Published with support from the Commission on Higher Education (CHEd) Acta Manilana Volume 64 (2016)  ISSN: 0065–1370

Official Publication of the Research Center for the Natural and Applied Sciences UNIVERSITY OF SANTO TOMAS ACTA Manilana Volume 63 (2015)  ISSN: 0065–1370

EDITOR-IN-CHIEF Fortunato B. Sevilla III, Ph.D. MANAGING EDITOR Mafel C. Ysrael, Ph.D. SECTION EDITORS Christina A. Binag, Ph.D. (Chemistry) John Donnie A. Ramos, Ph.D. (Biology) Ma. Carlota B. Decena, Ph.D. (Mathematics) Marilyn C. Mabini, Ph.D. (Engineering) EDITORIAL ASSISTANT Christopher D. Purugganan

INTERNATIONAL ADVISORY BOARD Salvador S. Alegret Ramaier Narayanaswamy Universitat Autonoma de Barcelona, Spain The University of Manchester, United Kingdom Geoffrey A. Cordell Cheong Nge University of Illinois at Chicago, U.S.A. Agency for Science, Technology and Research, Singapore Kevin D. Croft Takashi Okamoto University of Western Australia, Australia Nagoya City University, Japan Henri J. Dumont Stephen G. Pyne Ghent University, Belgium University of Wollongong, Australia Scott G. Franzblau Roger W. Read University of Illinois at Chicago, U.S.A. University of New South Wales, Australia Mary J. Garson Giorgio Sberveglieri The University of Queensland, Australia University of Brescia, Italy Hasuck Kim Steven L. Stephenson Daegu Gyeongbuk Institute of Science and Technology, University of Arkansas, U.S.A. Republic of Korea David Mark Taylor Nordin Hj. Lajis National University of Singapore, Singapore Universiti Putra Malaysia, Malaysia Shueh-Lin Yau Arben Merkoci National Central University, Taiwan, R.O.C. Catalan Institute of Nanoscience and Nanotechnology, Spain

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GRAPHICAL ABSTRACTS

Molecular confirmation and taxonomy of the Rubiaceous Mycetia 1–7 Article apoensis (Elmer) Govaerts John Christopher C. Villanueva, Remigio S. Callanta Jr., Jasmin Aei F. Neptuno, Maryneil A. Verin, Porferio S. Bangcaya, Vincent Louie D. Cabelin, & Grecebio Jonathan D. Alejandro

GC-MS metabolite profiling of the hexane extract and antimicrobial 8–16 Article characterization of the Philippine endemic Rubiaceae species Uncaria cordata var. circa, Psychotria luzoniensis, and Psydrax puberula Sarleen G. Castro, John Emmanuel V. Cid, Winnie Adrianne S. Ibañez, Grecebio Jonathan D. Alejandro, & Mario A. Tan

https://www.slideshare.net/lourensrobberts/antimicrobial-susceptibility-testing-2013 17–23 Comparison of cellular-based viability and apoptosis assays on Article doxorubicin treated colorectal adenocarcinoma cells Thomas Adrian Tiongson, Maria Charina Magsumbol, Mark Kevin Devanadera, & Myla R. Santiago

25–32 Fluorophore-labeled bioengineered glucose binding protein Article for measurement of transdermal glucose Cristina Tiangco, Sheniqua Brown, Fortunato Sevilla III, Govind Rao, & Leah Tolosa

33–39 Enantioselective chiral 2-pyridyl-2-imidazoline organocatalyzed Aldol Article reactions in brine Aileen Marice R. Baltazar, Simon Budde, Christian Faderl, Aldrick B. Verano, & Allan Patrick G. Macabeo

Cl- Na+ N N N - Na+ N N Cl H H OH O Na+ 1 1 R CHO O R - + Cl R2 R2 Na+ + - + - - Na Cl Na Cl Cl 67–74 A review of recent literature on repairable-item inventory systems Article Marilyn C. Mabini

REPAIR SUPPLIER FACILITY REPAIRABLE-ITEM INVENTORY MODELS

Single- Multi- WAREHOUSE Echelon Echelon scrap

Deterministic Stochastic Deterministic Stochastic Inventory policy: Inventory policy: Inventory policy: Inventory policy: USERS fixed lot size (s-1,s); (Q.r): fixed lot size (s-1,s); (Q.r); (s,S): service level base (s-1,s), based depot (s,S)

75–86 Reviving the Philippine shrimp industry: molecular diagnostics Article and therapeutics Maria Violeta R. Tare, Pocholo Mari T. Arabit, Mark Anthony G. Fran, David Angelo V. Guanzon, Jalizah Jaira E. Lim, Sharlaine Joi Ann B. Orense, Joselito A. Tabardillo Jr., & Mary Beth B. Maningas

87–98 Natural products-based discovery of antitubercular agents from Philippine Article medicinal plants — A review Allan Patrick G. Macabeo, Oliver B. Villaflores, Scott G. Franzblau, & Ma. Alicia M. Aguinaldo N MeO H OMe N O O HO Ten plant families H O N H MeO H O Explored seventeen plants O O OH OMe

Identified sixty compounds N OMe H O OMe OMe O OH MeO

N N O HO O >5 promising antitubercular hit compounds!!! Acta Manilana Volume 64 (2016) ISSN: 0065–1370 Manila, Philippines ACTA Manilana GUIDE TO CONTRIBUTORS

The ACTA MANILANA publishes papers dealing with any aspect of the natural sciences. Research papers, preliminary and short communications, and review papers are welcome. Papers submitted are considered with the understanding that they have not been published and are not under consideration for publication elsewhere. Every paper will be reviewed by at least two referees on whose recommendations the Editor’s decision on acceptance will be based. Script Requirement Two complete copies of the manuscript are required. They should be clearly typed double- spaced on an 8.5  11 inches paper. Original photograph must be included when applicable. An e-copy of the manuscript (using any of the following program: MSWord or Adobe PageMaker) should also be submitted. Please indicate the e-mail addresses of the corresponding author(s).

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Molecular confirmation and taxonomy of the Rubiaceous Mycetia apoensis (Elmer) Govaerts

John Christopher C. Villanueva1, Remigio S. Callanta Jr.1, Jasmin Aei F. Neptuno1, Maryneil A. Verin1, Porferio S. Bangcaya3, Vincent Louie D. Cabelin4, & Grecebio Jonathan D. Alejandro*1,2

1Department of Biological Sciences, College of Science; 2Research Center for the Natural & Applied Sciences, University of Santo Tomas, 1015 Manila, Philippines; 3College of Teacher Education, Biological Science Department, University of Antique, Tibiao Campus, 5707 Antique, Philippines; 4College of Arts and Sciences, Notre Dame of Dadiangas University, General Santos City, 9500 South Cotabato, Philippines

The little known endemic Philippine Rubiaceae Adenosacme apoensis Elmer was transferred to the genus Mycetia Reinw. based on herbarium specimens. Also, Mycetia apoensis was once thought as conspecific with M. cauliflora. To date, M. apoensis lacks comprehensive vegetative and reproductive descriptions to fully understand the species and be able to delineate from other members of Mycetia. To verify the generic affiliation of the species with more certitude, two chloroplast markers (rps16 intron and trnL-F region) were sequenced from the recent collections at Mt. Apo, Davao. Bayesian analysis of the combined plastid (rps16 and trnL-F) dataset strongly supported (PP = 1.0) the inclusion of M. apoensis in the genus Mycetia and resolved M. cauliflora as its sister-taxa. A comprehensive description and botanical illustrations of M. apoensis as well as its conservation status based on IUCN criteria are here provided.

Keywords: Adenosacme, Mycetia, cpDNA, rps16, trnL-F, Philippine endemic, conservation

INTRODUCTION [4], Isertieae [5], to the recently Argostemmateae [6]. In its current tribe, the genus Argostemma Mycetia Reinw. of the family Rubiaceae is Wall. is resolved as its sister-taxa [7]. composed of about 45 species of small shrubs characterized by the corky white bark of older Currently, there are four Mycetia species branches, the yellow or rarely white flowers and [Mycetia apoensis (Elmer) Govaerts, Mycetia the fungus-like, snowy white, juicy berries [1]. cauliflora Reinw., Mycetia javanica (Blume) The genus is distributed in tropical and Reinw. Ex Korth., and Mycetia mindanaensis subtropical Asia [2] and underwent several tribal (Elmer) Govaerts] known to be present in the affiliations from Mussaendeae [3], Hedyotideae Philippines [8]. Two of the Philippine endemic species (M. apoensis and M. mindanaensis) *To whom correspondence should be addressed were previously under the genus Adenosacme [email protected] / Wall. but transferred to Mycetia based on [email protected]

© 2016 UST Research Center for the Natural and Applied Sciences, Manila, Philippines Villanueva JCC et al.Acta Manilana 64 (2016)

morphology of herbarium specimens [9]. 17]. In all PCR runs, one sample was run with Moreover, Elmer [10] noted the close water instead of DNA as a negative control to relationship between M. apoensis and M. test for contamination. Amplified DNA was cauliflora. There is a need to re-examine the purified with the QiaQuick PCR purification kit morphology of the incompletely known M. (Qiagen). All sequences were retrieved by the apoensis. commercial services of Macrogen, Korea.

In a recent botanical assessment of the CodonCode Aligner v.3.0.1 was used to Thomasian Angiosperm Phylogeny & assemble and manually edit the forward and Barcoding Group (TAPBG) at Mt. Apo, Brgy. reverse sequences. Subsequently, the Ilomavis, Kidapawan City, the flowering and sequences were assembled using Seaview v4.5.2 fruiting branches of M. apoensis were spotted for alignment and the excision of unnecessary from low to mid elevation of the mountain. The bases. Additional DNA sequences were TAPBG took the opportunity to study the plant retrieved from Genbank (http:// to: 1) confirm its generic affiliation inferred from www.ncbi.nlm.nih.gov/) representing members two chloroplast makers (rps16 intron and trnL- of the tribe Argostemmateae and species from F region); 2) resolve whether M. apoensis is closely related tribes such as Anthospermeae, conspecific with M. cauliflora; and, 3) provide Danaideae, Dunnieae, Knoxieae, Paederiae, comprehensive descriptions, botanical Putorieae, Rubieae, Spermacoceae, and illustration as well as the conservation status of Theligoneae [18]. Colletoecema and Luculia M. apoensis. were used as outgroups for character polarity. A Bayesian analysis of the aligned sequences MATERIALS AND METHODS was conducted using the software Mr. Bayes v3.2.2 [19]. The best performing evolutionary Two samples of M. apoensis (coded as 14-505 model was determined using MrModelTest v.2.3 and 14-510) were collected at Mt. Apo National [20] under three model selection criteria: a) Park, through the Kidapawan-Magpet trail Akaike Information Criterion (AIC) [21]; b) AICc running through the Ilomavis Campsite (second order criterion of AIC); and, c) the (1,800 masl) and the Ko’ong Campsite Bayesian Information Criterion (BIC) [22]. (2,000 masl). Field photographs of the collected Bayesian analysis was performed with a sample plants were taken. Leaf samples were placed in frequency of 1000, 4 parallel chains and 10 bags containing silica gel for DNA analysis [11]. million generations. Vegetative and reproductive branches were likewise collected for herbarium specimens. RESULTS AND DISCUSSION Preservation of reproductive parts was done by placing the parts in a plastic tube filled with Sequence characteristics and variation. The commercial 70% ethyl alcohol. combined (rps16 intron and trnL-F region) analysis included 38 sequences. Four new Silica-dried leaf samples were subjected to DNA sequences of M. apoensis from the two extraction following the protocol of Qiagen molecular markers are newly published here. DNeasy Plant Mini Kit (Qiagen, Germany). The Matrix lengths of the two markers are 1,390 base rps16 intron was amplified and sequenced using pairs (bp) for the trnL-F marker and 1205 bp for rps16-1F/rps16-2R [12] while the trnL-F region the rps16 intron. Although the rps16 intron data was done using the primer pair c/f [13, 14]. PCR set has the shorter matrix length, it yielded the reactions were performed on a Biometra T- highest number of informative characters (216 Gradient in volumes of 25 mL following the PCR bp) compared to the trnL-F region (206 bp). The parameters and mixture of Alejandro et al. [15– aligned combined data set consisted of 2,596 bp

2 Molecular confirmation and taxonomy of the Rubiaceous

and a total of 422 informative characters. Argostemma with high support (PP = 1.0). Both Alignment was without difficulty due to low Neohymenopagon and Mouretia possessed genetic variation across the two cpDNA regions. persistent calyx lobes on the fruit that is also common in Argostemma and Mycetia [18]. Phylogenetic position of Mycetia apoensis. The two sampled M. apoensis nested in a subgroup Our combined tree (Fig. 1) suggests that M. together with M. cauliflora. These two Mycetia apoensis is closely related to M. cauliflora with species are sister to another subgroup high support (PP = 1.0). Sequence variation containing M. gracilis, M. javanica, and M. between M. apoensis and M. cauliflora is 7.70% malayana (Fig. 1). All five included Mycetia for the trnL-F region but 0.00% for the rps16 species formed a monophyletic clade with region. The relatively high divergence in the strong support (PP = 1.0) (Fig. 1). This conforms trnL-F region indicates that the two species are with the transfer of Adenosacme apoensis made not conspecific in contrast to Elmer [10]. Based by Davis et al. [9]. Similar to the findings of on morphology, M. apoensis is distinct from M. Rydin et al. [18], our combined tree suggests cauliflora in having more slender and somewhat that Neohymenopogon Bennet and Mouretia longer calyx teeth [23], longer petiole and calyx, Pit. are closely related with the tribe smooth yellowish gray bark, scurfy leaf, petiole Argostemmateae along with Mycetia and and peduncle surfaces, 9–12 lateral nerve pairs,

Colletoecema dewevrei Outgroup Luculia grandifolia Carpacoce sp. 0.99 1.00 Anthospermum herbaceum 1.00 Phyllis nobla Anthospermeae Opercularia vaginata 1.00 Leptopdermis potaninii 1.00 Serissa foetida Paederieae Spermadictyon suaveolens 0.99 Valantia hispida 1.00 1.00 1.00 Sharardia arvensis 1.00 Rubieae Galium album 1.00 Kelloggia galioides 0.89 - Putorieae 0.53 Plocama pendula - Theligoneae Theligonum cynocrambe - Paederieae Saprosma fruticosa Mouretia larsenii 1.00 Argostemma gracile 0.97 Argostemma brachyantherum 1.00 0.62 1.00 Mycetia cauliflora 1.00 Mycetia apoensis 14-505 1.00 Mycetia apoensis 14-510 Argostemmateae 0.86 0.87 Mycetia gracilis 1.00 Mycetia javanica Mycetia malayana Neohymenopogon parasiticus 1.00 Dunnia sinensis - Dunnieae 0.76 Placopoda virgata 1.00 1.00 Trainolepsis mandrarensis Paratriaina xerophila Knoxieae Parapentas silvatica 1.00 Danais fragrans 1.00 Danais comorensis Danaideae Schismatoclada farahimpensis 1.00 Kohautia caespitosa 1.00 Manostachya ternifolia Spermacoceae 1.00 Dentella repens Pentodon pentandrus Figure 1. Majority rule consensus tree inferred from the combined rps16 and trnL-F sequence data. Numbers above nodes are Bayesian posterior probabilities. The highlighted text indicates Mycetia apoensis.

