Identification of Vitex Negundo L. (Lagundi) Accessions From Brgy. Mambugan, Antipolo City, Rizal, and From Ateneo De Manila University, Brgy. Loyola Heights, Quezon City Through Dna Barcoding Using Chloroplast and Nuclear Dna Markers

Date of Award

5-2022

Document Type

Thesis

Degree Name

Master of Science in Biology

First Advisor

Vivian A. Panes, PhD

Abstract

Vitex negundo or “lagundi”, a known and recognized medicinal plant in the Philippines, recently had an increased demand for its reported medicinal properties and potential to cure mild respiratory symptoms of COVID-19. Leaf morphology is also challenging, particularly for Vitex negundo in that there are accessions which are palmately compound with three (3) leaflets, while there are other accessions that are palmately compound with five (5) leaflets. Moreover, adulteration of the raw materials for herbal medicinal products has been reported. To address these problems, the need to establish a barcode database specifically for Philippine medicinal plants is important to aid in the identification of the plant species for raw materials, and for the regulation of the herbal medicinal products sold in the market. This study aims to barcode and identify thirteen (13) lagundi accessions acquired from a farm located in Brgy. Mambugan, Antipolo City, Rizal, and from within the campus of Ateneo de Manila University, Brgy. Loyola Heights, Quezon City, using chloroplast (matK, rbcL) and nuclear (ITS2) DNA markers.

Genomic DNA from all accessions was successfully extracted through potassium ethyl xanthogenate or PEX method, and amplified using the DNA markers through polymerase chain reaction or PCR. Amplicons were sent to Macrogen, Inc. for sequencing. Quality of DNA sequences was inspected through Geneious Prime 2022.0.2, and phylogenetic tree analyses were performed through MEGA 11 software. Origanum vulgare or “oregano” was chosen as the outgroup for this study.

Phylogenetic tree analyses (MEGA 11) for the rbcL sequences of V. negundo revealed that all thirteen (13) accessions grouped into one clade with bootstrap support values ranging from 54% to 100%, whereas the outgroup O. vulgare, grouped into a different clade. For matK sequences, all V. negundo accessions grouped in to one clade with bootstrap support values ranging from 55% to 100%, while the outgroup grouped into a separate clade. For ITS2 sequences, all V. negundo accessions grouped together, and separated from O. vulgare accessions, with bootstrap values that ranged from 57% to 100%. Pairwise percentage sequence divergence values for ITS2 sequences ranged from 0.5% to 34%, which averaged to 11%. Percentage divergence for ITS2 sequences is the highest among the three DNA markers (rbcL – 0.7%; matK – 3%). Overall results show that using ITS2 as the DNA barcode has the highest discrimination power, followed by matK, then rbcL. Although rbcL has the lowest variable in discriminating species (0.7%), this is the first study to analyze and determine the DNA barcode for the rbcL gene of the Philippine accessions of Vitex negundo. For V. negundo species, ITS2 and matK has proven to be more variable, hence the higher discriminatory power to elucidate among species.

The DNA barcodes acquired in this study is not enough to identify V. negundo up to its species level, but have the potential to narrow down other known Vitex species which then establish their relatedness through their known medicinal properties. The DNA extraction method can be adapted for successfully extracting Philippine medicinal plants for future studies.

This study can also serve as baseline for establishing a database for the Philippine medicinal plants, and for authenticating the herbal medicinal products (HMPs) sold in the market. Future studies may include other varieties of lagundi and barcode accessions from Visayas and Mindanao regions as well. The number of accessions for this study has been made limited due to the COVID-19 pandemic.

Keywords: Barcoding, herbal medicinal products (HMPs), DNA Markers

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