al. (2021) also recommended
the use of ITS and trnH-psbA for DNA-based herbal teas’
authentication. Moreover, Alfeche et al. 2019 used SRM
DNA barcode library for Antidesma bunius (bignay), where
only three of 11 HMPs were authentic using combined
dataset. Despite the strengths of DNA barcoding, it cannot
determine the active compound available in the HMPs.
Additionally, multiple species may have such compounds,
and the actual plant material used may be adulterated.
Figure 3. Maximum-Likelihood tree reconstruction for combined (ITS-trnH-psbA) barcode data of A. Blumea and B. Ehretia derived
HMPs and wild samples
A
A
A
B
B
B
LOLA et al. – Authentication of sambong and tsaang-gubat
1265
Establishment of the standard chromatographic profiles
for B. balsamifera and E. microphylla
Currently, HPLC is the golden standard used for
authenticating herbal medicines due to its efficiency in
quality control. This method determines the active compound
present in the HMPs. An active compound/bioactive
compound is important since it is responsible for the
product’s effectiveness in treating or alleviating the effects
of a certain illness/disease. Moreover, the long-term use of
herbal products relies on maintaining the constant levels of
such compounds (Zarsuelo et al. 2018). Qualitative solute
identification via comparison of retention data of the active
compound is done to screen the HMPs (Liang et al. 2004).
Further, they stated that one or two pharmacologically
active compounds can be used for screening the quality and
authenticity of HMPs using HPLC.
B. balsamifera contains QN and E. microphylla includes
RA. QN was determined as an active flavonoid in the
leaves of B. balsamifera (Jirakitticharoen et al. 2022). It
offers a wide range of functions such as anti-inflammatory
and antioxidant properties. QN was also proven as an
important active compound involved in the diuretic properties
of sambong in treating patients with kidney stones (Rasonabe
et al. 2023). E. microphylla leaves have RA which is
attributed to its efficacy in controlling allergies (Shukla and
Kaur 2018). Some of the HMPs’ manufacturers also
indicate the active compounds present in their products, as
placed at the back of their boxes or labels just like QN and
RA, respectively. Therefore, chromatograms for QN and
RA showed great importance in authenticating B. balsamifera
and E. microphylla, respectively. The retention time for
QN in B. balsamifera was shown to be 20-30 minutes in
the studies of Toralba et al. (2015) and Jirakitticharoen et
al. (2022). Meanwhile, retention time for RA varies in
different plant samples such as 52-54 min in Ehretia tinifolia
(Monroy-Garcia et al. 2021) and 12.5-14.5 in selected Thai
plants (Chaowuttikul 2020). The established chromatographic
profiles were then used to authenticate HMPs (Figures 4.A-
D). In this study, the peak for RA was consistently seen at a
retention time of 16-18 minutes. Upon comparing the
chromatographic profiles of the active compounds to the
generated HPLC profiles of wild B. balsamifera and E.
microphylla, similar distinct peaks can be found. This
indicates that QN and RA are indeed present in B. balsamifera
and E. microphylla extracts, respectively.
Authentication of B. balsamifera and E. microphylla
derived HMPs following HPLC analysis
The HPLC chromatograms of the eight B. balsamifera
HMPs are shown in Figure 5A-5H, and a comparison with
the tR of the standard QN (25-27 min) indicates the presence
of QN in five of the eight HMPs not including BB2, BB4,
and BMP3. The absence of QN in BB4 corroborated with
the results of the DNA Barcoding, indicating that BB4 does
not contain B. balsamifera. DNA Barcoding has identified
BB2 and BMP3 as B. balsamifera; however, QN was not
detected based on the HPLC. This bioactive compound may
have been destroyed or deactivated after the manufacturing
process. With this said, HPLC must be used since it
determines the presence of the active compound and must
be corroborated with another authentication technique.
Comparison of the tR of the standard RA (16-18 min)
with those of the E. microphylla HMPs (Figures 6.A-F)
demonstrate the presence of RAs except in EMP1. This
corroborates with the results of the DNA Barcoding,
indicating EMP1 is Cymbopogon citratus, and not E.
microphylla.
Figure 4. Chromatogram of A. Standard QN; B. Wild B. balsamifera
(CB17-070); C. Standard RA; D. Wild E. microphylla (CB17-
067)
A
A
B
B
C
C
D
D
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26 (3): 1258-1270, March 2025
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Figure 5. HPLC chromatogram of the eight B. balsamifera derived HMPs: A. BB1; B. BB2; C. BB3; D. BB4; E. BMP1; F. BMP2; G.