3 Villanueva JCC et al.Acta Manilana 64 (2016) a style which is short tomentose and glabrous Taxonomy of Mycetia apoensis. This section towards the base and a subglobose fruit shape. provides the comprehensive vegetative and Comparative morphology of the two species is reproductive morphology as well as the presented in Table 1.

Table 1. Morphological comparison of M. cauliflora and M. apoensis (The significant differences are in bold fonts.)

Mycetia cauliflora Mycetia apoensis Habit Shrub Shrub Height 1–2 m 1–2 m Stem Thickness Finger thick 3.9–7.5 mm Color White Covered with a yellowish gray bark Stipule Texture Submembranous Submembranous Shape Lanceolate-ovate Triangularly acuminate to lanceolate Length 2.5–6 mm long 5.0–8.0 mm long, Surface Subglabrous Subglabrous Petiole Length 2–10 mm long 5.0–20 mm long Leaf blade Shape Lanceolate or oblanceolate Broadly and more or less oblanceolate Venation Reticulate Reticulate Length 125–160 mm 90–200 mm Width 35–70 mm 30–50 mm Apex Sharply pointed Acuminate Base Gradually tapered Attenuate Surface Glabrous, puberulous Glabrous, dirty brown scurfy on the nerves beneath Lateral nerve pairs 10–18 pairs 9–12 pairs Inflorescence Type Thyrses Loose cymose panicle Pedicel Length 15 mm 17–21 mm Calyx Shape Turbinate Elliptic, the base much constricted Length 5 mm long 7.0–8.0 mm long Lobes number 5 5 Lobes length 1–2 mm long 3.0–3.5 mm long Corolla Color Yellow Bright Yellow Shape Funnel-tubular Tubular Length 10 mm long 12–13 mm long Surface adaxial (above) Glabrous outside, rough inside Glabrous except the strigose hairs in the middle portion of the tube Anther Length/Width 1.5 mm long 2 mm long Style Length In long-styled form: 9–10 mm long 4–8 (–11) mm long In short-styled form: 2 mm long Surface Puberulous Glabrous towards the base, short tomentose Fruits Presence of calyx lobes Crowned by the calyx lobes Crowned by the calyx lobes Shape Oblong Subglobose Length 10–15 mm long 5–12.71 mm Width 8–10 mm wide 5–12.45 mm

4 Molecular confirmation and taxonomy of the Rubiaceous

Figure 2. Mycetia apoensis. (A) Habit, (B) fruits, and (C) flower. Scale bars in A, B, and C indicate 1 cm. Photos taken by Villanueva JCC and Alejandro GJD.

distribution, habitat and conservation status of M. apoensis.

Mycetia apoensis (Elmer) Govaerts, Fig. 2 and Fig. 3 Figure 3. Mycetia apoensis. (Elmer) Govaerts. (A) Type: flowering branch, (B) Inflorescence Philippines, Mindanao, District of Davao, (C) infructescence, (D) open corolla showing Todaya (Mt. Apo), v.1909, Elmer 10504 (K!). anther and style, (E) longitudinal section of fruit, and (F) cross section of ovary. From Lax shrub, 1–2 m tall; stem, 3.9–7.5 mm thick, Villanueva et al. 14-505 & 14-510 (USTH). terete, subglabrous and covered with a smooth Drawn by Diego N. yellowish gray bark; branches are sparingly rebranched and reclinate. Leaves thin, paler a whorl of persistent, dry, straw-colored, 2.0– green beneath, mainly horizontal, flat or only 3.0 mm long triangular bracts, bearing 1 or 2 the tips recurved, opposite, glabrous or dirty similar whorls, and about 11–14 mm long, brown scurfy, membranous or thinly pedicels similar in vestiture, very slender, 17– chartaceous, drying green, broadly or more or 21 mm long, subtended by similar bracts that less oblanceolate, entire margins, 9.0–20 cm long are chiefly in whorls of three’s, the larger ones and 3.0–5.0 cm wide just above the middle apex occasionally branched; calyx 7–8 mm long, 2.7– acuminate, base attenuate; nerves more 3.2 mm wide, elliptic, the base is much prominent beneath, 9–12 on each side of the constricted, rough puberulent or finely prominent midvein, reticulate crossbars quite scabrous, abruptly divided into five very thin, evident; petiole slender, sub-glabrous or 3.0–3.5 mm long calyx lobes which are triangular minutely scurfy brown, 0.5–2.0 cm in length, at the base and very narrowly lanceolate at the stipules submembranous and subglabrous, 5.0– apex; corolla bright yellow, 12–13 mm long, 8.0 mm long, 1.8–2.5 mm wide, entire, brown tubular, tube strigose adaxially, subglabrous when dry, triangularly acuminate to lanceolate. abaxially, 9–10 mm long; lobes 5 triangularly Infloresence a loose cymose panicle, oblong, glabrous adaxially and subglabrouse descending usually in the leaf axis, once or twice abaxially, valvate; stamens 5, just below the rebranced, 3.5–5.0 cm long and wide; peduncle throat; filament glabrous, 0.5–0.6 mm long, subcompressed, dirty brown scurfy, arising from adnate to the corolla; anthers oblong, 2.0 mm

5 Villanueva JCC et al.Acta Manilana 64 (2016)

long, dorsifixed, acute at apex, style slightly Health, Research and Development for the exceeding the corolla, 9–10 mm and two-forked research funding. at apex although occasionally easily separating clear to the narrowed base, glabrous toward the REFERENCES base, short tomentose otherwise; fruit [1] Gideon O, Tjitrosoedirdjo S, & Veldkamp J. Notes subglobose 5–12.71 mm long 5–12.45 mm wide, on Mycetia. Floribunda 1988; 1:17–20. slightly scabrous, two-celled, juicy, snow-berry [2] Wu ZY, Raven PH, Hong DY (Eds.). Flora of white, sunken at apex and surmounted by the China, Vol. 19 (Cucurbitaceae through five persistent calyx teeth; seeds 0.5 mm across, Valerianaceae, with Annonaceae and dark brown, angularly flattened, very numerous Berberidaceae), pp. 242–247. (St. Louis: Science in two dense masses. Press [Beijing] and Missouri Botanical Garden Press, 2011). [3] Schumann K. Rubiaceae, Die Natürlichen Distribution and habitat: Restricted to Mt. Apo Pflanzenfamilien. In: Engler A & Prantl K (Eds.) National Park, Davao and Mt. Hibok Hibok, Ergänzungshef (Vol. 4), pp. 1–156. (Leipzig: Camiguin; from 1200–1300 masl in a very moist Engelmann, 1891). densely forested flats. [4] Verdcourt B. Remarks on the classification of the Rubiaceae. Bulletin du Jardin Botanique de Conservation status of Mycetia apoensis. This l’État 1958; 28:209–290. species is restricted to Mt. Apo National Park at [5] Robbrecht E. Supplement to the 1988 outline of 1200–1300 masl and Mt. Hibok Hibok, Camiguin. the classification of the Rubiaceae, index to the genera. In: Robbrecht E (Ed.) Advances in It was also reported that the same species was Rubiaceae Macrosystematics. Opera Botanica collected in Mindoro [10]. Based on the IUCN Belgica 1994; 6:173–196. Red List Categories and Criteria [24], M. [6] Alejandro GJD & Liede S. The Philippine apoensis is categorized as Vulnerable [VU Rubiaceae genera: Updated synopsis in intkey B2ab(i)]; B2, area of occupancy estimated to be databases of the delta system. Blumea 2003; less than 2,000 km2 (area of Mt. Apo: 550 km2 48:261–277. [7] Bremer B. Phylogenetic studies within Rubiaceae and area of Mt. Hibok Hibok: 238 km2); a, and relationships to other families based on severely fragmented or known to exist at no more molecular data. Opera Botanica Belgica 1996; than 10 locations (M. apoensis: known to exist 7:33–50. at two locations); b(i), continuing decline, [8] Pelser P, Barcelona J, & Nickrent D (Eds.). Co’s observed, inferred or projected, in extent of Digital Flora of the Philippines, 2011. Retrieved occurrence (M. apoensis: a decrease in the extent from www.philippineplants.org on 16 July 2016. [9] Davis A, Govaerts R, & Rusham M. Nomenclatural of occurrence of this species is inferred due to changes in preparation for a World Rubiaceae the lack of recent records indicating its presence Checklist. Botanical Journal of the Linnean in Mindoro). Society 2008; 157:115–124. [10] Elmer A. Adenosacme. Leaflets of Philippine CONCLUSION Botany 1911; 3:1001–1003. [11] Chase MW & Hills HH. Silica gel: An ideal material Molecular and morphological analyses confirm for preservation of leaf samples for DNA studies. the identity and distinctness of M. apoensis Taxon 1991; 40:215–220. over M. cauliflora. [12] Oxelman B, Lide’N, M, & Berglund D. Chloroplast rps16 intron phylogeny of the tribe Sileneae (Caryophyllaceae). Plant Systematics and ACKNOWLEDGMENT Evolution 1997; 184:393–410. We thank the UST Research Center for Natural [13] Taberlet P, Gielly L, Pautou G, & Bouvet J. Universal primers for amplification of three non- and Applied Sciences for the use of facilities. coding regions of chloroplast DNA. Plant GJDA thanks the DOST-Philippine Council for Molecular Biology 1991; 17:1105–1109.

6 Molecular confirmation and taxonomy of the Rubiaceous

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7 Acta Manilana 64 (2016), pp. 9–16 Printed in the Philippines ISSN: 0065–1370

GC-MS metabolite profiling of the hexane extract and antimicrobial characterization of the Philippine endemic Rubiaceae species Uncaria cordata var. circa, Psychotria luzoniensis, and Psydrax puberula

Sarleen G. Castro1, John Emmanuel V. Cid1, Winnie Adrianne S. Ibañez1, Grecebio Jonathan D. Alejandro1,2, & Mario A. Tan*1,2

1College of Science and 2Research Center for the Natural and Applied Sciences University of Santo Tomas, 1015 Manila, Philippines

Recent studies showed that the Rubiaceae family, the 4th largest family among dicotyledonous flowering plants, contains compounds that exhibit antimicrobial properties. This study aims to characterize the antimicrobial activity of the extracts and determine the chemical constituents of the hexane extract from endemic Philippine Rubiaceae species Uncaria cordata var. circa, Psychotria luzoniensis, and Psydrax puberula. The crude leaves extract of each plant species were subjected to solvent-solvent extraction to obtain the hexane, chloroform, and butanol extracts. The sub-extracts of each plant were subjected to antimicrobial micro-plate dilution assay. Extracts from the three plants exhibited promising MIC and MBC activities against Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 25923. Characterization of the hexane extracts using GC-MS showed that these plants contained compounds that exhibited biological activities. The results provided in this study have shown that the three endemic plants are new sources of biologically-active secondary metabolites.

