Journal of Ethnopharmacology 274 (2021) 113909

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Genetic identification of medicinally used Salacia species by nrDNA ITS

sequences and a PCR-RFLP assay for authentication of Salacia-related
health foods
Shu Zhu a, *, Qundong Liu a, Jingyu He a, Naomi Nakajima b, S.P. Samarakoon c, Swe Swe d,
Khin Zaw d, Katsuko Komatsu a, **
a
Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan
b
Uchida Wakanyaku Ltd., 4-3-3, Higashi Nippori, Arakawa-ku, Tokyo, 116-8571, Japan
c
Department of Botany, University of Ruhuna, Matara, Sri Lanka
d
Department of Traditional Medicine, Ministry of Health & Sports, 47 Nay Pyi Taw, Myanmar

A R T I C L E I N F O A B S T R A C T

Keywords: Ethnopharmacological relevance: The roots and stems of several Salacia species have been used as traditional
Salacia medicines, especially in Ayurvedic medical system for the treatment of diabetes, rheumatism, gonorrhea,
Genetic identification amenorrhea, skin diseases, etc. Due to reported evidence supporting Salacia’s beneficial effects in early-stage
ITS sequence
diabetes and other lifestyle-related diseases, Salacia-based dietary supplements and health foods have been
trnK-rps16 sequence
PCR-RFLP assay
gaining popularity in Japan and other countries in recent years. However, due to the morphological similarities
between Salacia plants, particularly in the medicinally used parts (roots and stems), the authentication of the
botanical identities of Salacia-derived products is challenging.
Aim of this study: This study aims to develop a genetic approach to authenticate the medicinally used Salacia
species and to determine the botanical sources of the commercially available Salacia-derived products.
Materials and methods: The sequences of nuclear DNA internal transcribed spacer (ITS) and chloroplast trnK-rps16
region were determined and compared between 10 plant specimens from three medicinally used Salacia species
as well as 48 samples of commercial crude drugs. Moreover, a PCR-restriction fragment length polymorphism
(RFLP) assay was developed for rapid identification based on the ITS sequences.
Results: The plant specimens from the three medicinally used Salacia species showed three main types of se­
quences in both ITS (types I, II, III) and trnK-rps16 (i, ii, iii) regions. Combined the sequences of ITS and trnK-
rps16 regions, S. reticulata and S. oblonga had type I-i and type III-iii or similar sequences, respectively.
S. chinensis had type II-ii or II(536M)-i sequences. Forty-eight samples of commercial crude drugs were identified
based on ITS and trnK-rps16 DNA barcode. A convenient PCR-RFLP assay using Cac8I restriction enzyme was
established and applied to identify the botanical sources of health food products purchased from online retailers.
All the twelve samples were identified as S. chinensis.
Conclusion: The nrDNA ITS sequences provided useful information to authenticate Salacia species and to eluci­
date the phylogenetic relationship within the Salacia genus. Genetic identification results revealed that
S. chinensis and S. reticulata are the major sources of commercially available Salacia-products. Based on the ITS
sequences, a convenient PCR-RFLP assay was established for the identification of the medicinally used Salacia
species as well as their derived health food products.

1. Introduction species which are mainly distributed in tropical and subtropical Asia,
such as Sri Lanka, India, Thailand, Vietnam and southern China (Das­
The genus Salacia L. (Hippocrateaceae) includes approximately 120 sanayake and Clayto, 1996; Hooker, 1875; Wu, Raven and Hong, 2009).

* Corresponding author.
** Corresponding author.
E-mail addresses: szhu@inm.u-toyama.ac.jp (S. Zhu), katsukok@inm.u-toyama.ac.jp (K. Komatsu).

https://doi.org/10.1016/j.jep.2021.113909
Received 10 November 2020; Received in revised form 3 February 2021; Accepted 4 February 2021
Available online 12 February 2021
0378-8741/© 2021 Published by Elsevier B.V.
S. Zhu et al. Journal of Ethnopharmacology 274 (2021) 113909