BMP3; H. BMP4
Several reports displayed the importance of HPLC in
the authentication of HMPs. Orman et al. (2022) showed
the use of HPLC in assessing the quality of South African
HMPs. The study showed that using flavonoids for rapid
scanning could be performed when the active compound is
unknown. Moreover, Xu et al. (2021) utilized HPLC for
seven bioactive components of the “Arnebiae Radix”
HMPs. The results of the study indicated that HPLC allows
the simultaneous discrimination of the seven main
naphthoquinones. Meanwhile, the study of Custers et al.
(2017) emphasized that chromatographic fingerprinting is
capable of detecting target plants in illegal herbal products
even in complex samples.
However, there are limitations to HPLC due to the lack
of long-term reproducibility and false results caused by
deliberate adulteration of the illegal addition of a marker
compound to a medicinal product (Hassan 2012). In this
study, results showed the success of HPLC in authenticating
the presence of a single active compound. However, it must
be noted that alternative medicine is effective due to the
presence of the actual plant extract and its active
compound/s. Therefore, another method should accompany
HPLC to test the authenticity of the plant source material.
DNA barcoding and HPLC as golden standards in the
authentication of herbal medicinal products
Results obtained from DNA Barcoding showed
incongruencies with the results obtained from the
chromatographic profiles of HPLC. Therefore, the combination
of DNA barcoding and HPLC is necessary. DNA Barcoding
A
A
B
B
C
C
D
D
E
E
F
F
G
G
H
H
LOLA et al. – Authentication of sambong and tsaang-gubat
1267
is only limited to genotypic identification whereas HPLC is
only limited to the determination and isolation of chemical
constituents.
Several studies revealed that DNA Barcoding and
HPLC must be used together to authenticate HMPs. Xu et
al. (2021) utilized both DNA barcoding and HPLC in the
authentication of traditional Chinese medicine. They showed
that ITS was also a good marker for checking the authenticity
of the HMPs. Moreover, their study demonstrated that
HPLC chromatograms could not differentiate the available
species in the HMPs but only the presence of the active
compounds. Furthermore, HPLC results unveiled that even
though the actual plant material was present, the expected
active compound in some of the samples was not detected
(Abubakar et al. 2018). Similar results were identified in
the present study wherein QN and RA were not detected in
some of the HMPs. Meanwhile, HPLC determined the
effective chemical constituents. In their study, Ghorbani et
al. (2020) conducted the authentication of Ruscus hyrcanus
using trnH-psbA barcoding and HPLC-PDA analysis.
Their study illustrated the effectiveness of trnH-psbA,
similar to its use in the barcoding of B. balsamifera and E.
microphylla. Moreover, they used two saponin compounds
for HPLC. Anantha Narayana and Johnson (2019) stated
that DNA Barcoding can detect raw materials from HMPs,
but it cannot check chemical constituents or plant parts
used. To identify the actual material and ingredients of the
HMPs, it must be complemented with other techniques.
However, HPLC cannot give a complete picture of the true
plant present from the herbal products. If the safety of
HMPs relies on specific bioactive compounds, absence of
toxins, allergens, and admixed pharmaceuticals, then HPLC
is more relevant than DNA-based analysis. To ascertain
species substitution or adulteration, DNA Barcoding provides
better resolution than HPLC (Techen et al. 2014; Palhares
et al. 2015). Additionally, the review paper of Abubakar et
al. (2017) suggested the combination of DNA barcoding
and chromatographic fingerprinting since DNA barcoding
can identify the herbal plants used, and HPLC can check
the quality of the products. Another study by Zhang et al.
(2016) stated that DNA barcoding and HPLC can
discriminate between species in Chinese HMPs; however,
DNA barcoding is more useful in terms of confirming the
identities of medicinal materials from various sources.
With the amount of available data that support the use of
these competing techniques, DNA barcoding coupled with
HPLC is hereby highly recommended for the authentication
of Philippine herbal products.
Figure 6. HPLC chromatogram of the six E. microphylla derived HMPs: A. EM1; B. EM2; C. EM3; D. EM4; E. EMP1; F. EMP2
A
A
B
B
C
C
D
D
E
E
F
H
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Possible reasons, such as clear mistake in vernacular
names between indigenous systems of medicine and local
dialects, similarity in morphological features of other species
of plants, and absence of authentic raw plant material can
be attributed to product adulteration (Srirama et al. 2017).
It is also possible that product substitution will occur given
that there is no regulatory tool in place or commercial test
for product authentication. Bearing in mind the consequences
of adulteration in the drug trade, labors have been prepared
to precisely identify and classify medicinal plants in
commercially available herbal products (Kool et al. 2012).
