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Tuklas Lunas Drug Discovery and Development program of the Philippine Council for Health Research and Development (PCHRD) Discovery of new drugs from natural substances Develop a database that will store information on all researches done on natural products (Drug Discovery Database)

Discovering Philippines Biodiversity With Possible Biological Activities

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Discovering Philippines Biodiversity with Possible Biological Activities

Tuklas LunasDrug Discovery and Development program of the Philippine Council for Health Research and Development (PCHRD)

Discovery of new drugs from natural substances

Develop a database that will store information on all researches done on natural products (Drug Discovery Database)

Discovery of new drugs from natural substances that can be developed up to the pre-clinical stageIn response to the call of the PCHRD this study was conducted

1Discovering Philippines Biodiversity with Possible Biological ActivitiesPANALIGAN, Jolo M., HIZON, Raphael Luis M., LOPEZ, Joseph Apollo D., NUCOM, Nicole Elena I. and ALEJANDRO, Grecebio Jonathan D.

Department of Biologcial Sciences, College of Science, University of Santo Tomas, Espaa Blvd., 1015 Manila, Philippines

BataanBataan is considered a hilly and mountainous province with a narrow plain on the eastern side.Mt. Natib is the highest peak of the Natib Caldera System.

Two interesting species under the family Rubiaceae were collected in Mt. Natib, Bataan.

Mt. Natib, peak of the Natib Caldera SystemMt. Samat, taken from the eastern plainsRubiaceaefourth largest family of angiosperms worldwide,

13,000 species in 660 genera; 80 of which are found in the Philippines.

Economic importance as in major crops such like coffee, medicine and ornamentals.Coffee plant Coffea arabica

Ixora sp.

Cinchona sp.Cinchona alkaloid quinine that is used against malaria

Economically important as exemplified by4Uncaria Schreb.Tribe Naucleea members are known to be economically important in some regions (gambir) of the world and used as medicine in others (cats claw). The biological activities associated with Uncaria includes cytotoxicity, anti-inflammatory, anti-viral, immunostimulation, anti-oxidant, CNS related response, vascular hypotensive, mutagenicity and antibacterial properties (Heitzman et. al., 2005).Characteristic hooks of Uncaria

Pyrostria Comm. ex Juss.Pyrostria is a genus from the tribe Vanguerieae, some members of the tribe have been known to cause Gousiekte, a plant-induced cardiotoxicosis in ruminants (Verstraete, 2011).

A member of Pyrostria has been found to have cytotoxic and anti-parasitic properties (Beniddir et. al., 2012).

PICTURES OF PYROSTRIA

Pyrostria sp.Molecular dataITS (nrDNA)Internal Transcribed Spacer or ITS region of the nuclear ribosomal DNA is the most widely known source of molecular character variation for phylogenetic interference (Baldwin, 1992).easily amplified and has high degree of variation between closely related species (Soltis et. al., 1998) (Alejandro et.al., 2005).

trnL-F (cpDNA)One of the most widely utilized non-coding regions in plant systematics (Quandt et. al. 2004).cpDNA sequences are a chief source of characters

Baldwin, B. 1992. Phylogenetic Utility of the InternalTranscribed Spacers of Nuclear Ribosomal DNAin Plants: An Example from the Compositae. Mol.Phylogenet. Evol. 1992, 1, 316.

Soltis, D., Soltis, P. and Doyle, J. Molecular Systematicsof Plants II (DNA Sequencing). Kluwer AcademicPublishers 1998. Norwell, Massachusetts

Alejandro, G.J.D.; Razafimandimbison, S. and Liede-Schumann, S. Polyphyly of Mussaenda inferredfrom ITS and trnT-F and its implication forgeneric limits in Mussaendeae (Rubiaceae). Am.J. Bot. 2005, 92 (3), 544557.7Statement of the ProblemLimited data are known concerning the biodiversity of Mt. Natib.

The collected species of Rubiaceae show deviating characteristics when compared to its closest relatives.

A call for phytochemical and pharmacological analysis has been raised by the biological activities associated with the genera mentioned.