Table 1 components have been elucidated (Paarakh, Patil and Thanga, 2008;
Plant materials. Stohs and Ray, 2015). A series of sulphoniums including salacinol,
Species Code Wild Collection place Date of kotalanol, ponkoranol and salaprinol, and their de-O-sulphonated
No. or collection compounds as neosalacinol, neokotalanol, neoponkoranol and neo­
Cult. salaprinol have been isolated from Salacia species (Yoshikawa et al.,
Salacia Sr-S1 Cult. University of Ruhuna, 2008.9. 1997; Akaki et al., 2014; Nakamura, Matsuda & Yoshikawa, 2011). In
reticulata Mapalana, Kamburupitiya, recent years, dietary supplements and health foods made from Salacia
Wight Sri Lanka plants have been gaining popularity in Japan and other countries due to
Sr-S2 Cult. University of Ruhuna, 2008.9.
Mapalana, Kamburupitiya,
reported evidence supporting Salacia’s beneficial effects in early-stage
Sri Lanka diabetes and other lifestyle-related diseases (Jayawardena et al., 2005;
Sr-S3 Cult. University of Ruhuna, 2008.9. Williams et al., 2007; Jeykodi et al., 2016; Shimada et al., 2014). The
Mapalana, Kamburupitiya, roots and stems of three Salacia plants, S. reticulata, S. oblonga and
Sri Lanka
S. chinensis, are considered to be the main sources of the raw materials
Sr-S4 Wild Denagama Estate, 2008.9.
Hukamana, Mathara District, for medicinal uses and for health food products (Stohs and Ray, 2015;
Southern Prov., Sri Lanka Matsuda et al., 2005). However, authentication of the botanical identi­
S. oblonga Wall. So-S1 Cult. Athgandara Estate, 2008.9. ties of Salacia-products is a challenging task due to the morphological
ex Wight & Imaduwa, Galle District, similarities of the medicinally used roots and stems of Salacia plants.
Arn. Southern Prov., Sri Lanka
So- Wild Horudugoda Estate, 2008.9.
Moreover, health food products are always in the forms of powder,
S2a Imaduwa, Galle District, shred, tea bags, etc., which complicates the authentication of their
Southern Prov., Sri Lanka source.
S. chinensis L. Sc-T1 Cult. Chulalongkon University, 2009.5. The internal transcribed spacer (ITS) sequence of the nuclear ribo­
Thailand
somal DNA (nrDNA) has been demonstrated to have high discriminatory
Sc-M1 Wild Band Bway Gone village, 2019.11.
Than-lyin District, Yangon power to differentiate closely related species, therefore is recommended
Region, Myanmar as a core DNA barcode for flowering plants (Kress et al., 2005; China
Sc-M2 Wild Shwe Due village, Sanda-wut 2018.11. Plant BOL Group, 2011) and also widely used for the identification of
group, Myeik District, crude drugs (Sukrong et al., 2007; Zhu et al., 2015). In addition, the
Tanintharyi Region,
sequences of noncoding regions from the chloroplast DNA (cpDNA),
Myanmar
Sc-M3 Wild Kya-khat-ta-pin village, Yay- 2018.11. which exists only in plants, are also widely used to infer phylogenetic
phyu township, Tanintharyi relationship at lower taxonomical levels and to discriminate species
Region, Myanmar (CBOL Plant Working Group, 2009). Dev et al. (2015) have compared
a
This specimen was obtained from a local traditional medicine doctor, being the sequences of nrDNA ITS2 region and cpDNA rbcL, matK and
locally called Kothala-Himbutu. trnH-psbA regions of Salacia species collected in West India and proposed
ITS2 sequence as a suitable DNA barcode for this genus. However,
The roots and stems of several species in this genus have been used as specimen sampling from a wider geographical region is required to
traditional medicines, especially in Ayurvedic medical system for the validate these findings.
treatment of diabetes, rheumatism, gonorrhea, amenorrhea, skin dis­ In order to objectively and accurately authenticate the botanical
eases, etc. (Ambasta, 1986; Jayameera, 1981; Chuakul et al., 1997). origins of Salacia specimens and to survey the commercially available
Over the past few decades, scientific evidence has accumulated to Salacia-derived products, the DNA sequences of nuclear ITS region and
demonstrate its anti-diabetic, anti-obesity and hepatoprotective activ­ chloroplast trnK-rps16 region of medicinally used Salacia species as well
ities, its underlying mechanism has been proposed, and its active as crude drugs were determined and compared. Furthermore, a

Table 2
Comparison of ITS sequences from Salacia species with the variable sites indicated.

1) The numerals above the sequence indicate the alignment position.

2) Asterisk (*) indicates the nucleotide is the same as that in the top line; hyphen (− ) indicates the aligned gap.
3) Y: additive peaks comprising C & T; S: additive peaks comprising C & G; R: additive peaks comprising G & A; M: additive peaks comprising C & A.
4) CAGG in italic indicates that two kinds of sequences, one with “CAGG” as a repeat unit of its upstream sequence and the other without this 4-bp repeat
unit at this position, exist simutanuously in the same individual.
5) DNo. indicates the crude drug samples as summarized in Table 4 and S1.