Keywords: Rubiaceae, Psychotria, Uncaria, Psydrax, MIC, MBC, GC-MS

INTRODUCTION anthraquinones, giving rise to new nature- oriented drugs with anti-inflammatory, The Rubiaceae family comprises of 659 genera mutagenic, antiviral, analgesic, antibacterial, and and approximately 13,000 species, making it one antioxidant effects on vascular diseases [2]. of the largest in number among vascular flora Majority of the phytochemical and [1]. This plant family is known to produce pharmacological analyses have been done from bioactive metabolites with great the genera Uncaria, Psychotria, Hedyotis, pharmacological potential, such as iridoids, Ophiorrhiza, and Morinda [2]. The Philippine indole alkaloids, terpenoids, flavonoids, and Rubiaceae is currently composed of 80 genera, having one of the largest number of indigenous species, a number of endemic species, and five *To whom correspondence should be addressed [email protected] endemic genera (Antherostele Bremek.,

© 2016 UST Research Center for the Natural and Applied Sciences, Manila, Philippines Castro SG, Cid JEV, Ibañez WAS, Alejandro GJD, & Tan MAActa Manilana 64 (2016)

Greeniopsis Merr., Sulitia Merr., Villaria Rolfe, flavonoids and their glycosides, coumarins, and Kanapia Arriola & Alejandro) [2]. Three sterols, quinolic acid, phenolics, and tannins [9]. genera from the family of Rubiaceae are the focus of this study, namely Psydrax, Psychotria, We herein report the determination of the and Uncaria with their respective endemic secondary metabolites present in the hexane species namely Psydrax puberula, Psychotria extract of Psydrax puberula, Psychotria luzoniensis, and Uncaria cordata var. circa. luzoniensis, and Uncaria cordata var. circa with the aid of GC-MS, and to assess the anti- Psydrax is a genus found throughout the Old microbial properties of their crude and World tropics from Africa, throughout southern semi-crude extracts. and Southeast Asia, to Australia [3]. This genus is yet to be studied for their anecdotal METHODOLOGY therapeutic uses, except for Psydrax amplifolia, The plant materials. Fresh leaves P. puberula which was used against diarrhea [4]. Literature were collected from Mt. Mingan, Dingalan, search revealed a dearth on the phytochemical Aurora in March 2015. Voucher specimens were investigation on the Psydrax species. deposited at the UST-Herbarium, Research Cyanogenic glucosides, iridoid dimers, Center for the Natural and Applied Sciences, flavonoids, and triterpenoids [5] have been with Accession #15-528. previously identified from this genus [5]. Fresh leaves of U. cordata var. circa were Psychotria is one of the largest genera in the collected from Orani, Bataan in August 2015. A Psychotrieae tribe, and is well-known to be a voucher specimen was deposited at the UST rich source of the secondary metabolites. Among Herbarium with Accession #12261-62. these compounds, the presence of various types of alkaloids including monoterpene indole, Fresh leaves of P. luzoniensis were collected quinoline, isoquinoline, and benzoquinolizidine from Ilocos Norte in August 2015. A voucher type have been well-established [6]. Non- specimen was deposited at the UST Herbarium alkaloidal compounds such as terpenoids, with Accession #12259-60. steroids, phenolics, flavonoids, coumarins, cyclic peptides and aliphatic compounds have Extraction of the plant materials also been isolated from various Psychotria species [6]. The presence of 112 Psychotria Psydrax puberula: Air-dried, ground leaves species in the Philippines had been documented (2 kg) were percolated with a total volume of and all of them are endemic in the country [7]. 22.4 L of methanol for three consecutive days. The collected methanol extracts were The genus Uncaria contains approximately 34 concentrated under reduced pressure to afford species, which is distributed in tropical regions, the crude extract. A portion of the marc (160 g) such as Southeast Asia, Africa and Southeast was dissolved in 200 mL of distilled water and America. Mostly, the hooks of this species are partitioned with hexane (1100 mL). The collected applied to treat wounds and ulcers, fever, hexane layer was dried with anhydrous sodium asthma, rheumatism, hyperpyrexia, sulfate and concentrated under reduced pressure hypertension, headaches, gastrointestinal to afford the hexane extract (Pp-H, 20.0 g). illness, and bacterial and fungal infections [8]. Chemical studies on the different Uncaria Uncaria cordata var. circa: Air-dried, ground species have elaborated the presence of leaves (900 g) were extracted with 13 L of 1:1 alkaloids, terpenoids and their glycosides, MeOH:DCM for three consecutive days. A 45 g

10 GC-MS metabolite profiling of the hexane extract

of the crude extract was obtained after concentration of 2 mg/mL of the extracts of the concentration under reduced pressure. The three plants was used for GC-MS analysis. For crude extract was subjected to the same GC-MS, a concentration of about 2 mg/mL of partitioning procedure using hexane (700 mL) the extracts was analyzed using a Perkin-Elmer to afford the semi-crude extract (Uc-H, 0.030 g). gas chromatograph model Clarus 680 coupled to a Perkin Elmer mass spectrophotometer model Psychotria luzoniensis: Air-dried, ground leaves Clarus SQ8T, equipped with an HP5 column (20 (175 g) were subjected to exhaustive extraction m × 0.32 mm ID; 0.25 m) programmed from 50°C using 4 L of 1:1 DCM: MeOH. The crude extract (5 min) to 300°C at 50°C/min and 5 min hold. The was subjected to the same procedure for carrier gas was helium (1.0 mL/min); injection partitioning using hexane (900 mL) to afford the was set in the split mode (1/10). Injector semi-crude extract (Pl-H, 10 g). temperatures were 250°C. Mass spectral data was acquired in the scan mode in the 33450 m/ Antimicrobial assay. The minimum inhibitory z range. concentration (MIC) and minimum bactericidal concentration (MBC) of the different extracts RESULTS AND DISCUSSION were determined using micro plate dilution assay. Extracts were diluted with DMSO to a GC-MS analysis. GC-MS is one of the best concentration of 1 mg/mL, placed in microwells, techniques to identify the constituents of then serially diluted (1:2) into eight wells to a volatile matter, whether it may be long-chained or branched hydrocarbons, alcohols, esters, and final volume of 100 L for each test organism. Final concentrations of the extracts were 500, acids [10]. There are twelve identified compounds in hexane extract of P. puberula, 250, 125, 62.5, 31.25, 15.62, 7.81, 3.9 g/mL. The following bacteria were used for the assay: Pp-H (Table 1), four in P. luzoniensis, Pl-H (Table Staphylococcus aureus ATCC 25923, 2), and four in U. cordata var. circa, Uc-H (Table Escherichia coli ATCC 25922, Enterococcus 3). These components were compared with the faecalis ATCC 49212 and Pseudomonas NIST 11 MS Search 2.0 library, characterized and identified, along with their retention time and aeruginosa ATCC 27853. A 100 L of bacterial suspension (1.5 × 108 CFU/mL) was added to respective biological activity. The compounds each well and incubated at 37°C for 24 h. The identified have 98% similarity search. The concentration in the last well with no growth as structures of the compounds are presented in determined by the absence of turbidity after 24 h Fig. 1. was reported as the MIC. All wells with no growth were then sub-cultured into nutrient Compound 6 was found in the hexane extract of agar (NA) plates to determine the MBC. The P. puberula and U. cordata var. circa. lowest concentration of extract which did not Interestingly, the abundant compounds show bacterial growth in the NA plates after identified in the GC-MS are the steroids - 24 h was reported as the MBC. All setups were sitosterol (13.78%) and betulin (10.57%) for P. done in triplicate for each of the compounds. puberula, (3,22E)-Ergosta-5,22-dien-3-ol DMSO was used as the negative control and (12.98%) for P. luzoniensis, and (3,22E)- Ciprofloxacin as the positive control. Ergosta-5,22-dien-3-ol, acetate (10.05%) for U. cordata var. circa. Of the 20 identified GC-MS analysis. GC-MS technique was used compounds by GC-MS, 10 were found to have in this study to identify the components present biological activities (Fig. 1). Diverse significant in the extract. The analysis was carried out at biological activities were noted which include the Research Center for Natural Sciences, anti-inflammatory (compounds 3, 4, 5, 7, 10, 11), University of Santo Tomas, Manila. A anti-microbial (3), anti-tumor (7, 11, 15),

11 Castro SG, Cid JEV, Ibañez WAS, Alejandro GJD, & Tan MAActa Manilana 64 (2016)

Table 1. Chemical constituents identified in the hexane sub-extract of Psydrax puberula (PpH)

RT (min) Peak Area % Compound Biological Uses 14, 18-[4-methyl-3-oxo-(1-oxa-4-azabutane- 8.1 0.37 1,4-diyl),(5)-Pregnane-3,20--diol (1) Remedy for heart diseases, suppress cancer 8.5 1.13 Digitoxin (2) growth [11] Antimicrobial, diuretic, anti-inflammatory, 8.9 3.45 Ethyl isoallocholate (3) antiasthma [12] 9 2.34 Methyl palmitate (4) Anti-inflammatory [13] Anti-inflammatory [14], antioxidant, hypocholesterolemic, nematicidal, pesticidal, 9.17 1.78 Palmitic acid (5) hemolytic, antiandrogenic, hemolytic, 5-alpha reductase inhibitor [15] 2-[2-(2-(2-pentylcyclopropyl) 9.45 1.26 methylcyclopropyl)methyl] cyclopropyl

Cyclopropanebutanoate (6) 9.65 2.01 Oleic acid (7) Antitumor, anti-inflammatory [16] 9.75 2.21 Methyl-6-cis,9-cis,11-trans-octadecatrienoate (8) 10.45 1.03 -monoolein (9) 10.66 13.78 -sitosterol (10) Immunomodulator/Anti-inflammatory [17] Anti-cancer, anti-tumor, Anti HIV; Anti carcinomic; Anti feedant; Antiflu; Anti inflammatory, Antitumor; Antiviral; 11.46 10.67 Betulin (11) Aphidifuge, Cytotoxic; Hypolipemic; Prostaglandin-Synthesis-Inhibitor, Topoisomerase-Inhibitor [17] (2-(3-acetoxy-4,4,14-trimethylandrost-8-en-17- 12.28 2.34 protein tyrosine phosphatase inhibitor [18] yl)-propanoic acid (12)

Table 2. Chemical constituents identified in the hexane sub-extract of Psychotria luzoniensis (PlH)

RT (min) Peak Area % Compound name Bioactivity/Uses 1.11 0.89 2-butyne-1,4-diol (13) 8.99 2.46 Z,Z-3,15-octadecadien-1-ol acetate (14) Flavour, fungicide, pesticide, antioxidant, 9.39 1.25 (Z)- methyl ester 9-octadecenoic acid (15) anti carcinogen, peroxisome proliferator [19] 9.44 12.98 (3,22E)-Ergosta-5,22-dien-3-ol (16)

Table 3. Chemical constituents identified in the hexane sub-extract of Uncaria cordata var. circa (UcH)

RT (min) Peak Area % Compound name Bioactivity/Uses (11E)-10,13,13-Trimethyl-11-tetradecen-1-yl 8.9 2.34 acetate (17)

2-[2-(2-(2-pentylcyclopropyl) 9.04 1.76 methylcyclopropyl)methyl] cyclopropyl Cyclopropanebutanoate (6)

9-octadecenoic acid (Z)-, 2-hydroxy-1- 9.37 1.27 (hydroxymethyl)ethyl ester (18) antitumor, cytotoxic, rheumatoid arthritis 9.42 10.05 (3,22E)-Ergosta-5,22-dien-3-ol, acetate (19) and immune promoter [20]

12 GC-MS metabolite profiling of the hexane extract

O

O

OH OH OH

O O HO

OH

O O O

OH HO (2) (11)

OH O

OH O O

O

O (3) (12)

HO OH O

O

(4) O O

OH O

(5) (15) O

OH (7)

O

(10) HO O (19) Figure 1. Chemical structures of bioactive compounds identified by GC-MS.

cytotoxicity (2, 11, 15, 19), anti-oxidant (5, 15), micro-plate dilution technique. Table 4 shows and enzyme inhibitor (5, 11, 12). The bioactive the results of the extracts and using DMSO as compounds are sterols (2, 3, 10, 19), terpenes negative control and Ciprofloxacin as the (11, 12) or long-chain carboxylic acids or esters positive control. DMSO showed no activity (4, 5, 7, 15). (>500 g/mL) while ciprofloxacin exhibited 3.91 g/mL activity. The other compounds identified in the GC-MS include long-chain hydrocarbon esters (6, 8, 9, Results indicate that the extracts of P. puberula 14, 17, 18), steroid (16), alkyne alcohol (13), showed antimicrobial activity against all the and an alkaloid (1). (Fig. 2). organisms. Over-all, P. puberula extracts displayed an inclination of antimicrobial activity Antimicrobial assay. The antimicrobial activity towards the Gram-positive bacteria (S. aureus). utilizing the MIC and MBC values of the The extracts of U. cordata var. circa also showed different extracts were determined using the promising activity (MIC) against the three

13 Castro SG, Cid JEV, Ibañez WAS, Alejandro GJD, & Tan MAActa Manilana 64 (2016)

Table 4. MIC and MBC of the crude and sub-extracts

Microorganism Extracts* MIC μg/mL MBC μg/mL Pp-CR 125 250 Pp-H 250 250 Pp-B 125 250 Pp-C 250 250 Uc-H 31.25 125 Escherichia coli ATCC 25922 Uc-C 31.25 125 Uc-B 31.25 >500 Pl-H >500 >500 Pl-C >500 >500 Pl-B 62.5 125 Pp-CR 62.5 125 Pp-H 125 125 Pp-B 125 125 Pp-C 125 125 Uc-H 62.50 125 Pseudomonas aeruginosa ATCC 27853 Uc-C 62.50 125 Uc-B 62.50 >500 Pl-H >500 >500 Pl-C >500 >500 Pl-B 62.50 125 Pp-CR 31.25 125 PpH 31.25 62.5 PpB 31.25 250 PpC 62.50 250 Uc-H 31.25 125 Staphylococcus aureus ATCC 25923 Uc-C 31.25 62.50 Uc-B 31.25 >500 Pl-H >500 >500 Pl-C >500 >500 Pl-B 62.5 125 *Pp – Psydrax puberula, Pl – Psychotria luzoniensis, Uc – Uncaria cordata var. circa, H – hexane, C – Choloform, B – n-butanol, CR – Crude extract

O O

O

O OH HO

(13) N

O (1)

O

OH O O

OH O O (9) (6)

O O

O O

(8) (14)