Besides conventional approaches, such as morpho-taxonomic
keys, wood anatomy, histological, and histochemical
techniques, DNA-based techniques have just been certified
as simple, and reliable tools to authenticate the medicinal
plant material used in natural health products (Seethapathy
et al. 2014). The practice of substituting the original active
crude drug in part, or in whole with other ingredients, is
known as adulteration. Adulteration can be free form or
inferior in therapeutic and chemical properties to that of the
original drug replaced with the purpose of increasing
revenues. Owing to having inexpensive prices of herbal
products and their accessibility, this became the topic of
adulteration (Seethapathy et al. 2014). The main topic in
standardizing these herbal products is the authentication of
raw botanical materials. It is disparagingly vital to
authenticate a plant product and detect any adulteration
throughout the manufacturing process for adulteration can
result in an unsuccessful and ineffective product, or worse,
can even harm the consumer. To properly identify plant
source material of these HMPs, modern techniques such as
DNA barcoding and HPLC can be utilized effectively in
identification of medicinally important plant species. Thus,
this study can serve as a benchmark for other HMP’s
evaluation for possible product adulteration.
The Philippines, being a third-world country, has
economic systems that require advancement. This concern
trickles down even in the process of acquiring quality
healthcare products. This encourages the masses to lean
towards cost-effective healthcare, such as cheap traditional
and alternative medicines. The increase in awareness of the
use of HMPs happened during the campaign done by the
country’s Department of Health (DOH) through “Traditional
Health Program” that encourages the use of 10 medicinal
plants which includes sambong and tsaang-gubat (Catublas
2016). Although the use of modern medicine may happen
at the same time, HMPs have maintained their popularity
for cultural and historical reasons (Tolentino et al. 2019).
In fact, the Philippines has one of the largest numbers of
registered herbal drugs (>2,000) worldwide (Sahoo et al.
2010). Moreover, the Department of Trade and Industry
(DTI) in the country revealed that the export market value
of natural health products, mostly HMPs, is an estimated
153 million USD as of 2011 (Zarsuelo et al. 2018). However,
substitutions, contaminants, and adulterations of alternative
medicines were determined. The studies of Alfeche et al.
(2019), Olivar et al. (2016), and Pedales et al. (2016)
proved that there is a need for a standard protocol in quality
control of medicinal products as they unmasked the
rampant adulteration in the Philippine herbal market. The
Philippine Food and Drug Administration (FDA) 2018 also
stated that there is an increasing number of violations
regarding the manufacturing, sales, and distributions of
HMPs. With these said, the success of this study reiterates
that a standard protocol for herbal product authentication
must be applied. Should this be implemented, quality control
protocols must take note of the most efficient barcoding
loci and the most active chemical component of the target
plant before conducting authentication methods. The use of
a concatenated barcoding approach (using at least two loci)
is highly recommended to elucidate species identity.
Therefore, the Traditional and Alternative Medicine Act
(TAMA) should integrate both techniques in herbal product
authentication of sambong and tsaang-gubat and other
high-valued herbal medicinal products for the safety of all
its consumers.
In conclusion, this is the first study that used both DNA
Barcoding and High-Performance Liquid Chromatography
(HPLC) in the authentication of Philippine HMPs, specifically
on B. balsamifera and E. microphylla. DNA barcoding
using ITS-trnH-psbA datasets authenticated the plant source
material while HPLC confirmed the presence of active
compounds, quercetin and rosmarinic acid from the HMPs.
Both analyses revealed cases of substitution. These HMP
samples deviated from the standard DNA barcoding and
HPLC profiles of both the wild samples and the other tested
HMPs. The use of genomic data is direct; it determines the
plant component of a sample. Therefore, HMPs subjected
to DNA barcoding protocols are expected to match that of
their marketed labels. The presence of a different species
after BLAST search and SRM analysis would support the
possibility of adulteration of the marketed HMPs.
HPLC detects the presence of active compounds from
wild samples and HMPs. This further presented the possible
degradation of active components due to manufacturing
processes and environmental conditions. The determination
of active compounds is necessary in HMPs since it is
responsible for the product’s effectiveness. However, multiple
species can have the same compounds, and the actual plant
material utilized may be adulterated. The coupling of DNA
barcoding and HPLC, therefore, is highly recommended to
ensure the safety of consumer health. To promote the safety
and well-being of Filipino consumers, it is necessary to
amend the Traditional and Alternative Medicine Act
(TAMA) and integrate provisions on DNA barcoding and
HPLC analysis protocols for the authentication of herbal
products.
ACKNOWLEDGEMENTS
The authors would like to acknowledge the DOST
Philippine Council for Health, Research and Development
(DOST-PCHRD) for the GD Alejandro research fund, and
the DOST-Science Education Institute for the Accelerated
Science and Technology Human Resource Development
(DOST-SEI-ASTHRD) for the scholarships of AA Lola,
SA Binag, and NG Alfeche. The authors would also like to
acknowledge the private individuals who provided them
with the wild samples used for this study. Lastly, the
LOLA et al. – Authentication of sambong and tsaang-gubat
1269
researchers are grateful to the Bureau of Plant and Industry
(BPI), Manila, Philippines for providing samples from their
collections.
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