Objectives of the StudyGeneral ObjectiveTo properly identify the collected species from Mt. Natib, Bataan and elucidate the bioactive components of both plant specimens.Specific ObjectivesThis study specifically aimed to:Conduct morphological assessment on the Uncaria and Pyrostria collected.Compare the morphological data set collected with the type specimens and protologues of Philippine endemic Uncaria and Pyrostria. Provide comprehensive descriptions and botanical illustrations of the collected samples.Extract, amplify, sequence the trnL-F and ITS regions of the collected Uncaria and Pyrostria.Perform phytochemical analysis to identify the constituents of both Uncaria and Pyrostria.

Significance of the StudyThis study contributed to the Taxonomy and Systematics of Rubiaceae. additional information needed for a comprehensive treatment of the tribe by discovering a new species of Uncaria.

Acknowledging a new species of Pyrostria helped in protecting the biodiversity of the genus. This study also helped preserve the biodiversity of Philippine Rubiaceae.

The exposition of the components of both plants served as a stepping stone for future researchers to identify which bioactive components of our material have pharmacological and medicinal applications.

This study was also helpful in the priority of the PCHRD to create the Drug Discovery Database and in their project named Tuklas Lunas.

All of the gathered information is useful to scientists, undergraduate and graduate students, researchers or anyone who is interested in studying the Rubiaceae of Philippines.

This study improved the understanding of the Philippine Rubiaceae and Philippine Flora. 10MethodologyCollectionDNA PurificationVegetative morphological assessmentDNA SequencingReproductive morphological assessmentHerbarium of Plant SpecimensAgarose gel ElectrophoresisDNA AmplificationDNA ExtractionDNA Sequence AnalysisGrinding of Plant specimensTests to elucidate the componentsThin Layer ChromatographyEvaporation of FractionsPartitioning of Crude ExtractEvaporation of Crude ExtractExtractionMorphological analysisMolecular Analysis Phytochemical AnalysisBasic Local Alignment Search Tool (BLAST)

Morphology of Uncaria lustrosa

It is a scandent shrub. The branches are strigose and angular. The stipules are bifid. The pubescent petiole ranges from 1 to 2.5 cm. The ovate to elliptic leaves ranges from 10 to 13 cm in width and 15 to 20 cm in length and is glabrous on the dorsal side while pubescent on the ventral side. The apex is broadly acuminate with a rounded base. The 8 to 10 pairs of arcuate to anastomosing lateral nerves arise from each side of the midrib and is more visible in the abaxial side of the blade. The inflorescences are arranged in globose. The 8 to 9.5 cm pubescent peduncle arises from pubescent recurved hooks. The 7 to 8 mm infundibuliform calyx tube is pubescent inside and outside. The 1.1 to 1.3 mm infundibuliform corolla tube is pubescent. The corolla lobe 4 to 5 mm The anther is 0.5-1 mm.Morphological ComparisonUncaria lustrosaUncaria cordataleavesGlabrous and lustrous upper surface, strigose lower surfaceThickly coriaceouspubescentshapeOval-ovateBroadly ovatebaseroundedRounded to deccurent acuminatemarginentireentireLeaf size15-18cm10-15cmLeaf ApexObtuse acuminateAcuteinflorescence5 cm diameter axillarySolitaryCalyxTubular, pubescentObtuse, pubescentReticulationsprominentfinepetiole1.5-18 cm1.5-2 cmPedicel1.6-1.9cm-PeduncleOpposite, 7.5 to 9cm-Lateral vein85Uncaria lustrosa Panaligan, Lopez and Alejandro, sp. nov.Using the results from the BLAST search, we identified the generic affiliation of our material to be with Uncaria.

Upon meticulous comparison of our material with its closest relative, we have found that our material is novel.

We propose a new species of Uncaria endemic to the Philippines.