14 GC-MS metabolite profiling of the hexane extract

O

O

(17)

OH H O

OH H H O

(16) (18) HO Figure 2. Other compounds identified from the GC-MS

bacteria. MBC values of U. cordata var. circa that all three plants may contain compounds showed, however, that the butanol extract did with interesting activities and could be a new not exhibit good activity (>500 g/mL) as source of biologically-active materials. compared with the hexane and chloroform extracts. The observed antimicrobial activity of ACKNOWLEDGMENT U. cordata var. circa is consistent with the other The Philippine Council for Health Research and species of the genus Uncaria [2]. Analysis of Development is gratefully acknowledged for the the antimicrobial activity of the P. luzoniensis financial grant. extracts may indicate that the compounds responsible are polar in nature. This was in REFERENCES accordance with the observed MIC and MBC values of both the hexane and chloroform [1] Alejandro GD & Liede S. The Philippine Rubiaceae extracts (>500 g/mL) versus the butanol extract. genera: updated synopsis in INTKEY databases Based on the identified metabolites by GC-MS, of the DELTA system. Blumea 2003; 48(2): 261– only ethyl isoallocholate (3) have a reported anti- 277. [2] Martins D & Nunez CV. Secondary Metabolites microbial activity. from Rubiaceae Species. Molecules 2015; 20(7):13422–13495. The antimicrobial results obtained from the three [3] Davis AP, Govaerts R, & Bridson DM. New Philippine endemic Rubiaceae species warrant combinations in Madacascan Vanguerieae phytochemical investigation into the (Rubiaceae) for the genera Psydrax, Pyrostria, compounds responsible for the activity. and Rytigynia. Blumea 2007; 52(1):139–145. [4] Laurente OS, de Guzman GQ, Bangcaya PS, & CONCLUSION Alejandro GJD. Inventory and Assessment of Flowering Plants in Mt. Kingfisher Park, Coron, The Rubiaceae family continuously proves Palawan, Philippines. Journal of Science 2014; itself to be rich in secondary metabolites with 4(8):512–516. [5] (a) Rochenback J, Nahrstedt A, & Wray V. great pharmacological activity. In this study, 20 Phytochemistry 1992; 31:567–570; (b) Joubouhi components were characterized from the hexane C, Mabou F, Tebou P, Ngnokam D, Harakat D, & extracts of the endemic Rubiaceae P. puberula, Nazabadioko L. Phytochemistry Letters 2015; P. luzoniensis, and U. cordata var. circa. It has 13:348–354. also been proven that these three plants have antimicrobial properties effective against both gram-positive and gram-negative bacteria. Through GC-MS analysis, it was also discovered

15 Castro SG, Cid JEV, Ibañez WAS, Alejandro GJD, & Tan MAActa Manilana 64 (2016)

[6] (a) Calixto NO, Pinto MEF, Ramalho SD, Burger [13] Saeed N, El-Demerdash E, Abdel-Rahman H, MCM, Bobey AF, Young MCM, Bolzani VS, & Pinto Algandaby M, Al-Abbasi F, & Abdel-Naim A. Anti- AC. The Genus Psychotria: Phytochemistry, inflammatory activity of methyl palmitate and ethyl Chemotaxonomy, Ethnopharmacology and palmitate in different experimental rat models. Biological Properties. Journal of the Brazilian Toxicology and Applied Pharmacology 2012; Chemical Society 2016; 27(8):1355–1378; (b) 264:84–93. Yang H, Zhang H, Yang C, & Chen Y. Chemical [14] Aparna V, Dileep KV, Mandal PK, Karthe Constituents of Plants from the P, Sadasivan C, & Haridas M. Anti-Inflammatory Genus Psychotria. Chemistry & Biodiversity Property of n-Hexadecanoic Acid: Structural 2016; 13(7):807–820. Evidence and Kinetic Assessment Chemical [7] Davis AP, Govaerts R, Bridson DM, Ruhsam M, Biology & Drug Design 2012; 80(3):434–439. Moat J, & Brummitt NA. A Global Assessment of [15] Patil A & Jadhav V. GC-MS Analysis of Bioactive Distribution, Diversity, Enedmism, and Taxonomic Components from Methanol Leaf Extract Effort in the Rubiaceae. Annals of the Missouri of Toddalia asiatica (L.). International Journal Botanical Garden 2009; 96(1):68–78. of Pharmaceutical Sciences Review and [8] Zhang Q, Zhao J, Xu J, Feng F, & Qu W. Medicinal Research 2014; 29:18–20. uses, phytochemistry and pharmacology of the [16] Parthipan B, Suky MGT, & Mohan VR. GC-MS genus Uncaria. Journal of Ethnopharmacology Analysis of Phytocomponents in Pleiospermium 2015; 173:48–80. alatum (Wall. ex Wight & Arn.) Swingle, [9] (a) Heitzman M, Neto C, Winiarz E, Vaisberg A, & (Rutaceae). Journal of Pharmacognosy and Hammond G. Phytochemistry 2015; 4:216–222. Ethnobotany, phytochemistry and pharmacology of [17] Nagalakashmi M & Murthy K. Phytochemical Uncaria (Rubiaceae). Phytochemistry 2005; Profile of Crude Seed Oil of Wrightia 66:5–29; (b) Ndagijimana A, Wang X, Pan G, tinctoria R.BR. and Wrightia arborea (DENNST.) Zhang F, Feng H, & Olaleye O. A review on indole MABB. by GC-MS. International Journal of alkaloids isolated from Uncaria rhyncho- Pharmaceutical Sciences Review and Research phylla and their pharmacological studies. 2015; 31:46–51. Fitoterapia 2013; 86:35–47. [18] Venkatachalam M, Singaravelu G, Govindaraju [10] Albinjose J, Jasmine E, Selvankumar T, & K, & Ahn, JS. PTP 1B inhibitory action of a Srinivasakumar KP. Bioactive Compounds of phytochemical propanoic acid, 2-(3-acetoxy- Tinospora Cordifolia by Gas Chromatography- 4,4,14-trimethylandrost-8-en-17-yl). Current Mass Spectrometry (GC-MS). International Science 2013; 105(6):827–831. Journal of Multidisciplinary Research and [19] Rajeswari R & Rani S. GC-MS Analysis of Development 2015; 2(1):88–97. Phytochemical Compounds in the Ethanolic [11] Einbond LS, Wu H, Sandu C, Ford M, Mighty J, Extract of Root of Lawsonia Inermis Linn. . Antonetti V, Redenti S, & Ma H. Digitoxin International Journal of ChemTech Research enhances the growth inhibitory effects of 2014; 7:389–399. thapsigargin and simvastatin on ER negative [20] Baraza LD, Joseph CC, Moshi MJ, & Nkunya MHH. human breast cancer cells. Fitoterapia 2016; Chemical constituents and biological activity of 109:146–154. three Tanzanian wild mushroom species. [12] Muthulakshmi A, Jothibai MR, & Mohan VR. Anti- Tanzania Journal of Science 2007; 33:1–7. inflammatory activity of methyl palmitate and ethyl palmitate in different experimental rat models. Journal of Applied Pharmaceutical Science 2012; 2(2):69–74.

16 Acta Manilana 64 (2016), pp. 17–23 Printed in the Philippines ISSN: 0065–1370

Comparison of cellular-based viability and apoptosis assays on doxorubicin treated colorectal adenocarcinoma cells

Thomas Adrian Tiongson1, Maria Charina Magsumbol1, Mark Kevin Devanadera1,2,3, & Myla R. Santiago*1,2,3

1Department of Biochemistry, Faculty of Pharmacy, 2Research Center for Natural and Applied Sciences, 3The Graduate School, University of Santo Tomas, 1015 Manila, Philippines

There are various assays that can be performed to measure cell viability and apoptosis, which is why in this study, comparison of some common assays, MTT and resazurin reduction assays for cell viability and, Flow cytometry and caspase 3/7 for cell apoptosis, were performed in Doxorubicin (DOX) treated colorectal adenocarcinoma cells. These cell- based assays were analyzed in order to determine which of the paired methods are more efficient in measuring cell viability and apoptosis at various concentrations of DOX and varied incubation times. Results showed that incubation time has no effect on both cell viability assays (MTT and resazurin). Resazurin was observed to be more sensitive than MTT in measuring cell viability while MTT is more specific in determining cytotoxicity. In terms of cell apoptosis, caspase 3/7 is more specific and sensitive because of its enzyme substrate reaction. However, annexin V was more sensitive than caspase 3/7 in detecting early apoptosis, late apoptosis and discrimination of necrotic cell from apoptotic cell in DOX-treated colorectal adenocarcinoma cells. Depending on application, cellular based assays for both viability and apoptosis have to be routinely standardized as different factors may affect its result such as culture mediums, buffers, cell density, pH, incubation temperature, humidity, chemical constituents, drug dosage, and incubation time.

Keywords: MTT, resazurin, flow cytometry, caspase 3/7, luminescence assay

INTRODUCTION of cell proliferation in a population. It is useful in the assessment of the potency of cytotoxic Cell viability and cytotoxicity assays are being drugs, such as anticancer agents. often used in cytotoxicity test of chemicals being Measurements of cell viability can be used to acted upon the cell culture. correlate cell number and cell behavior [1]. There are various techniques used for the detection Cell viability assays measure the number of of cell viability, based on many cell functions healthy cells in a sample and indicate the extent including, cell adherence, enzyme activity, permeability of the cell membrane, etc. Among *To whom correspondence should be addressed [email protected] the commonly used procedures are the MTT

© 2016 UST Research Center for the Natural and Applied Sciences, Manila, Philippines Tiongson TA, Magsumbol MC, Devanadera MK, & Santiago MRActa Manilana 64 (2016) assay and the resazurin (Alamar blue) assay. Doxorubicin, also known as doxorubicin These methods are based on the change in the hydrochloride, is a chemotherapeutic drug used color of the reagent dye due to mitochondrial in treatment of numerous cancers, like breast, enzymes excreted by living cells [8–11]. ovarian, bladder, etc. [4]. It inhibits the growth of solid tumors. This drug belongs to a class of Cell apoptosis is best characterized as a medications called anthracycline antibiotics morphological distinct form of cell death. made from the fungus Streptomycin. In Apoptosis, ever since, has been recognized as chemotherapy, one of the most widely-used a distinctive mode of “programmed” cell death, drugs for the treatment of colorectal cancer is in which elimination of cells is genetically DOX. determined. This event occurs normally as a homeostatic mechanism during development EXPERIMENTAL and aging for the sole purpose of maintenance of cell populations in tissues. Also, apoptosis Cell culture. Colorectal adenocarcinoma cells can occur as a defense mechanism such as in were donated by Globetek Science Foundation immune reactions and when a disease damages and were cultured on a tissue culture flask using the cells [3]. Several methodologies have been Minimum Essential Media (MEM) with Fetal applied for the detection of apoptosis based on Bovine Serum (FBS) and antibiotic-antimycotic. the various events accompanying programmed The flask containing the cells were incubated at cell death. Among the common techniques are 37°C humidified incubator with 5% CO2. The cells caspase analysis which detects the release of were viewed in a phase contrast microscope and active caspase enzymes through a luminescence observed for 90% confluence and complete method and flow cytometry analysis based on attachment of cells prior to assay. the detection of alteration in the membrane of Sample treatment. Cells from the flask were apoptotic cells through the staining dye annex trypsinized using trypsin-EDTA solution and the in V. detached cells were centrifuged. Cell pellets were In this study, two cell viability assays (MTT collected and mixed with supplemented MEM. and resazurin) were paired and two apoptosis A cell suspension in MEM was mixed with trypan assays (flow cytometry and caspase 3/7 blue and the mixture was placed in a luminescence) were paired to compare the hemacytometer for cell counting. Prior to seeding sensitivity and specificity of these methods in all of samples were done in triplicates. The measuring cell viability and apoptosis in counted cells were diluted, seeded into a 96- colorectal adenocarcinoma cells treated with well plate, and incubated for 24 h prior to assay. doxorubicin (DOX). These cell-based assays After seeding, the cells were treated with were analyzed in order to determine which of Doxorubicin HCl (DOX) with 50 g/mL, 25 g/ the methods are more efficient in measuring cell mL, 12.5 g/mL, and 6.25 g/mL and were viability and apoptosis at various incubated for 24 h prior to assay. concentrations of DOX and varied incubation MTT assay. The cells treated with DOX at times. Sensitivity and tumor-resistance against different concentrations and untreated cells were cytotoxic drug (DOX) is an important factor on mixed with 5 mg/mL MTT solution (3-(4,5- determining the effectivity of a certain cytotoxic dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium drug and its effect on a certain cell line (colorectal bromide) [1]. The wells treated with MTT dye adenocarcinoma). were incubated for 4 h and sterilized DMSO was introduced to dissolve formazan crystals and

18 Comparison of cellular-based viability and apoptosis assays

mixed well. Absorbance was measured at 570 nm colorectal cancer cells was done based on the for MTT using a Corona Microplate reader SH- manufacturer’s protocol (Promega, USA). 1000 (Hitachi, Japan) [9]. After 24 h of incubation Caspase-Glo™ 3/7 Reagent was mixed with equal at room temperature, the absorbance of the 96- volume of Caspase-Glo™ 3/7 Buffer. Blank well plate was measured and compared with sample which contain the reagent and the cell results with 4-hour incubation. culture medium, the negative control which constitute the reagent, cell culture medium with Resazurin reduction assay. Cells treated with cancer cells, and the test sample which DOX and untreated cells were mixed with composed of the reagent, cell culture medium 0.15 mg/mL concentration of resazurin that has with treated cancer cells. Caspase-Glo™ 3/7 was been dissolved in Dulbecco’s Phosphate Buffer mixed equally with the cell culture medium Saline (DPBS) with pH 7.4 and filter sterilized. containing the untreated and treated cancer The 96-well plate containing the cells and dye cells; it was mixed at 300 rpm for 30 s and was incubated for 4 and 24 h at 37°C in a incubated at room temperature for 3 h. After

humidified incubator with 5% CO2. [7]. incubation, the mixtures were read using Absorbance was read at 560 nm using a Corona Promega Luminometer and caspase activity or Microplate reader SH-1000 (Hitachi, Japan). apoptosis rate was measured in terms of Relative Light Unit (RLU). Flow Cytometry analysis using Annexin V. Muse™ Annexin V & Dead Cell Kit (Merck To compute for apoptotic rate of untreated cells, Millipore, Germany) was used for the apoptotic % apoptosis = (RLU of untreated / cell cell assay following the manufacturer’s protocol. concentration) × 100; while for cells treated with Flow cytometry is used to analyze the physical DOX, % apoptosis = ([RLU of treated cells – and chemical characteristics of particles in a fluid RLU of untreated cells] / RLU of treated cells) × as it passes through at least one laser. 100. Fluorescently labelled cell components are excited by the laser to emit light at varying RESULTS AND DISCUSSION wavelengths. A 24-h cultured treated and untreated cells were placed in a microcentrifuge Cellular-based viability assay. The colorectal tubes and mixed with Muse™ Annexin V & adenocarcinoma cells were viewed under a Dead Cell Reagent (Merck Millipore, Germany). phase contrast microscope before and after its The samples with the reagent were mixed, placed treatment with DOX. It was observed that the in dark, and incubated at room temperature for cancer cells had undergone lysis after the 20 min. After incubation, samples were treatment with the DOX (Fig. 1). Cellular lysis introduced to the flow cytometer (Muse™ Cell Analyzer, Merck Millipore, Germany). The data from the flow cytometer were gathered and analyzed.