2
S. Zhu et al.
convenient PCR-restriction fragment length polymorphism (RFLP) assay
was developed for the identification of Salacia species and their derived
health food samples based on the differences in ITS sequences.
2. Materials and methods
2.1. Materials
Ten specimens of the three medicinally used Salacia species
(Table 1), including four specimens of S. reticulata and two specimens of
S. oblonga collected in Sri Lanka, and four specimens of S. chinensis, one
collected from Thailand and three from Myanmar, were used in the
present study. The specimens collected in Sri Lanka were identified by
Prof. Samarakoon S.P. at the University of Rufuna, Sri Lanka; the spec­
imens collected in Thailand were identified by Prof. Sukrong S. at the
Chulalongkorn University, Thailand; and the specimen collected in
Myanmar was identified by Ms. Swe S., the senior botanical researcher
and the director of the Research & Development Division, Department of
Traditional Medicine, Ministry of Health and Sports, Myanmar,
respectively.
Forty-eight crude drug samples were genetically analyzed to identify
their botanical origins, including two samples obtained in Sri Lanka (one
from a local market and another from a local institute), and others from
Japanese markets which were imported from Sri Lanka, India, and
Thailand as the raw material for the manufacture of health food prod­
ucts (Table S1). One or two individuals of each sample, and an overall
total of sixty individuals were analyzed. In addition, twelve samples of
Salacia-tea or Salacia-products were purchased from online retailers
(Table S2), and their botanical identity was determined using the PCR-
RFLP assay developed in our laboratory.
2.2. Genomic DNA extraction and PCR amplification of the two targeted
DNA loci
Total DNA was extracted from 40-50 mg of dried leaf or 80–100 mg
of stem or root by using DNeasy Plant Mini Kit (Qiagen, Germany) with a
few modifications (Zhu et al., 2011) to the protocol provided by the
manufacturer. The extracted total DNA was detected by electrophoresis
on a 1% agarose gel and then was used as a template in the following
PCR amplification.
The entire ITS and trnK-rps16 regions were amplified by PCR using
two pairs of primers flanking the two DNA regions. The primers used for
amplification of the ITS region were ITS-1F (5’-TCC ACT GAA CCT TAT
CAT TTA G-3’) and 18S-25S-3’R (5’-CCA TGC TTA AAC TCA GCG GGT-
3’) (Zhu et al., 2015). In the case of crude drug samples, two shorter
fragments were amplified instead of the larger full-length ITS segment.
Primer sets ITS-1F and 5.8S-77R (5’-GCA ATT CAC ACC AAG TAT CGC
ATT-3’) for the ITS1 region, and In-5.8S-5’F (5’-TCT CGC ATC GAT GAA
GAA CG-3’) and 18S-25S-3’R for the ITS2 region were used. The primers
for the trnK-rps16 region were S-trnK-5F (5’-TAC TCT ACC ATT GAG
TTA GCA AC-3’) and S-rps16-3R (5’-AAA GGT GCT CAA CCT ACA AGA
AC-3’). PCR amplification was performed using 10–100 ng of total DNA
as a template in 25 μl of reaction mixture consisting of 1 × PCR buffer for
KOD-Plus, 1.0 mM MgCl2, 0.2 mM of each dNTP, 0.4 U KOD-Plus DNA
polymerase (Toyobo, Japan), and 0.25 μM of each primer. A Takara
thermal cycler TP-600 (Takara, Japan) was used to carry out PCR
amplification using the cycling conditions: initial denaturation at 95 ◦ C
for 5 min, followed by 35 cycles of denaturation at 95 ◦ C for 30 s,
annealing at 52 ◦ C for 30 s, extension at 68 ◦ C for 50 s, and then final
extension at 68 ◦ C for 10 min. A 2 μl aliquot of the PCR product was
examined on a 1.0% agarose gel by electrophoresis, and the remaining
product was purified using Wizard SV Gel and PCR Clean-Up System
(Promega, U.S.A).
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Journal of Ethnopharmacology 274 (2021) 113909
2.3. Sequencing and phylogenetic analysis
Sequencing reaction using the purified PCR products as template was
carried out using ABI PRISM Bigdye Terminator v3.1 Cycle sequencing
kit (ThermoFisher, U.S.A.) with each of the four primers (ITS-1F, 5.8S-
77R, In-5.8S-5’F and 18S-25S-3’R) for ITS region and S-trnK-5F and S-
rps16-3R for trnK-rps16 region. The sequence was determined directly
by ABI Prism 3100-Avant Genetic Analyzer (ThermoFisher, U.S.A.) and
analyzed by sequencing analysis Software (v5.3 Patch 1). The analyzed
sequences were assembled, and the consensus sequence of each sample
was finally constructed. Then the obtained DNA sequences were aligned
and compared using Multalin software (http://multalin.toulouse.inra.
fr/multalin/). The nucleotide sequences obtained in this study were
deposited in the International Nucleotide Sequence Database (INSD:
DDBJ/EMBL/GenBank) with the accession numbers LC556193-
LC556200 for the ITS sequences and LC556334-LC556341 for the
trnK-rps16 sequences.
The phylogenetic tree was reconstructed using the Neighbor-Joining
(NJ) method (Saitou and Nei, 1987) by the MEGA X (Kumar et al., 2018)
software. The evolutionary distances were computed using the
Tamura-Nei method (Tamura and Nei, 1993) and the tree was drawn to
scale, with branch lengths in the same units as those of the evolutionary
distances. Bootstrap analysis (Felsenstein, 1985) was performed with
1000 replications to estimate the confidence of the topology of the ob­
tained tree.
2.4. Cloning of PCR product of ITS region from the sample D2
The PCR product of the ITS region from the sample D2 was subjected
to subcloning. The entire ITS region was amplified from total DNA using
the primer set ITS-1F and 18S-25S-3’R and then was ligated into the
pTA2 vector by using TArget Clone-Plus Kit (Toyobo, Japan) as follows.
First, an A-attachment reaction was performed prior to the ligation re­
action since the PCR products generated by the KOD-Plus polymerase
are blunt-end products. The A-attachment mixture consisting of 9 μl PCR
products and 1 μl 10 × A-attachment mix was incubated at 60 ◦ C for 1 h.
Second, 10 μl of the ligation mixture containing 5 μl 2 × ligation buffer,
1 μl pTA2 vector (50 ng/μl), 1 μl T4 DNA ligase, and 2.5 μl A-attached
PCR products was prepared and incubated for 30 min at room temper­
ature. Subsequently, 2 μl of the ligation mixture was added to 50 μl of
thawed ECOSTM X competent E. coli DH5α cells (NipponGene, Japan)
and vortexed for 2 s to mix. The solution was incubated on ice for 5 min,
then shifted to 42 ◦ C for 45 s to achieve the transformation. The trans­
formation liquid was plated on a LB/Amp plate (1% polypeptone, 0.5%
yeast extract, 1% NaCl, 1.5% agar, 50 μg/ml ampicillin, 40 μg/ml X-gal
and 72 μg/ml isopropyl β-D-1-thiogalactopyranoside) and incubated at
37 ◦ C for 12–16 h. Then, ten of the single white colonies were selected
and separately inoculated into 2 ml of liquid LB/Amp medium (1%
polypeptone, 0.5% yeast extract, 1% NaCl and 100 μg/mL ampicillin).
After incubation at 37 ◦ C for 22 h, the bacteria were collected by
centrifugation at 4000 rpm for 15 min and plasmids were purified using
QIAprep Spin Miniprep Kit (Qiagen, Germany). The purified plasmid
was used for direct sequencing.
2.5. PCR-RFLP assay for identification
Based on the nucleotide sequence differences among the three
medicinally used Salacia species, a restriction enzyme Cac8I (New En­
gland Biolabs Japan Inc., Japan) was selected and used for the PCR-RFLP
assay. Cac8I recognizes a 6-bp sequence unit GCN^NGC and cuts the DNA
at the center of this target sequence. The primer pair ITS-1F and 5.8S-
77R for the ITS1 region was used to amplify a 380 bp fragment; the
primer pair In-5.8S-5’F and 18S-25S-3’R for the ITS2 region was used to
amplify a 420 bp fragment. The thermal cycling conditions used were
identical to those indicated in section 2.2. The resulting PCR amplicons
from the respective specimens of each species and the health food
S. Zhu et al. Journal of Ethnopharmacology 274 (2021) 113909