Uncaria lustrosa

Basic Local Alignment Seach Tool (BLAST)

Pyrostria glandulosa Shrub 1.5 m high; branches glabrous. Stipules 3-7 x 0.2-0.5mm. Leaf blade elliptic-lanceolate, 3.5-4.8 x 1-2.2 cm, dark green above, pale green below, bothsidesglabrous; apex acuminate; base attenuate to decurrent; marginsentire and glabrous; lateral nerves 3-5 on eachside of the midrib; petioles 4-7 x 0.5-1 mm, glabrous. Bracts persistent on bothsides; Flower not seen; Fruits axillary; 5-7 x 4-5 mm, green whenyoung, glabrous, pedicel 3-5 mm, glabrous.

20Morphological ComparisonCanthium glandulosum

Pyrostria glandulosa

Baseattenuateattenuate / decurrentMarginentireentireApexNarrowly acuminateacuminateLeaf ShapeLanceolateelliptic / lanceolateLeaf size5-8 x 1.9-3.4 cm3.5-4.8 x 1-2.2 cmVenationarcuatearcuateHairabsentabsent# of lateral nerves3-5 pairs3-5 pairsPetiole8 mm3-6 mm PeduncleaxillaryaxillaryFruitpyriformpyriformPyrostria glandulosa (Merill) Hizon, Panaligan and Alejandro, comb. nov.The Genus Canthium is now restricted to species having supra axillary spines.

Our material having no supra axillary spines is grouped under Pyrostria and is supported by its possession of persistent bracts that encloses the infloresences, which is a unique feature in Pyrostria.

We propose a new combination Pyrostria glandulosa (Merill) Hizon, Panaligan and Alejandro, comb. nov

Persistent bracts of Pyrostria

Phytochemical Screening for UncariaSample DragendorffPotassium ferricyanide ferric chlorideBorntragerVanillin sulfuric acidAluminum - ChlorideMagnesium AcetateCrude Extract-++ (coumarin)++-Hexane Fraction---++-DCM Fraction-++ (coumarin)++-Butanol Fraction-+-++-Components detected: Phenols, tannins, flavonoids, coumarins, anthrones, higher alcohols, steroids, essential oils

Dragendorff test for alkaloidsPotassium phenols, tannins, flavonoidsBorntrager coumarins, anthraquinones, anthrones, phenolsVanillin higher alcohols, phenols, steroids, essential oilsAluminum chloride flavonoids, steroidsMg acetate - anthraquinones23Phytochemical Screening for PyrostriaSample DragendorffPotassium ferricyanide ferric chlorideBorntragerVanillin sulfuric acidAluminum - ChlorideMagnesium AcetateCrude Extract-++++-Hexane Fraction-++++-DCM Fraction-++++-Butanol Fraction-++++-Components detected: Phenols, tannins, flavonoids, coumarins, anthrones, higher alcohols, steroids, essential oils

Dragendorff test for alkaloidsPotassium phenols, tannins, flavonoidsBorntrager coumarins, anthraquinones, anthrones, phenolsVanillin higher alcohols, phenols, steroids, essential oilsAluminum chloride flavonoids, steroidsMg acetate - anthraquinones

24Isolation of VomifoliolVomifoliol is isolated from the DCM fraction of Uncaria lustrosa.

Vomifoliol is a characteristic terpene from family Rubiaceae which may be useful as a chemotaxonomic marker .

Vomifoliol was reported to be involved in the regulation of stomatal aperture and showed moderate activity against Klebsiella oxytoca.

Structure of VomifoliolGram negative bacteria that is closely related to k. pneumoniae

25RecommendationsAdditional pharmacological tests need to be conducted on the crude extract of Uncaria lustrosa for pharmacological applications.

Further isolation and identification of the other bioactive constituents

Chemical and pharmacological analysis on Pyrostria glandulosaConclusionsThe collected specimens were identified to be under Uncaria and Pyrostria by comparing morphological data set with type specimens and protologues of representative species of both genera.

Comprehensive descriptions were provided.

The sequences from the amplified DNA of both plant specimens were used to identify their generic affiliation.

The isolation of vomifoliol showed that Uncaria lustrosa, an endemic plant, is a new biologically-active material worth pursuing for further chemical and pharmacological analysis. 27