Caspase 3/7 analysis using luminescence. Luminescence caspase assay is used to determine quantitatively cell apoptosis. Caspases helps stimulate the cascade of AB signaling events that activates cell death. Cell Figure 1. Micrograph analysis of (A) untreated health assay using Caspase Glo™ 3/7 for colorectal adenocarcinoma cells and (B) apoptosis screening of the drug on the doxorubicin treated colorectal adenocarcinoma cells.

19 Tiongson TA, Magsumbol MC, Devanadera MK, & Santiago MRActa Manilana 64 (2016)

was observed as small particles present on the reaction may be due to the sensitivity of the culture plate with orange coloration because of dye to react on viable cells and with that sudden the orange color absorb by the cell from the change on the percentage inhibition with respect DOX. Figure 1 only shows that DOX is very to the MTT assay, it can be noted that the toxic even at low concentration on colorectal resazurin dye is more sensitive to other factors adenocarcinoma cells. than the MTT dye.

In vitro cytotoxicity assay of colorectal Although resazurin reduction assay was adenocarcinoma cells was done by treating it observed to be more sensitive than MTT, the with various concentrations of DOX (6.25–50 g/ lowest concentration of DOX exhibited an mL) for 24 h. Inhibition of cell viability was irregular low inhibition rate (Fig. 2). According measured using MTT and resazurin reduction to some protocols, exposing cells to resazurin assay at 24 and 48 h incubation. As seen in for several hours or days changes the cell Fig. 2, each concentrations of DOX have morphology but does not exhibit toxic effect on inhibition greater than 50%. Therefore, the IC50 the cells suggesting interference with normal of DOX was noted to be less than 6.25 g/mL. cell function. The cell exposure to this dye possibly depletes reduced forms of nucleotides In addition, as DOX concentration increases, resulting in other effect rather than cytotoxic the inhibition of the drug against the viability of effects [7]. A comparison on the reaction of the colorectal adenocarcinoma cells increases metabolic indicators such as MTT and resazurin for both parameters of incubation time (Fig. 2). has been analyzed and it was noted that the Incubation time of 4 and 24 h resulted no MTT dye can be reduced by NADPH, FADH, significant difference (p>0.05) for both MTT FMNH, and NADH but not with cytochrome c and resazurin cytotoxicity assay (Table 1) using which is inversely proportional to the resazurin, paired t-test. This indicates that incubation time in which it reacts with other metabolic reaction has no effect on measuring of cell viability products including cytochromes to monitor all though both assays have relatively similar the reducing environment within a living system results, but on the lowest concentration, [12]. resazurin assay showed a sudden increase on percentage inhibition (Fig. 2). Sudden increase Statistical analysis of both colorimetric assay on the percent inhibition using colorimetric used paired t-test to analyze the differences between the two assay procedures. There are drastic differences between the assays in both 100 – 4h incubation the 4 h and 24 h incubation of MTT and with MTT 80 – assay resazurin assays shown in Fig. 2. It has been 24 h incubation noted that incubation time of each dye has a with MTT 60 – assay 4h incubation % Inhibition 40 – with Resazurin assay Table 1. Comparison of Paired t-Test 24 h 20 – incubation of MTT and Resazurin Assay with Resazurin assay Paired Samples Correlations 0 – 6.25 1.25 25 50 Correlation Sig. DOX Concentration ( g/mL)  Pair 1 MTT4 & MTT24 .974 .026 Figure 2. Comparison of cytotoxicity assay (MTT Pair 2 RES4 & RES24 .999 .001 vs. resazurin) of colorectal adenocarcinoma cells Pair 3 MTT4 & RES4 –.425 .575 against free DOX at different concentrations and Pair 4 MTT24 & RES24 –.204 .796 incubation times.

20 Comparison of cellular-based viability and apoptosis assays great correlation and significant within the group impairment reaction. However, MTT is more assay but are not correlated and are insignificant specific in determining cytotoxicity since it only between MTT assay and resazurin assay reacts on certain metabolic products and is not regardless of the incubation time as shown in affected by other reducing agents within the Table 1. living system unlike the resazurin dye which is affected by all the reducing agents present in In cytotoxicity assays, the following parameters the cell and the color is easily bleached out due have to be greatly considered such as assay to prolonged incubation time, environmental procedure, cell line to be tested and the end- pH, and temperature [8, 10, 12]. Also, resazurin point determination wherein the correlation may not accurately show cellular proliferation between measurements, limitation of the assay rates because of the non-linear correlation of procedure and cell viability are taken into the dye reduction and cell density which may considerations [8, 12, 14]. Assays to be used in be more useful in screening for cytotoxicity than cytotoxicity are very sensitive on cell type like assessing cell proliferation [12]. cell lines or primary cells, adherent or floating cells, and sometimes depending on the cell Cellular based apoptosis assay. For the morphology, cycle and metabolism. These apoptosis methods, flow cytometry using bioassays may be affected by different factors annexin V indicated both early and late apoptotic such as culture mediums, buffers, cell density, rates of the cell line (Fig. 3); while the caspase pH, micro-titer plate, incubation temperature, 3/7 assay indicated the total cell apoptosis humidity, chemical constituents, drug dosage, through Relative Light Unit (RLU) proportioned and incubation time [10, 12]. to its caspase activity (Fig. 4). Highest apoptotic rate for DOX-treated cells using flow cytometry Based on the results, both MTT assay and and caspase assay were observed at resazurin assay are sensitive in terms of assay concentrations 50 g/mL and 25 g/mL, procedure and its reaction to the metabolic respectively. It is assumed that the reading at

A C POPULATION PROFILE APOPTOSIS PROFILE POPULATION PROFILE APOPTOSIS PROFILE 4- 4- 4- 4- Dead Late Apop./Dead Dead Late Apop./Dead 0.05% 70.35% 2.10% 91.05% 3- 3- 3- 3-

2- 2- 2- 2- VIABILITY VIABILITY

CELL SIZE INDEX 1- 1- CELL SIZE INDEX 1- 1- 6.65% 22.95% 0.65% 6.20% Live Early Apop. Live Early Apop. 0 - 0 - 0 - 0 - 01234 01234 01234 01234 Live ANNEXIN V Apoptotic Live ANNEXIN V Apoptotic Live ANNEXIN V Apoptotic Live ANNEXIN V Apoptotic

B D POPULATION PROFILE APOPTOSIS PROFILE POPULATION PROFILE APOPTOSIS PROFILE 4- 4- 4- 4- Dead Late Apop./Dead Dead Late Apop./Dead 0.70% 94.30% 0.00% 95.35% 3- 3- 3- 3-

2- 2- 2- 2- VIABILITY VIABILITY

CELL SIZE INDEX 1- 1- CELL SIZE INDEX 1- 1- 0.25% 4.75% 0.20% 4.45% Live Early Apop. Live Early Apop. 0 - 0 - 0 - 0 - 01234 01234 01234 01234 Live ANNEXIN V Apoptotic Live ANNEXIN V Apoptotic Live ANNEXIN V Apoptotic Live ANNEXIN V Apoptotic Figure 3. Apoptosis profile of colorectal adenocarcinoma cells that were (A) untreated, (B) treated with 12.5 g/ mL DOX, (C) treated with 25 g/mL DOX, and (D) treated with 50 g/mL DOX.

21 Tiongson TA, Magsumbol MC, Devanadera MK, & Santiago MRActa Manilana 64 (2016)

120 – Table 2. Comparison of Paired t-Test of Annexin V Flow Cytometry Caspase Glo 3/7 Annexin V Flow Cytometry and Caspase 100 – Paired Samples Correlations 80 – Correlation Sig. Flow Cytometry Pair 1 .921 .079 60 – and Caspase

% Apoptosis 40 –

20 – detection of activity vary since the reaction is

0 – cell density dependent [12]. Untreated 1.25 25 50 DOX Concentration (g/mL) Figure 4. Apoptosis profile of colorectal Annexin V used to detect phospholipids adenocarcinoma cells treated with free DOX at specifically the phosphatidylserine that can be different concentrations. found on the cell membrane. Assay procedure using annexin V was very sensitive since it the highest concentration via caspase assay detects early apoptosis when the cell membrane shifted its apoptotic rate downward was because is slowly disintegrating, late apoptosis when the the cell could have started undergoing cell cell membrane is completely disintegrated, and death, causing it to decrease its caspase level discrimination of necrotic cell from apoptotic cell activity, hence, decreasing also its RLU or [13, 14]. From the results obtained, it is evident apoptotic rate. that flow cytometry was the more reliable method to use for the apoptotic profile as it As seen in Table 2, both caspase 3/7 and flow showed stages of cell apoptosis when treated cytometry are not significantly correlated in with DOX at different concentrations. terms of apoptotic rate. Caspase 3/7 assay is used as a method of detecting apoptotic activity REFERENCES because of the proteins role in apoptosis [1] Stoddart M. Cell Viability Assays: Introduction. pathway. Caspase-3 has been identified as one Methods in Molecular and Cellular Biology 2011; of the proteins that trigger apoptosis on the 740:1–2. upstream part of the cascade system while [2] Borra RC, Lotufo MA, Gagioti SM, Barros FDM, & caspase-7 was detected to be active in Andrade PM. A Simple Method to Measure Cell promoting apoptosis on the downstream part Viability in Proliferation and Cytotoxicity Assays. Brazilian Oral Research 2009; 23(3):255–262. of the cascade system. Both proteins were used [3] Elmore S. Apoptosis: A Review of Programmed to critically detect the occurring apoptosis on Cell Death. Toxicologic Pathology 2007; the cell system upon lysis has also seen in Fig. 35(4):495–516. 1. Using caspase 3 and 7 were very specific and [4] Goncalves M, Figueira P, Maciel D, Rodrigues J, sensitive because it uses enzyme-substrate Shi X, Tomas H, & Li Y. Antitumor Efficacy of reaction and liberated caspase in the solution Doxorubicin-loaded Laponite/Alginate Hybrid Hydrogels. Macromolecular Bioscience 2014; from the lyse cells were easily detected due to 14(1):110–120. the liberation of fluorescent substance. [5] Trebunova M, Laputkova G, Slaba E, Lacjakova However, limitations on the use of caspase 3/7 K, & Verebova A. Effects of Docetaxel, have to be noted such as the substrate was Doxorubicin and Cyclophosphamide on Human temperature sensitive; substrate should be Breast Cancer Cell Line MCF-7. Anticancer immediately used after reconstitution as it slowly Research 2012; 32:2849–2854. loses its activity if not preserved properly and

22 Comparison of cellular-based viability and apoptosis assays

[6] Manchun S, Dassc CR, Cheewatanakornkoola [11] O’Brien J, Wilson I, Orton T, & Pognan F. K, & Sriamornsaka P. Enhanced Anti-Tumor Effect Investigation of the Alamar Blue (Resazurin) of pH-responsive Rextrin Nanogels Delivering Fluorescent Dye for the Assessment of Doxorubicin on Colorectal Cancer. Carbohydrate Mammalian Cell Cytotoxicity. European Journal Polymers 2015; 126:222–230. of Biochemistry 2000; 267(17):5421–5426. [7] Riss TL, Moravec RA, Niles AL, Benink HA, [12] Rampersad SN. Multiple Applications of Alamar Worzella TJ, Minor L, Storts D, & Reid Y. Cell Blue as an Indicator of Metabolic Function and Viability Assays. In: Sittampalam G, Coussens N, Cellular Health in Cell Viability Bioassays. Sensors & Nelson H (Eds.) Assay Guidance Manual, pp. 2012; 12:12347–12360. 1–23. (2013). [13] Shutte B, Nuydens R, Geerts H, & Ramaekers F. [8] Perrot S, Dutertre-Catella H, Martin C, Warnet Annexin V Binding Assay as a Tool to Measure JM, & Rat P. A New Nondestructive Cytometric Apoptosis in Differentiated Neuronal Cells. Assay Based on Resazurin Metabolism and an Journal of Neuroscience Methods 1998; 86: 63– Organ Culture Model for Assessment of Corneal 69. Viability. Cytometery Part A. 2003; 55A:7–14. [14] Rieger AM, Nelson KL, Konowalchuck JD, & [9] Mosmann T. Rapid Colorimetric Assay for Cellular Barreda DR. Modified Annexin V/Propidium Iodide Growth and Survival: Application to Proliferation Apoptosis Assay for Accurate Assessment of and Cytotoxicity Assays. Journal of Cell Death. Journal of Visualized Experiments Immunological Methods 1983; 65:55–63. 2011; 50: e2597. [10] Wang P, Henning S, & Heber D. Limitations of MTT and MTS-Based Assays for Measurement of Antiproliferative Activity of Green Tea Polyphenols. PLoS ONE. 2010; 5(4):1–10.