Fig. 1. Phylogenetic tree of Salacia species constructed by the Neighbor-Joining method based on the ITS sequence.
The bold-faced sequences were determined in the present study for which the corresponding specimens, crude drug code number.,
and the sequence types are shown in Tables 1, 2 and 4.

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S. Zhu et al. Journal of Ethnopharmacology 274 (2021) 113909

samples were digested with Cac8I at 37 ◦ C for 4 h. The digested frag­

ments were detected by electrophoresis on a 4% agarose gel (E-gel 4%
(HR), ThermoFisher Scientific, USA).

3. Results and discussion

3.1. nrDNA ITS sequence of plant specimens and phylogenetic

relationship of Salacia species inferred from the ITS sequence

The ITS sequence of all plant specimens was bidirectionally deter­

mined to eliminate any chance of ambiguity. The entire ITS sequence
length ranged from 650 to 658 bp in the three medicinally used Salacia
species, including ITS1, 5.8S rRNA gene and ITS2 regions. Detailed
comparison between respective specimens of the same species and be­
tween the species (Table 2) revealed the discriminatory nature of the ITS
sequences among the three Salacia species, although intra-species vari­
ation occurred. Four specimens of S. reticulata showed the same type I
sequence except for one additive site (position 579th) in sample Sr-S4.
Four specimens of S. chinensis collected from Thailand and Myanmar
revealed almost identical type II or II(536M) sequences, which differed
from the type I sequence of S. reticulata by four specific nucleotides at
positions 33rd and 112th-114th, and a 4-bp insertion (CAGG, as a repeat
unit of its upstream sequence) at positions 621st-624th (Table 2). Two

2) Asterisk (*) indicates the nucleotide is the same as that in the top line; hyphen (− ) indicates the aligned gap.
specimens of S. oblonga showed almost the same type III sequence except
for one additive site at the aligned position 112th in sample So-S2 (type
III(112Y)). The type III sequence of S. oblonga differed significantly from
those of S. reticulata or S. chinensis, with 30 sites of nucleotide differences
detected.
Comparison of trnK-rps16 IGS sequences from Salacia species with the variable sites indicated.

