TJPS 2017, 41 (Supplement Issue): 213

Molecular Analysis of Bacopa Plants Based on matK Chloroplast Genome

Chayapol Tungphatthong1,*, Jutharat Somnuek1, Kornkanok Ingkaninan2, Suchada Sukrong1

1 Department of Pharmacognosy & Pharmaceutical Botany, CU.D.HIP, Faculty of Pharmaceutical Sciences,

Chulalongkorn University, Bangkok 10330, Thailand 2 Department of Pharmaceutical Chemistry & Pharmacognosy, Faculty of Pharmaceutical Sciences, Naresuan

University, Phitsanulok 65000, Thailand

* Corresponding author: Chayapol Tungphatthong Tel.+66(0)80598956 E-mail: chayapol.tun@student.chula.ac.th

Keywords: Bacopa monnieri, Plantaginaceae, matK, DNA fingerprint

Introduction Nowadays, the Thai public health policy promotes people to use Thai medicinal plants in the

primary health care. Medicinal plants have been use worldwide for centuries to maintain health and to

treat diseases [1]. Bacopa monnieri has been used in Ayuravedic Materia Medica for century. This

plants has been used for memory enhancer, anti-inflammatory, analgesic, antipyretic, sedative and

antiepilepsy [2] and also used as an ingredient in traditional Thai medicine for antipyretic of measles

and chickenpox. Bacoside A, the putative bioactive component of B. monnieri, was found to be a mixture

of four saponins comprising of bacoside A3, bacopaside II, Jujubogenin isomer of bacopasaponin C

and bacopasaponin C. [3]. In Thailand, there are three species of plants in the genus Bacopa

(Plantaginaceae) including B. monnieri (L.) Wettst (phrom mi), B. caroliniana (Walter) B. L. Rob. ExH

(lan phai lin) and B. floribunda (R. Br.) Wettst. (phak sam lan) [4]. Morphological characteristic

resemblant of plants in the genus Bacopa makes it difficult to identify and confound to use.

Chemical analysis is the conventional method used for authentication of medicinal plants.

However, phytochemical composition was affected from geographic location, seasonal variations,

storage conditions and processing method [5]. Recently, DNA barcoding has been proposed as an

alternative method for identification and authentication of medicinal plants in herbal products. DNA

barcodes are consist of multi-loci of DNA regions such as matK, rbcL, trnH-psbA and ITS [6] . matK,

the chloroplast genome, was purposed to be the one of core barcodes to identify angiosperm at species

levels [7]. In our study, we aim to obtain full length matK nucleotide sequences from three bacopa plants

for analysis the polymorphic sites.

Methods Plant materials Herbarium specimens of three Bacopa plants were collected from various sites of Thailand

(Table1). All of specimens were prepared and collected in the Museum of Natural Medicines, Faculty

of Pharmaceutical Sciences, Chulalongkorn University.

Genomic DNA extraction Genomic DNA was isolated from 200 mg of the fresh leaf samples using DNeasy® Plant Mini

Kit (Qiagen®). Plant Tissues were disrupted by mortar and pestle using liquid nitrogen. All genomic

DNA solutions were stored in -20°C until further analysis.

Thai Journal of Pharmaceutical Sciences (TJPS) The JSPS-NRCT Follow-Up Seminar 2017 and

33rd International Annual Meeting in Pharmaceutical Sciences

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TJPS 2017, 41 (Supplement Issue): 213

Table 1 Plant materials

ID Code Species Geographic region location Voucher ID

BM01 B. monnieri Chulalongkorn University, Bangkok CU-MN 20170125

BM02 B. monnieri Nakhon Pathom CU-MN 20170127

BM03 B. monnieri Naresuan University,Phitsanulok CU-MN 20170128

BC01 B. caroliniana Chulalongkorn University, Bangkok CU-MN 20170132

BC02 B. caroliniana Naresuan University,Phitsanulok CU-MN 20170133

BF01 B. floribunda Sakhon Nakorn CU-MN 20170137

PCR amplification and sequence determination of the matK gene

matK gene was chosen for amplification using universal primers (matK aF, matK8R, trnK

3914F and trnK 2R in Table 2). The PCR amplification was performed in 50 μL of reaction mixture with

GoTaq® Flexi DNA Polymerase (Promega, USA) (5X PCR buffer, 25 mM MgCl2, 2.5 mM of each dNTP,

10 mM of each primer, 5U Taq polymerase) and 10–100 ng of total DNA as a template. PCR

amplifications were carried out in a C1000TM Thermal Cycler (Bio-Rad, USA) using cycling conditions

at 96°C for 3 min, followed by 30 cycles of 96°C for 1 min 30 sec, 59°C for 1 min and 72°C for 2 min

and final extension at 72°C for 10 min. The PCR products were examined electrophoretically using

1.0% agarose gels. All PCR was sequenced in both directions using primers as mentioned. The

sequencing process was performed by capillary sequencing (Bioneer, South Korea)

Sequence analysis of matK DNA

The raw DNA sequences were verified, compiled and multialigned using Geneious® Program.

The polymorphism sites were observed and can be developed into many types of molecular marker

for authentication.

Table 2 matK universal primers were used for amplifying and sequencing.