23 Acta Manilana 64 (2016), pp. 25–32 Printed in the Philippines ISSN: 0065–1370

Fluorophore-labeled bioengineered glucose binding protein for measurement of transdermal glucose

Cristina Tiangco1,3, Sheniqua Brown1, 2 Fortunato Sevilla III3, Govind Rao1,2, & Leah Tolosa*1,2

1Center for Advanced Sensor Technology, 2Department of Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County, Baltimore, MD, 21250 3The Graduate School, University of Santo Tomas, 1015 Manila, Philippines

Measurement of glucose is an important component of care in the intensive care unit. Current methodologies for glucose monitoring include enzymatic-based laboratory analyzers, point of care testing and continuous glucose monitoring systems that all require breaking the skin. This study focuses on the development of a glucose biosensor to measure transdermal glucose (TG) by noninvasive passive diffusion. The H152C glucose binding protein (GBP) labeled with the polarity-sensitive dye BADAN (6-bromoacetyl-2- dimethylaminonaphthalene) was prepared and characterized with the aim of producing a biosensor with an operating range at micromolar levels compatible with TG concentrations. The BADAN-labeled GBP exhibited a large fluorescence intensity increase (228%) upon addition of a saturating concentration of glucose. The glucose binding constant was calculated

to be (Kd) 1.124 ± 0.2361 M. The biosensor has a linear operating range of 0.030–0.460 M, making it suitable for monitoring transdermal glucose. The use of the biosensor was demonstrated by measuring TG collected by passive diffusion of glucose through the skin of healthy adult subjects. Results showed that the H152C GBP labeled with BADAN is capable of measuring TG and can be used for noninvasive glucose sensing.

Keywords: glucose binding protein, biosensor, BADAN, transdermal glucose

INTRODUCTION negative bacteria such as E. coli. Unlike enzymes, GBP does not require other reactants Current FDA-approved blood glucose devices and co-factors. It exhibits high binding are based on biocatalytic elements such as sensitivity, specificity and affinity to glucose. glucose oxidase [1–5], glucose dehydrogenase The binding equilibrium is straightforward, [6] and hexokinase [7]. An alternative simple, reversible, and generates no reactive by- technology for glucose sensing employs products. Signal transduction arises from a bioaffinity elements such as the glucose binding change in conformation of the protein upon protein (GBP) [8–13]. GBP is a member of a binding with glucose, and could be quantified diverse group of transport proteins from Gram- through a polarity-sensitive fluorescent dye, *To whom correspondence should be addressed which can differentiate the conformations of [email protected] GBP.

© 2016 UST Research Center for the Natural and Applied Sciences, Manila, Philippines Tiangco C, Brown S, Sevilla F III, Rao G, & Tolosa LActa Manilana 64 (2016)

The binding constant (Kd) of GBP for glucose is Modified E. coli (BL21 DE3 strain) containing in the micromolar level, making GBP suitable for the plasmid (GBP H152C-pET303 CT His) was the detection of low glucose concentrations used as source of GBP. It was constructed in such as in transdermal glucose flux in permeated the laboratory by molecular cloning of E. coli skins. The capability of GBP to measure (BL21 DE3 strain). Pre-constructed mutant (GBP transdermal glucose has been demonstrated [14, H152C) was suspended in water and underwent 15]. Transdermal glucose (TG) measurement has transformation with BL 21 competent cells. gained interests due to its noninvasiveness in Clones were taken for the mutant and glycerol nature and its potential in medical diagnosis on stocks were made and inoculated. challenged patients. Preparation of H152C GBP mutant. The Efforts have been made to increase the binding H152C GBP was expressed, separated and constant to millimolar levels by studying purified as described previously [10, 11, 13] with different mutants of GBP to enable methods that some modifications. The GBP was can complement blood glucose levels [16, 17]. A overexpressed at the periplasmic space by GBP mutant (L255C) had been applied for growing the modified E. coli cells in LB media. measurement of TG [14, 15]. In this paper, a The cell growth was measured at the end of 3 h bioengineered GBP (H152C) was prepared and by determining its optical density at 600 nm using characterized as a recognition element for TG the spectrophotometer (Agilent technologies). measurement. This mutant has a polyhistidine At 0.4 optical density, expression of GBP was tag that allows for quicker purification and induced by adding 500 L of 1 M isopropyl-2- provides a handle for protein immobilization. D-thiogalactopyranoside (IPTG) and grown for 4–5 h at 37°C at 225 rpm agitation. EXPERIMENTAL Bacterial cells were lysed through sonication Materials. D-Glucose, NaH2PO4, Na2HPO4, for 10 min in a beaker containing ice using the NaH2PO4.H2O, N,N-dimethylformamide, KH2PO4 probe sonicator (Fisher Scientific Sonic and Coomassie Brilliant Blue solution were Dismembrator Model 500). Affinity acquired from Sigma-Aldrich (St. Louis, MO). chromatography was performed in two columns BADAN (6-bromoacetyl-2-dimethylamino- packed with 5 mL of equilibrated Ni-NTA naphthalene) was obtained from Molecular agarose resin (Thermo Scientific). Probes (Eugene, OR). Slide-A-lyzer dialysis casettes were obtained from Thermo Scientific Sodium dodecyl sulfate polyacrylamide gel (Rockford, IL). Sephadex G-25 fine were from GE electrophoresis (SDS-PAGE) was performed to Healthcare (Bio Sciences, AB). Luria-Bertani (LB) verify the size, presence and purity of GBP. The medium, ampicillin, isopropyl-2-D- concentration of the total protein of the isolated thiogalactopyranoside (IPTG), guanidium GBP was determined using Bradford Protein chloride (GuHCl), Tris-hydrochloride and urea Assay. The highly pure fractions were dialysed were from Fisher Scientific. LB medium using 20 mM phosphate (NaH2PO4 and supplemented by antibiotics (100 g/mL  Na2HPO4) buffer, pH 7.4 at 4°C. ampicillin) was used to allow growth of cells. His Pur Ni-NTA resin and tris(2-carboxyethyl) The GBP was labeled with environmentally phosphine (TCEP) were obtained from Thermo sensitive fluorophore, BADAN at the single Scientific (Rockford, IL). All chemicals were used cysteine mutation at position 152 following the without further purification. Plasmids were manufacturer’s recommended labeling created and purchased from Life Technologies. procedure. The fluorophore was covalently

26 Fluorophore-labeled bioengineered glucose binding protein

attached to the cysteine mutation and known to 20, 50, 100, and 200 M. Fluorescence intensities be responsive to glucose [11, 16]. Unreacted of GBP in the presence of glucose were plotted BADAN was removed by size exclusion against glucose concentration (M) to attain chromatography column, Sephadex G-25 fine the calibration curve. resin. The extent of labeling was determined from the GBP concentration and optical density of The mean fluorescence readings were calculated protein with dye. The following equation was from the four readings. Measurements are used for the calculation of the extent of labeling reported as normalized fluorescence intensity (1): calculated as: mole dye Ax MWprotein Extent of labelling = * 100 = * mg * 100 mole protein  protein conc ( mL )

( − ) where Ax is the absorbance value of the dye, BADAN at the absorption maximum where the F and F0 were the fluorescence wavelength,  is the molar extinction coefficient intensities of GBP in the presence and absence of the dye at the absorption maximum, which is of glucose, respectively. 20000 and MW is the molecular weight of protein, which is 33310 g/mol. The Varian Cary Eclipse fluorescence spectrophotometer was used to measure the The purified protein was dialysed in 100 mM fluorescence spectra. BADAN was excited at phosphate buffer, pH 7.5. The final product was 390 nm with a slit width of 5 nm. The emission 0.22 m filter-sterilized and stored at 4°C. spectra were measured in the range of 400– 650 nm with the maximum fluorescence emission

Fluorescence measurements. The fluorescence (Emmax) occurred approximately at 540 nm, using intensities of BADAN-labeled GBP in the the following conditions: high PMT detector absence and presence of D-glucose in 1 PBS voltage, average read time 0.10 s, excitation filter buffer were measured on a 96-well SpectraMax auto and emission filter open. plate reader (Molecular Devices, Sunnyvale, CA). BADAN was excited at 400 nm, with the Reusability (Reversibility) of the glucose binding protein. To check the possibility of maximum fluorescence emission (Emmax) at 550 nm. 200 L of GBP solution was added to BADAN-labeled GBP as a reversible biosensor, each of the designated wells. Glucose standards previously used GBP was recovered after the or samples (5.0 L) were then added to each of assay and studied for its reusability. To remove the wells. The plate was gently shaken for 5 s, the bound glucose, the used GBP solution was and the intensities were measured 4 times. All injected into a Slide-A-lyzer dialysis cassette, measurements were made following the same which was then dialyzed in 1× PBS buffer for 6 h instrumental conditions: excitation wavelength in a cold room. Afterwards, the 1× PBS buffer 400 nm, emission wavelength 550 nm, excitation was replaced with fresh buffer and the GBP was and emission slit width 5 nm, PMT detector dialyzed for another 6 h. The GBP solution was voltage 750 V, and average time 0.1 s. transferred from the dialysis cassette to a sterile glass vial and filtered-sterilized. The recovered Standard solutions were prepared by dissolving GBP solution was tested with glucose standard

D-glucose (>99.5% purity) from Sigma-Aldrich solution and the results were compared with the (St. Louis, MO) in 1 PBS, pH 7.4. A 1 mM fresh GBP solution. The dialysis and recovery glucose solution, pH 7.5 was used as a stock steps were repeated several times to see whether solution. The stock solution was then diluted the GBP response remains the same. to the following concentrations: 1, 2, 4, 6, 8, 10,

27 Tiangco C, Brown S, Sevilla F III, Rao G, & Tolosa LActa Manilana 64 (2016)

Determination of the binding constant of GBP. measurement were collected from healthy adult

The binding constant Kd of the GBP and its subjects who had been fasting overnight. Two maximum glucose binding were calculated by TG samples were collected during the fasting fitting the data of fluorescence intensities (a.u.) stage at about 30 min interval. After taking 75 g at different glucose concentrations (M) to glucose, samples were then collected from the binding-saturation curves using Prism 6 same subjects at 30 min time intervals for 2 h. (GraphPad Software, San Diego, CA, USA). These steps capture the increase and steady decrease of glucose levels in the subject’s body Measurement of transdermal glucose. Samples after consuming glucose. for transdermal glucose measurement were collected from healthy adult subjects, Measurement of blood glucose. The blood specifically from the skin of the fingers. The glucose (BG) levels of the subjects were sampling protocol involved washing the hands measured concurrently with the transdermal with soap and water and followed by a distilled glucose. Corresponding BG levels were water rinse. The finger was then submerged in determined using a commercially available an agitated 20 mM phosphate buffer in a beaker glucometer (ReliOn™ Confirm glucose meter). for 5 min. The buffer was kept at constant The BG level was measured on the same finger temperature of 37°C using a jacketed beaker and where TG was collected. continuous circulating water bath. Buffer temperature was monitored using an electronic RESULTS AND DISCUSSION thermometer. The washing step was followed H152C GBP preparation. The H152C mutant by drying of the finger for 2 min with filtered air. of the glucose binding protein used in the Washing and drying steps are important to experiments contained a polyhistidine tag, which remove the residual glucose from the stratum allows for ease of purification. It was isolated corneum. This was to ensure that the collected from the lysate of the bacterial cells of modified sample is the glucose diffusing through the skin E. coli by means of sonication and affinity at time of collection. The five-minute increased chromatography. The SDS-PAGE of the purified temperature wash was also optimized [20]. GBP showed two thick bands at the 34 kDa Collection of the transdermal glucose sample position consistent with the size of the GBP [8] was done by inverting a 1.5 mL Eppendorf tube (Fig. 1). containing 250 L of 20 mM phosphate buffer

at room temperature on the same finger for 5 min. Protein GBP H152C with His-tag The total sampling time was 12 min. The sample Ladder 12345

was collected and stored at –80°C. About 20 L – 250 kD TG samples were added to 200 L of the GBP solution. The fluorescence intensities were – 150 kD measured as previously described for the – 100 kD – 75 kD glucose standard solutions and the change in intensities were calculated against GBP without – 50 kD – 37 kD samples. The TG concentrations in the samples GBP were calculated from the calibration curve. – 25 kD – 20 kD – 15 kD To induce larger blood glucose changes, – 10 kD subjects were subjected to an oral glucose tolerance test. Samples for transdermal glucose Figure 1. SDS-PAGE results showing the presence of H152C GBP