Based on our sequence analysis of the three species and the available
data for a number of ITS sequences from Salacia species in GenBank, a

3) The underlined sequences indicate a sequence unit presented at the indel positions.
phylogenetic tree was constructed using the NJ method as shown in
Fig. 1. Two major clades supported by high bootstrap value were
resolved in the NJ tree, one consisting of Old-World species and the
other of New-World species. This result is consistent with the previous

4) DNo. indicates the crude drug samples as summarized in Table 4 and S1.
report by Coughenour et al. (2011) and provides a more precise reso­
lution. The Old World clade was then divided into two subclades, in

1) The numerals above the sequence indicate the alignment position.

which four African taxa formed one subclade, clearly separated from the
Asian species. The major subclade consisting of Asian species was
further clustered into two groups, one consisting of S. reticulata and
S. chinensis, and the other of S. fruticosa, S. oblonga and S. macrosperma.
Monophyletic clustering of S. reticulata and S. chinensis suggested these
two are closely related sister species. These two species also share
similar morphology when compared with other species, although
discernable from each other by the petal shape and fruit size. (Dassa­
nayake and Clayto, 1996). Monophyletic clustering of S. oblonga and
S. macrosperma suggested these two are closely related sister species.
The type III or type III(112Y) sequences of S. oblonga determined in the
present study were different from the sequences (Accession No.
KF881913, KF881914 and KF881915) of S. oblonga in GenBank, thereby
suggesting that they belonged to different subgroups. S. fruticosa was
placed sister to the S. oblonga-S. macrosperma cluster based on the
observed sequence similarity.

3.2. cpDNA trnK-rps16 sequence of plant specimens

Table 3

Dev et al. (2012) have reported that the sequences of cpDNA rbcL and
matK regions are much more stable and less informative than the trnH-
psbA sequence. In a preliminary experiment, four cpDNA loci (trnL-trnF,
petB-petD, trnK-rps16, and trnH-psbA regions) were sequenced to inves­
tigate their discriminatory ability for Salacia species (data not shown).
Of the four loci tested, the trnK-rps16 sequence was found to exhibit high
interspecies divergence, and thus was used in the present study. The four
S. reticulata specimens (Sr-S1,2,3,4) had identical type-i sequence. While
the four S. chinensis specimens showed two types of sequences. Three
specimens collected in Myanmar (Sc-M1,2,3) showed the same type-i
sequence as that of S. reticulata. A specimen (Sc-T1) collected in

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S. Zhu et al. Journal of Ethnopharmacology 274 (2021) 113909

Thailand revealed type ii sequence that differed from the type-i
sequence at three positions 165th, 590th, and 606th (Table 3). The two
specimens of S. oblonga (So-S1,2) had type iii and iii’ sequences which
showed high similarity, differed only by a site of nucleotide difference
(position 134th) and two indels (at positions 357th and 424th-445th). The
types i and ii shared similar sequences; they were different from the type
iii/iii’ by more than four sites of nucleotide differences and three sites of
indels.
The findings from the sequence analysis of the trnK-rps16 region
were mostly consistent with that of the ITS region. Therefore, pooling
the sequence data of ITS and trnK-rps16 regions reveal that S. reticulata
and S. oblonga from Sri Lanka had type I-i or I(579S)-i, and type III-iii or
III(112Y)-iii’ sequences, respectively. Additionally, S. chinensis from
Thailand and Myanmar had type II-ii and II(536M)-i sequences,
respectively. Due to the interspecies conservation and intra-species
variation of the trnK-rps16 region of Salacia species, the ITS sequence
is more informative for the authentication of Salacia plants.
3.3. Identification of crude drug samples based on the sequences of
nrDNA ITS region and cpDNA trnK-rps16 region
Sequences of the nrDNA ITS and cpDNA trnK-rps16 regions of all the
48 crude drug samples (Table S1) were determined and compared with
those obtained from the plant specimens (Tables 2 and 3) to identify
their botanical origins. The sequence types as well as the identification
results of the crude drug samples are summarized in Table 4. In cases
where two individuals in each sample were analyzed, an almost iden­
tical sequence was detected in both individuals, indicating the