Primer name Primer Sequence (5´->3´) Tm

matK aF TGG GTT GCT AAC TCA ATG G 54.5

matK 8R AAC TAG TCG GAT GGA GTA G 54.5

trnK 3914F CTA TAT CCA CTT ATC TTT CAG GAG 58.1

trnK 2R AAA GTT CTA GCA CAA GAA AGT GCA 57.6

Results Complete matK nucleotide sequences of B. monnieri, B. caroliniana and B. floribunda were

successfully amplified and sequenced by universal primers. The sequence data was assembled by

Geneious® Program. The full length of matK sequences are 1,521 bp for B. caroliniana and 1,536 bp

for both B. monnieri, and B. floribunda. The complete matK sequenes were aligned by ClustralOmega.

Ninety-one polymorphic regions were observed among the three taxa sequences. Fifteen indels were

observed at position 348-353 (six indels) and position 1528-1536 (nine indels).

Fifty polymorphic regions (37, 49, 126, 136, 155, 162, 179, 267, 268, 291, 334, 348, 349, 350,

351, 352, 353, 417, 459, 540, 599, 624, 644, 649, 652, 710, 719, 759, 761, 807, 864, 881, 929, 930, 954, 971, 985, 1029, 1090, 1104, 1154, 1525, 1528, 1529, 1530, 1531, 1532, 1533,1534,1535 and

1536) of the alignments are useful for discrimination B. caroliniana from the others. Twenty-six

polymorphic regions (31, 242, 248, 432, 666, 687, 778, 822, 857, 938, 939, 984, 1092, 1200, 1216,

1284, 1308, 1329, 1352, 1356, 1358, 1445, 1448, 1470, 1473 and 1510) of the alignments are useful

for discrimination B. monnieri from others. Twelve polymorphic regions (288, 316, 354, 441, 446, 633,

750, 771, 779, 1074, 1081 and 1206) of the alignments are useful for distinguish B. floribunda from the

others. Interestingly,only one polymorphic region (211) can be separate the three Bacoap spp.(Figure 1)

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TJPS 2017, 41 (Supplement Issue): 213

Discussion Molecular analysis technique was become a new solution aiding the problem of conventional

authentication. The chloroplast matK gene, the core barcode for land plants, were used for

discriminate the plant at species level [7].

Interestingly, DNA sequences of Bacopa data are very limited available in GenBank database

but B. monnieri . Unfortunately, data of matK sequences for B. caroliniana and B. floribunda do not

exist in the database. As we know, this is the first study of nucleotide sequences of B. floribunda. The

polymorphic sites observed in Figure 1, can be discriminated three Bacopa spp. Polymorphic data will

be developed as method for crude drug and herbal products authentication.

Figure 1 The polymorphic region of matK chloroplast DNA alignments of Bacopa monnieri, B.

floribunda and B. caroliniana. Underscore (_) reveals an indels. Vertical Numbers above the

polymorphic sites are their position in multiple sequence alignment. The first position corresponds to

the first nucleotide position of matK gene.

Conclusion In conclusion, all three matK nucleotide sequences of Bacopa were obtained. The variations of

nucleotide sequence can be used for authentication and phylogenetic analysis. In the future, the

polymorphic regions were developed for convenient and faster DNA marker for authentication of the

medicinal plant B. monnieri.

Acknowledgement This project was partially supported by Ratchadapiseksomphot Endowment under Outstanding

Research Performance Program, Chulalongkorn University. We are grateful to Chulalongkorn

University Drug and Health Products Innovation Promotion Center (CU.D.HIP) for providing facilities.

We also thank Assoc. Prof. Thatree Phadungcharoen for identification of plants samples and

Piroonrat Dechbumroong for her assistant.

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TJPS 2017, 41 (Supplement Issue): 216

Reference 1. Ganie SH, Upadhyay P, Das S, Sharma MP. Authentication of medicinal plants by DNA markers. Plant

Gene.2015; 4:83–99.

2. Russo A, Borrelli F. Bacopa monniera, a reputed nootropic plant: an overview. Phytomedicine. 2005;

12(4):305–17.

3. Deepak M, Sangli GK, Arun PC, Amit A. Quantitative determination of the major saponin mixture

bacoside A in Bacopa monnieri by HPLC. Phytochem. Anal. 2005;16:24–9.

4. Office of the forest herbarium, department of national parks, wildlife and plant conservation. Thai plant

name Tem Smitinand. revised. Bangkok; National office of buddism press. 2014.

5. Upton R, Graff A, Jolliffe G, Länger R, Williamson E. American Herbal Pharmacopoeia: Botanical

Pharmacognosy – microscopic Character- ization of botanical Medicines. Boca Raton: CRC Press;

2011

6. Li M, Cao H, But PPH, Shaw PC. Identification of herbal medicinal materials using DNA barcodes. J

Syst Evol 2011; 49(3): 271-283.

7. CBOL Plant Working Group. A DNA barcode for land plants. Proc. Natl. Acad. Sci. 2009;106:12794–7.

8. Parveen I, Gafner S, Techen N, et al. DNA Barcoding for the identification of botanicals in herbal

medicine and dietary supplements: strengths and limitations. Planta Med. 2016;82(14):1225–35.

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