28 Fluorophore-labeled bioengineered glucose binding protein

The protein had a total concentration of 8.5 M increase in fluorescence intensity upon binding and the calculated labeling extent of protein- with glucose is due to the increased bound BADAN was found to be 83.17%. hydrophobicity around this site brought about by the closing of the two globular domains of Fluorescence of BADAN-labeled GBP. When the protein around glucose [18]. the GBP labeled with BADAN was excited at 400 nm, the maximum emission spectra occurred The fluorescence intensity of the BADAN- at approximately 540 nm. Addition of saturating labeled GBP increased with increasing glucose concentration of glucose (42 mM) to GBP concentrations, varying significantly at low resulted in an increase in fluorescence intensity concentrations and only slightly at the higher of 228%. Figure 2 shows the change in the concentrations of 0.674 and 1.085 M glucose fluorescence spectra of the protein in the (Fig. 3). A linear behavior occurred at the absence and in the presence of glucose. concentration range of 0.030–0.460 M (combine Fig. 4 and Fig. 5). Within this range, BADAN, a polarity sensitive dye, was reacted the GBP displayed good sensitivity (1.68 M–1) to the cysteine mutation at position 152 near to glucose concentration and is most useful for the glucose-binding site in GBP. The observed

1000 – 8000 – GBP H152C (0 mM glucose) 42 mM glucose 800 – 7000 –

600 – 6000 – 400 –

Intensity (a.u) 5000 – 200 – Fluorescence Intensity (a.u) 0– 4000 – 300 400 500 600 700 0.0 0.5 1.0 1.5 2.0 2.5 Wavelength (nm) Total Glucose Concentration (M) Figure 2. Emission spectra of GBP labeled with Figure 4. Fluorescence intensity of GBP as a BADAN showing the increase in fluorescence function of total glucose concentration (M) intensity upon addition of glucose (n = 4)

800 – GBP H152C 1.0 – 0.025 M glucose 600 – 0.071 M glucose 0.163 M glucose 0.8 – 0.296 M glucose 0.467 M glucose 400 – 0.674 M glucose 0.6 – 1.085 M glucose 0.4 – Intensity (a.u) 200 – 0.2 –

0– Normalized Intensity 0.0 – 400 450 500 550 600 650 0.1 0.2 0.3 0.4 0.5 –0.2 – Wavelength (nm) Total Glucose Concentration (M) Figure 3. Emission spectra of BADAN-labeled GBP Figure 5. Linear range of calibration curve (n = 4) with increasing glucose concentrations

29 Tiangco C, Brown S, Sevilla F III, Rao G, & Tolosa LActa Manilana 64 (2016)

glucose measurement. The limit of detection of nonlinear regression and the best values for

the assay (based on 3 s) is 0.030 M. “Fmax and Kd can be determined.

Binding constant of the GBP and calibration Reusability of the glucose binding protein. curve. The binding constant, Kd of GBP H152C- Reusability tests showed that the calibration BADAN was found to be 1.124±0.236 M curves did not change significantly after several

(Fig. 4). The apparent Kd was slightly lower recoveries (Fig. 6). These tests only proved that compared from previous work, 2.35 mM [18]. The the GBP could be used many times, which could binding constant, can be calculated by fitting be considered as a potential reversible and experimental results from GBP response reusable sensor [19]. corresponding to different glucose concentrations to the binding isotherm (2): Transdermal glucose measurement. Prior to the collection of TG sample, the finger was subjected to washing and drying in order to remove the

existing glucose in the stratum corneum. These ∆ [] ∆ = steps ensure that the collected glucose is the ( + []) where F is the normalized signal change at any TG diffusing through the skin. Previous study ligand concentration, Fmax is the normalized showed that the temperature of the washing signal change at saturating ligand buffer and drying step are important parameters. concentration, [S] is the concentration of the An increase in temperature of washing buffer ligand in free state and Kd is the binding decreased the washing time required - from an constant. This equation along with equations initially 15-min wash at room temperature [15] to for single binding equilibria and mass balance 5-min at 37°C [20]. Table 1 shows the optimized produce the following equation (3): parameters for sampling and analysis.

The TG concentrations in the collected samples ∆ were determined from a previously prepared =1+ ∆ calibration curve (Fig. 5). The results of the TG ∆ []− [] ∆ measurements for samples collected at different where [E]t and [S]t are the total concentrations of GBP and glucose respectively. The times during fasting and after intake of glucose experimental results can be analyzed with are shown in Fig. 7 together with the results of BG measurements. It could be seen that TG levels follow the same trend as the BG. However,

0.6 – TG concentration had an average lag time of 49 min for individuals 1 and 2. A peak value of

0.4 – Table 1. Optimized Parameters Freshly prepared for Noninvasive Transdermal Glucose After 1st recovery Sampling and Analysis 0.2 – After 2nd recovery After 3rd recovery

Normalized Intensity Parameter Optimized value Wash time 5 min [20] 0.0 – PBS Buffer temperature 37C [20] 0 50 100 150 200 250 Drying time 2 min [15] Glucose Concentration (M) Sampling time 5 min [15] Volume of PBS buffer 250 L [14, 15] Figure 6. Reusability test of the BADAN-labeled Volume of GBP 200 L

H152C GBP (n = 4)

30 Fluorophore-labeled bioengineered glucose binding protein

Oral Glucose Tolerance Testing (Non-diabetic Individual 1) to account for the lag time. However, the results 135 – 0.25 130 for individual 3 did not show a lag time, which M)

125 – 0.20  can be associated with the age of individual. 120 These results showed a correlation exists – 0.15 115 between TG and BG. 110 – 0.10 105 CONCLUSION

100 – 0.05 Blod glucose conc (mg/dL) A BADAN-labeled mutant of GBP (H152C) was 95 Skin glucose conc (M) Blood Glucose Conc. (mg/dL) synthesized, prepared and characterized. It had 90 – 0.00 0 50 100 150 200 a Kd in the micromolar levels indicating its Time (min) capability to detect low TG concentrations. Oral Glucose Tolerance Testing (Non-diabetic Individual 2) Fluorescence intensities increased markedly 130 – 0.40 upon addition of glucose. There is a correlation – 0.35 120 M)between ( Glucose Transdermal the measured transdermal glucose and  – 0.30 blood glucose levels with a lag of TG behind 110 – 0.25 blood glucose. 100 – 0.20 – 0.15 ACKNOWLEDGMENT 90 – 0.10 80 The authors acknowledge Science, Technology, Blod glucose conc (mg/dL) – 0.05 Skin glucose conc ( M) Glucose ( Transdermal Research and Innovation for Development

Blood Glucose Conc. (mg/dL)  70 – 0.00 (STRIDE) of United States Agency for 0 50 100 150 200 Time (min) International Development (USAID) for Cristina Tiangco’s support. They would also like to Oral Glucose Tolerance Testing (Non-diabetic Individual 3) 170 – 0.30 thank KarunaSri Mupparapu for the GBP 160 preparation training, Chandrasekhar – 0.25 M) 150  Gurramkonda for the construction of H152C 140 – 0.20 plasmid, Sean Najmi and Chisom Nwaneri for 130 helping with the experimental work. 120 – 0.15 110 – 0.10 100 REFERENCES 90 – 0.05 Blod glucose conc (mg/dL) [1] Khan AY, Noronha SB, & Bandyopadhyaya R. 80 Skin glucose conc ( M) Glucose ( Transdermal

Blood Glucose Conc. (mg/dL)  70 – 0.00 Superior performance of a carbon-paste 0 50 100 150 200 250 electrode based glucose biosensor containing Time (min) glucose oxidase enzyme in mesoporous silica Figure 7. Comparison of TG and BG levels of powder. Advanced Powder Technology 2016; healthy adult subjects from their Oral Glucose 27:85–92. doi: 10.1016/j.apt.2015.11.003 Tolerance Tests (OGTT) (Individuals 1–3) [2] Barsan MM, Pifferi V, Falciola L, & Brett CMA. New CNT/poly(brilliant green) and CNT/poly(3,4- ethylenedioxythiophene) based electrochemical enzyme biosensors. Analytica Chimica Acta 0.24–0.37 M were observed for the TG 2016; 927:35–45. doi: 10.1016/j.aca.2016.04.049 concentrations and 122–152 for the BG levels. A [3] Hervás Pérez JP, López-Ruiz B, & López- lag is expected in skin glucose samples because Cabarcos E. Synthesis and characterization of of the time required for ingested glucose to microparticles based on poly-methacrylic acid circulate to the blood, the tissues and then with glucose oxidase for biosensor applications. through the skin. Further investigation is needed Talanta 2016; 149:310–318. doi: 10.1016/ j.talanta.2015.11.053

31 Tiangco C, Brown S, Sevilla F III, Rao G, & Tolosa LActa Manilana 64 (2016)

[4] Devasenathipathy R, Mani V, Chen SM, Huang [13] Fonin AV, Stepanenko OV, Povarova OI, Volova ST, Huang TT, Lin CM, Hwa KY, Chen TY, & Chen CA, Philippova EM, Bublikov GS, Kuznetsova IM, BJ. Glucose biosensor based on glucose oxidase Demchenko AP, & Turoverov KK. Spectral immobilized at gold nanoparticles decorated characteristics of the mutant form GGBP/H152C graphene-carbon nanotubes. Enzyme and of D-glucose/D-galactose-binding protein labeled Microbial Technology 2015; 78:40–45. doi: with fluorescent dye BADAN: influence of 10.1016/j.enzmictec.2015.06.006 external factors. Peer J 2014; 2:e275. doi: [5] Pahurkar VG, Tamgadge YS, Gambhire AB, & 10.7717/peerj.275 Muley GG. Glucose oxidase immobilized PANI [14] Ge X, Rao G, Kostov Y, Kanjananimmanont S, cladding modified fiber optic intrinsic biosensor Viscardi RM, Woo H, & Tolosa L. Detection of for detection of glucose. Sensors and Actuators trace glucose on the surface of a semipermeable B-Chemical 2015; 210:362–368. doi: 10.1016/ membrane using a fluorescently labeled glucose- j.snb.2014.12.125 binding protein: a promising approach to [6] D ’auria S, Cesare N Di, Gryczynski Z, Gryczynski noninvasive glucose monitoring. Journal of I, Rossi M, & Lakowicz JR. A Thermophilic Diabetes Science and Technology 2013; 7:4– Apoglucose Dehydrogenase as Nonconsuming 12. Glucose Sensor. Biochemical and Biophysical [15] Kanjananimmanont S, Ge X, Mupparapu K, Rao Research Communications 2000; 274:727–731. G, Potts R, & Tolosa L. Passive Diffusion of doi: 10.1006/bbrc.2000.3172 Transdermal Glucose: Noninvasive Glucose [7] Hussain F, Birch DJS, & Pickup JC. Glucose Sensing Using a Fluorescent Glucose Binding sensing based on the intrinsic fluorescence of Protein. Journal of Diabetes Science and sol-gel immobilized yeast hexokinase. Analytical Technology 2014; 8:291–298. doi: 10.1177/ Biochemistry 2005; 335:137–143. doi: 10.1016/ 1932296813519994 j.ab.2005.01.016 [16] Khan F, Saxl TE, & Pickup JC. Fluorescence [8] Ge X, Tolosa L, & Rao G. Dual-labeled glucose intensity- and lifetime-based glucose sensing binding protein for ratiometric measurements of using an engineered high-Kd mutant of glucose/ glucose. Analytical Chemistry 2004; 76:1403– galactose-binding protein. Analytical 1410. doi: 10.1021/ac035063p Biochemistry 2010; 399:39–43. doi: 10.1016/ [9] Amiss TJ, Sherman DB, Nycz CM, Andaluz SA, j.ab.2009.11.035 & Pitner JB. Engineering and rapid selection of a [17] Saxl T, Khan F, Ferla M, Birch D, & Pickup J. A low-affinity glucose/galactose-binding protein for fluorescence lifetime-based fibre-optic glucose a glucose biosensor. Protein Science 2007; sensor using glucose/galactose-binding protein. 16:2350–2359. doi: 10.1110/ps.073119507 Analyst 2011; 136:968–972. doi: 10.1039/ [10] de Lorimier RM, Tian Y, & Hellinga HW. Binding C0AN00430H and signaling of surface-immobilized [18] Pickup JC, Khan F, Zhi Z-L, Coulter J, & Birch reagentless fluorescent biosensors derived from DJS. Fluorescence intensity- and lifetime-based periplasmic binding proteins. Protein Science glucose sensing using glucose/galactose- 2006;15:1936–1944. doi: 10.1110/ps.062261606 binding protein. Journal of Diabetes Science [11] Khan F, Gnudi L, & Pickup JC. Fluorescence- and Technology 2013; 7:62–71. doi: 10.1177/ based sensing of glucose using engineered 193229681300700108 glucose/galactose-binding protein: A comparison [19] Lam H, Kostov Y, Rao G, & Tolosa L. Low-cost of fluorescence resonance energy transfer and optical lifetime assisted ratiometric glutamine environmentally sensitive dye labelling strategies. sensor based on glutamine binding protein. Biochemical and Biophysical Research Analytical Biochemistry 2008; 383:61–67. doi: Communications 2008; 365:102–106. doi: 10.1016/j.ab.2008.08.018 10.1016/j.bbrc.2007.10.129 [20] Brown S. Transdermal Glucose Sensor for [12] Scognamiglio V, Aurilia V, Cennamo N, Ringhieri Neonates Using Glucose Binding Protein P, Iozzino L, Tartaglia M, Staiano M, Ruggiero G, (Master’s Thesis). (University of Maryland Orlando P, Labella T, Zeni L, Vitale A, & D’Auria Baltimore County, 2015) S. D-galactose/D-glucose-binding Protein from Escherichia coli as Probe for a Non-consuming Glucose Implantable Fluorescence Biosensor. Sensors 2007; 7:2484–2491. doi: 10.3390/ s7102484