Table 4
Identification results of the crude drug samples based on the ITS and trnK-rps16 sequences.
Code Name of sample Producing location Sampling Type of ITS seq. (individual Type of trnK-rps16 seq. (individual Identity (according to DNA
No. size 1,2) 1,2) sequence)

D1 Salacia Sri Lanka 1 I i Salacia reticulata

D5 Salacia Sri Lanka 1 I i S. reticulata
D20 Salacia Sri Lanka 2 I, I i, i S. reticulata
D34 Salacia Sri Lanka 2 I, I i, i S. reticulata
D37 Salacia Sri Lanka 2 I, I i, i S. reticulata
D41 Kothala- Sri Lanka 2 I, I i, i S. reticulata
Himbutu
D42 Salacia Sri Lanka 2 I, I i, i S. reticulata
D43 Salacia Sri Lanka 1 I i S. reticulata
D44 Salacia Sri Lanka 1 I i S. reticulata
D3 Salacia Sri Lanka 1 II i(ins.) S. chinensis
D4 Salacia Sri Lanka 1 II i(ins.) S. chinensis
D2 Salacia Sri Lanka 1 Mix i(ins.) Hybrid (S. reticulata × S.
chinensis)
D19 Salacia Sri Lanka 1 Mix i(ins.) Hybrid (S. reticulata × S.
chinensis)
D40 Kothala- Sri Lanka 1 IV iv S. oblonga
Himbutu

D10 Salacia India 1 I i S. reticulata

D21 Salacia Maharashtra, India 2 I(251R), I(251R) i(43C), i(43C) S. reticulata
D25 Salacia India 1 I i(43C) S. reticulata
D27 Salacia India 1 I i(43C) S. reticulata
D28 Salacia Maharashtra, India 2 I, I /, i S. reticulata
D29 Salacia India 2 I, I i, i S. reticulata
D32 Salacia Maharashtra, India 1 I i S. reticulata
D51 Sapta rangi India 2 I(251A), I(251R) i, i S. reticulata
D6 Salacia India 1 II / S. chinensis
D7 Salacia India 1 II / S. chinensis
D8 Salacia India 1 II / S. chinensis
D11 Salacia India 1 II i(ins.) S. chinensis
D12 Salacia India 1 II i(ins.) S. chinensis
D13 Salacia India 1 II i(ins.) S. chinensis
D14 Salacia India 1 II i(ins.) S. chinensis
D15 Salacia India 1 II i(ins.) S. chinensis
D16 Salacia India 1 II i(ins.) S. chinensis
D17 Salacia India 2 II, II i(ins.), i(ins.) S. chinensis
D18 Salacia India 2 II, II i(ins.), i(ins.) S. chinensis
D22 Salacia India 2 II, II i(ins.), i(ins.) S. chinensis
D23 Salacia India 2 II, II /, i(ins.) S. chinensis
D26 Salacia India 1 II i(ins.) S. chinensis
D31 Salacia India 2 II, II i(ins.), i(ins.) S. chinensis
D33 Salacia Maharashtra, India 1 II i(ins.) S. chinensis
D38 Salacia Andhra Pradesh, 2 II, II i(ins.), i(ins.) S. chinensis
India
D39 Salacia Maharashtra, India 2 II, II i(ins.), i(ins.) S. chinensis
D45 Salacia India 1 II i(ins.) S. chinensis
D46 Salacia India 2 II, II i(ins.), i(ins.) S. chinensis
D50 Ponkoranti India 2 II, II i(ins.), i(ins.) S. chinensis
D52 Salacia India 2 II, II i(43C), i(ins.) S. chinensis
D9 Salacia India 1 IV iv(388T) S. oblonga
D24 Salacia India 2 IV, IV iv, iv S. oblonga

D30 Salacia Thailand 1 I / S. reticulata

D47 Salacia Thailand 1 II / S. chinensis

/: failed in PCR amplification or sequence determination.

6
S. Zhu et al.
respective samples were derived from a single botanical origin and not a
mixture.
Sixteen crude drug samples showed the type I ITS sequence and two
samples (D21 and D51) showed a similar sequence with one nucleotide
difference at position 251st (type I(251A/R)) (Table 2). Twenty-five sam­
ples had the type II sequence. Three samples (D9, D24, D40) had iden­
tical sequences, distinct from any sequence detected in plant specimens
in the present study, and were tentatively assigned as type IV. A BLAST
search in the GenBank revealed that the type IV sequence is completely
identical to the ITS sequence of S. oblonga (Accession No. KF881915).
Two samples (D2 and D19) showed a typical nucleotide additivity in the
ITS sequence at the variable sites between types I and II (Table 2). As the
nrDNA is of biparental inheritance, the nucleotide additivity detected in
the ITS sequence suggested a hybrid origin of the plants and provide
helpful information to infer the involved progenitors or lineages. Further
cloning analysis was conducted on the sample D2 and the results showed
that among the 10 colonies obtained, four had type I(33C) and the other
six had type II sequences.
Five types of trnK-rps16 sequences were detected in the crude drug
samples, except in five samples that failed in sequencing. Fourteen
samples showed the type i sequence and four samples had a similar
sequence with a nucleotide difference at site 43rd (type i(43C)). Twenty-
two samples showed identical trnK-rps16 sequence that had two repeats
of a 13-bp sequence unit at positions 479th-504th (type i(ins.)), and
differed from the type i that had only one unit of this 13-bp sequence
unit. Notably, these twenty-two samples had the type II ITS sequence.
The three samples (D9, D24 and D40) that had the type IV ITS sequences
revealed similar trnK-rps16 sequences as type iv or type iv(388T) (Table 3)
that were quite similar to the type iii’ sequence.
Based on the above results and also upon consideration of intra-
7
Journal of Ethnopharmacology 274 (2021) 113909
species variation in the sequences of both DNA regions, thirteen sam­
ples that had type I-i sequences and four samples (D21, D25, D27, D51)
that had the similar type I(251R)-i(43C), I-i(43C), or I(251A/R)-i sequences
were identified to be S. reticulata; twenty-one samples with type II-i(ins.)
or II-i(43C) sequence were identified to be S. chinensis; three samples with
type IV-iv or type IV-iv(388T) sequence were identified to be S. oblonga.
Two samples (D2, D19) with type Mix-i(ins.) are likely derived from a
hybrid plant of S. reticulata and S. chinensis. However, the genotype III-
iii found in the specimens of S. oblonga from Sri Lanka wasn’t detected in
the crude drug samples from either Sri Lanka or India. To resolve this
discrepancy, a broader geographical sampling of S. oblonga along with
field surveys in both countries is required.
These 48 crude drug samples were from the three main producing
countries. As for the 14 samples produced in Sri Lanka, about two-thirds
were derived from S. reticulata. Of the 32 samples produced in India,
two-thirds were derived from S. chinensis and one-fourth from
S. reticulata. The two samples produced in Thailand were derived from
S. reticulata and S. chinensis, respectively. These results clearly indicate
that S. chinensis and S. reticulata are the major sources of Salacia-
products, while S. oblonga is seldom the source of commercially avail­
able Salacia-derived products. In a previous report, Udayan and Pradeep
(2012) have doubted the presence of S. reticulata in India; however, their
survey was limited to the Kerala region of the country. The present re­
sults based on the genetic analysis of commercial samples suggest that
S. reticulata has distribution in India and is not rare. Two samples, D40
and D41, named Kothala-Himbutu were identified as S. oblonga and
S. reticulata, respectively. In a personal communication, Prof. Samar­
akoon S. P. from the University of Ruhuna asserted that all three species
(S. reticulata, S. chinensis and S. oblonga) have been used as
Kothala-Himbutu in Sri Lanka. The present study revealed through
Fig. 2. Sequence comparisons between the types
I–IV in the ITS1 region (a) and ITS2 region (b) indi­
cating the recognition site of the restriction enzyme
Cac8I, and the expected fragments after digestion
with Cac8I in the PCR-RFLP assay.
The number above sequence is the aligned position
corresponding to the sequence shown in Table 2. The
variable nucleotides among the species are indicated
in boldface. The underlined sequences indicate the
recognition site of Cac8I and the arrowhead indicates
the site of cleavage. The sequence indicated in grey is
the recognition site of Cac 8I found in all species. The
expected fragments indicated in grey are shorter than
90 bp and produce faint bands in agarose
electrophoresis.
S. Zhu et al. Journal of Ethnopharmacology 274 (2021) 113909

genetic identification that at least S. reticulata and S. oblonga are the

sources of the botanical origin of Kothala-Himbutu.

3.4. Development of PCR-RFLP assay for convenient identification of

three Salacia species and its application in the authentication of health
food products

Based on the ITS sequence information of the three Salacia species,

we developed a convenient PCR-RFLP assay to achieve a fast and ac­
curate authentication of health food products. A restriction enzyme
Cac8I that recognizes and cleaves DNA at the GCN^NGC sequence
enabled the specific ITS sequences of the respective species to be
digested into discriminatory fragment patterns. Considering that crude
drug samples always have highly degraded DNA, the ITS1 and ITS2
regions were amplified separately to facilitate the PCR-RFLP assay. Next,
the two amplicons were used in the subsequent enzymatic digestion,
which generated clear and unambiguous fragment profiles. The recog­
nition sites and the expected fragments after digestion of the respective
species with specific sequence types are summarized in Fig. 2. By using
the restriction enzyme Cac8I to digest the ITS1 amplicon, S. oblonga
could be clearly discriminated from S. reticulata and S. chinensis (Fig. 2a).
Furthermore, the digestion of the ITS2 amplicons allowed the three
species to be discriminated from each other unambiguously (Fig. 2b).
The results of the PCR-RFLP assay on the representative samples with
specific sequence types are shown in Fig. 3. Electrophoretic profiles of
the resulting fragments from the respective species were fully consistent
with the theoretical expectation. The ITS1 amplicons of S. reticulata and
S. chinensis were cleaved into two fragments of 175 bp and 207 bp
(Fig. 3a, Lanes 1, 2), and that of S. oblonga were cleaved into two frag­
Fig. 3. Electrophoregram of the PCR-RFLP assay on the three Salacia species.
ments of 276 bp and 104 bp (Fig. 3a, Lanes 3, 4). The ITS2 amplicons of (a) After digestion of the ITS1 amplicon with Cac8I; (b) After digestion of the
the respective species were cleaved into distinguishable fragment pro­ ITS2 amplicon with Cac8I. M: Ultra Low Range DNA Ladder; Lane 1:
files (Fig. 3b). Although the shorter fragments (<90 bp) were not clearly S. reticulata (Sr-S3, type I); Lane 2: S. chinensis (Sc-T1, type II); Lane 3: S. oblonga
visible, the longer fragments (>90 bp) displayed a key distinguishable (So-S1, type III); Lane 4: sample S. oblonga (D9, type IV); Lane 5: Salacia-tea
pattern. S. reticulata was characterized by the two bands of 195 and 149 (health food sample HF1); Lane 6: Salacia (health food sample HF8).
bp, S. chinensis by the two bands of 195 and 92 bp, S. oblonga (type III) by
the two bands of 199 and 143 bp, and S. oblonga (type IV) by the two claim that S. reticulata and S. oblonga are the major ones. In addition, a
bands of 230 and 189 bp (Fig. 3b). A combination of fragment profiles in convenient PCR-RFLP assay was established based on the ITS sequence
the ITS1 and ITS2 regions allowed the three species to be definitively for the identification of the medicinally used Salacia species as well as
discriminated. It is well known that the brightness of the DNA bands their derived health food products. This assay provides a useful tool for
corresponds to their lengths when the fragments are in equimolar the health food industry to ensure the botanical origin of raw materials
amounts. In the PCR-RFLP assay, all the resulting fragments after and also for the regulatory agencies to verify and monitor the Salacia-
digestion are in equimolar amounts, therefore, the smaller the length, products available to the consumers.
the weaker the band brightness. It is worth noting that the faintly
discernible bands of these fragments were consistent with the theoretical Declaration of competing interest
prediction.
The established PCR-RFLP assay was applied to identify the botanical The authors declare that they have no known competing financial
origins of the health food products purchased from online retailers. The interests or personal relationships that could have appeared to influence
results revealed that all the twelve samples were derived from the work reported in this paper.
S. chinensis although most of them were claimed to be S. reticulata or
S. oblonga (Table S2). The PCR-RFLP results of two health food samples Acknowledgement
(HF1, HF8) are shown in Fig. 3 (Lanes 5, 6). The PCR-RFLP results of all
samples were further confirmed by sequencing analysis of the entire ITS We greatly appreciate Prof. Suchada Sukrong at Chulalongkorn
region. University, Thailand for kindly providing the specimen of S. chinensis.
We are very grateful to Mr. Akifumi Nagatomo and Mr. Yoichi Matsuura
4. Conclusion at Morishita Jintan Co., Ltd., Japan for kindly providing many crude
drug samples. This work was supported in part by “Research and
The present study determined and compared the nrDNA ITS se­ development support program for feasibility demonstration of food in­
quences and the cpDNA trnK-rps16 sequences of the medicinally used dustry related technologies in East Asia” of Food Industry Affairs Bu­
Salacia species. The genetic information gained in this study is appli­ reau, Ministry of Agriculture, Forestry and Fisheries of Japan in
cable in the identification of crude drugs and health foods that are made 2010–2011, and in part by JSPS KAKENHI [Grant numbers
from Salacia plants, ensuring safe and efficient uses of these Salacia- JP21406004, JP15H05268 and JP18K06716] and by 2020 director’s
products. The ITS sequence is more informative than the trnK-rps16 Leadership Expenses, Institute of Natural Medicine, University of
sequence, and the latter may serve as a complemental barcode locus for Toyama.
genetic discrimination of Salacia species. Genetic identification results
revealed that S. chinensis and S. reticulata are the major sources of
commercially available Salacia-products, as opposed to the general

8
S. Zhu et al.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jep.2021.113909.
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