32 Acta Manilana 64 (2016), pp. 33–39 Printed in the Philippines ISSN: 0065–1370

Enantioselective chiral 2-pyridyl-2-imidazoline organocatalyzed Aldol reactions in brine

Aileen Marice R. Baltazar1, Simon Budde2, Christian Faderl2, Aldrick B. Verano1, & Allan Patrick G. Macabeo*1

1Laboratory for Organic Reactivity, Discovery and Synthesis (LORDS), Research Center for the Natural and Applied Sciences, University of Santo Tomas, 1015 Manila 2Institut für Organische Chemie, Universität Regensburg, Universitätsstrasse 31 D-93053 Regensburg, Germany

Among the known organocatalyst manifolds, the organocatalytic utility of 2- pyridylimidazolines has been less explored. In this study, a collection of chiral 2-pyridyl-2-imidazolines (picolinylimidazolines) were screened for organocatalytic activity towards enantioselective direct Aldol reaction. The chiral imidazolines (1a-1d) were prepared through conventional iodine-promoted oxidative condensation and cyclization of 2- picolinaldehydes 2 (and/or 2,6-pyridine dicarbaldehyde) with chiral 1,2-diamine derivatives 3 in excellent yields (>96% isolated yields). Organocatalytic parameters were optimized to determine the best Aldol reaction conditions that would induce enantioselectivity such as

catalyst loading, temperature, reaction time and solvent. Thus, using a C2-symmetric cyclohexane-based 2-pyridyl-2-imidazoline organocatalyst 1c, the best conditions that gave excellent enantioinduction of up to 99:1 enantiomeric ratio (er) and yield were observed with brine as the solvent, a 1-h reaction period at room temperature and 10 mol% as the minimum catalyst load. The reaction conditions were also amenable to a variety of benzaldehyde substrates with electron-donating and electron-withdrawing substituents. Our study demonstrates for the first time the asymmetric construction of -hydroxy carbonyl structures

using chiral C2-symmetric 2-pyridyl-2-imidazolines as organocatalysts.

Keywords: Aldol reaction, organocatalysis, picolinylimidazoline, -hydroxy carbonyl, asymmetric synthesis

INTRODUCTION simplifies transition states and reduces the number of diastereomeric reaction sites to The design of a new catalyst for asymmetric favorably effect high enantioselectivity [1]. reactions is one of the key challenges and a Asymmetric organocatalysis has become a very highly important task in organic synthesis. In rapidly growing field as a result of both the the design of a new catalyst scaffold, molecular novelty of the concept and the selectivities symmetry plays a powerful guideline as it attained by many organocatalytic transformations [2]. Among the main advantages *To whom correspondence should be addressed of this methodology is the stability of [email protected] / [email protected] intermediate species participating in the catalytic

© 2016 UST Research Center for the Natural and Applied Sciences, Manila, Philippines Baltazar AMR, Budde S, Faderl C, Verano AB, & Macabeo APGActa Manilana 64 (2016)

cycle toward water and oxygen and therefore organocatalysts 1 as additional organic reactions do not require special care with regard manifolds to facilitate the construction of - to the use of inert atmosphere or dry solvent hydroxy carbonyl architectures 4 via asymmetric [3]. In addition, the presence of hazardous metals Aldol reactions. is precluded in the reaction scheme making the methodology even more interesting from the EXPERIMENTAL environmental point of view [4]. As a realization General. All reagents were purchased from grows that organic molecules not only have ease chemical suppliers (Sigma, Merck) and were used of manipulation and “green” advantage but can without further purification. Colum also be very efficient catalysts, asymmetric chromatography was performed using Merck organocatalysis may begin to catch up with Silica Gel 60 (230–400 mesh). Visualization was spectacular advancements of enantioselective carried-out with UV using Mineralight Lamp transition metal catalysis. Multiband UV at 254 nm and 366 nm and vanillin- The tremendous development of Aldol reactions sulfuric acid followed by heating at 100°C until since 1872 has provided an array of synthetic colored spots appear. NMR analysis was information advancing the construction of well- performed in Bruker AMX 300 MHz. Samples defined asymmetric centers [5]. As a for NMR were dissolved in CDCl3 with TMS as consequence, the synthetic utility of this internal standard. Experiments include routine 1 13 reaction has grown interest in developing H and C. organocatalysts beneficial to the construction General procedure for the synthesis of of -hydroxy structures. Thus, as a contribution imidazoline organocatalysts 1a-1d. To a to expanding methodologies related to solution of 2-pyridine carbaldehyde asymmetric Aldol reactions, we explored the (picolinaldehyde) or 2,6 pyridine dicarbaldehyde utility of imidazolines in this study. Imidazolines 2 (1 equiv) in t-butyl alcohol (18.5 mL per are structural analogues of imidazoles which are 1.85 mmol) was added diamine derivative 3 (1.1 widely used for chiral ligand designs, and or 2.2 equiv). The obtained mixture was stirred various substituents can be introduced to the at room temperature and K CO (3 or 6 equiv) nitrogen atom for tuning steric environments 2 3 and I (1.25 or 2.50 equiv) were added and stirred and electron density [6]. There are only a few 2 at 70°C for 4 h. The mixture was quenched with asymmetric reactions with imidazolines as an saturated Na SO until iodine disappeared and organocatalyst such as the Diels-Alder 2 3 extracted with DCM (3×). The organic layer was reactions (as Bronsted acid catalysts) [7] and washed with saturated NaHCO and dried over the Morita Baylis-Hillman [8]. In this paper, we 3 anhydrous Na SO . After filtration, the solvent disclose the catalytic activity of several C - 2 4 2 was evaporated and chromatographed using 9:1 symmetric, chiral 2-pyridyl-2-imidazolines DCM-methanol to obtain 1.

NH2 I2, K2CO3 N + R2 N 3 2 R3 N R = H, 2 1 R o R2 R R N CHO t-BuOH, 70 C,4h HN NH HN 2 2 1 2 R2 R R = H, CHO R = -(CH2)4, Ph 2 3 1

Scheme 1. General scheme for the synthesis of picolinylimidazoline organocatalysts 1.

34 Enantioselective chiral 2-pyridyl-2-imidazoline

1 H NMR δ (300 MHz, CDCl3) 8.51 (d, 1H, J =4.6 Hz, Py-6-H), 8.13 (d, 1H, J = 7.8 Hz, Py- 3, 3-H), 7.83-7.75 (m, 1H, Py), 7.29 N N (m, 1H, Py), 3.20-3.09 (br m, 2H, 2 x CH) 2.31-2.19 (br, m, 2H, CH2), 1.83-1.72 (br m, 2H, CH2), 1.58-1.45 (br m, 2H, CH2), N 13 H 1.36- 1.24 (br m, 2H, CH ). C-NMR δ (100 MHz, CDCl3) 165.3, 148.8, 148.5, 136.7, 125.3, 122.2, 30.8 (CH), 25.0 (CH2) 1a [9].

1 N N H NMR (300 MHz, CDCl3) 1.34, m, 4H), 1.55 (m, 4H), 1.83 N (m, 4H), 2.28 (m, 4H), 3.36 (brm, 4H) 7.81 (t, J = 7.3 Hz, 1H), 13 N N 7.21 (d, J = 7.3 Hz, 2H); C NMR (100 MHz, CDCl3): 21.6, H H 22.9, 30.7, 23.5, 137.3, 147.8, 164.6 [9].

1b

N 1 N H NMR (300 MHz, CDCl3):  4.82 (d, J= 7.9 Hz, 1H), 5.13 (d, J= 8.4 Hz, 1H), 6.54 (s, 1H), 7.27-7.45 (m, 11H, aromatic), 7.82 N (ddd, J= 1.7, 7.7, 7.7 Hz, 1H, aromatic), 8.31-8.35 (m, 1H, H aromatic), 8.62-8.65 (m, 1H, aromatic); 13C NMR (125 MHz,

CDCl3):  77.2, 122.7, 125.3, 126.0-128.6(Ph), 136.6, 143.2, 148.4, 148.7, 162.5 [10].

1c

N N 1 N H NMR (300 MHz, CDCl3): 4.90, bs, 4H), 7.22-7.31 (m, 20H), 13 N N 7.89 (t, J = 7.8 Hz, 1H), 8.46 (d, J = 7.8, 2H); C NMR (100 H H MHz, CDCl3) 74.9, 125.5, 127.1, 128.3, 129.2, 136.1, 138.2, 142.9, 162.1 [11].

1d

Representative procedure for the Aldol with Shimadzu SPD-10AV. Flow rate was 1.0 mL/ reaction. To a mixture of imidazoline catalyst 1 min and the pump pressure was 664 psi. UV-VIS (10 mole %) and 4-nitrobenzaldehyde (1 equiv) Spectrophotometric Detector was set at 254 nm in brine (1 mL per 0.074 mmol) was added acetone and connected to a Shimadzu Communication (0.25 mL per 0.074 mmol) and stirred for 1 h at Bus Module CBM-102, with a CLASS GC-10 room temperature. Product formation was judged Workstation. Separation was done on a chiral by TLC (9:1 DCM:MeOH). Saturated sodium column Lux 5u Cellulose-2 (250 × 4.6 mm). bicarbonate and DCM was added and the Solvent systems was 70:30 n-hexane/ isopropyl organic layer separated and dried over alcohol. anhydrous sodium sulfate. The concentrated product was column chromatographed on silica RESULTS AND DISCUSSION gel and eluted with 1:1 petroleum ether-ethyl Pyridylimidazoline organocatalysts (1a–1d) were acetate to give the Aldol product 4. prepared through iodine-promoted Enantiomeric ratio (er) determination. condensation and oxidative cyclization from Enantiomeric ratio was determined on a commercially available picolinaldehydes, 2 Shimadzu Prominence LC-20AT Pump equipped (pyridine-2-carbaldehyde, 2a or pyridine-2,6-

35 Baltazar AMR, Budde S, Faderl C, Verano AB, & Macabeo APGActa Manilana 64 (2016) dicarbaldehyde, 2b and) and chiral diamines, 3 done and the results are presented in Table 1.

(1R,2R-cyclohexane diamine or 3a 1R,2R-1,2- The best catalyst was found to be the C2- diphenyl-1,2-ethylenediamine, 3b) in excellent symmetric 1c (Entry 10) giving the highest yield yields (Scheme 2). (78%) and excellent enantiomeric ratio (99:1) with high preference and selectivity to the formation The synthesis of picolinylimidazolines 1 in of the R-configurated -hydroxy Aldol product general follows a mechanism (Scheme 3) which (4a). proceeds initially by condensation of an aldehyde with a diamine to afford imine Brine and a 10 mol% catalyst 1c were observed intermediate 5 which in equilibrium, cyclizes to to be the best solvent and catalyst loading. No dihydroimidazoline 6 [12]. Iodine-promoted significant improvement in yield was obtained oxidative addition followed by elimination thus when the catalyst loading was increased to 15 proceeds to the target picolinylimidazolines 1. mol% while a longer reaction time was required when a 5 mol% catalyst was used. Lowering the The catalytic activity of 2-pyridyl-2-imidazolines temperature to 0°C did not improve the yield 1a–1d was evaluated in the direct and enantioselectivity. enantioselective Aldol reaction of 4- nitrobenzaldehyde and acetone. Optimization of The addition of NaCl in the reaction promotes catalyst loading, temperature and solvent were “salting in” effect accelerating the

NH2 N N + N N CHO NH2 H 3a 2a 1a (79%)

N N NH2 N + H N CHO NH2 2a 3b 1b (96%)

NH N N 2 N + N N OHC N CHO NH2 H H 2b 3a 1c (95%)

NH2 N N Ph N + Ph OHC N CHO N N H H NH2 Ph Ph 2b 1d (97%) 3b

Scheme 2. Synthesis of picolinylimidazolines 1a–1d.

Reaction conditions: I2 (1.25 equiv), K2CO3 (3 equiv), t-BuOH, 70°C, 4 h.

36 Enantioselective chiral 2-pyridyl-2-imidazoline

NH2 -H O H + R 2 N R N R N N R N CHO NH2 HN R NH2 2 3 5 R 6 I H I2 N -HI N N R N R (-HI) HN HN R R 1 Scheme 3. Proposed mechanism en route picolinylimidazoline organocatalysts 1.

Table 1. Optimization of organocatalyzed direct Aldol reaction conditions

OH O OH O CHO O 1c,brine rt, 1 h O N 2 O 2N O 2N 4a (R enantiomer) 4b (S enantiomer) Catalyst loading Isolated Entry Solvent Catalyst er (R:S) (mol %) Yield

1 CHCl3 1a 20 59 79:21 2 DCM 1a 20 35 57:43 3 H2O 1a 20 70 91:9 4 Brine 1a 20 60 94:6 5 CHCl3 1b 10 63 99:1 6 Brine 1c 10 53 70:30 7 DCM 1c 10 46 96:4 8 H2O 1c 10 65 91:9 9 Brine 1c 5 29 99:1 10 Brine 1c 10 78 99:1 11 Brine 1c 15 14 98:2 12 Brine 1d 10 58 94:6

organocatalyzed Aldol reaction in water by solutions. The hydrogen bonds thus formed, favouring the aggregation of organic species/ are less available to interact with the soluble ion intermediates in concentrated hydrophobic form of the salt. The same result was noted when pockets. Very polar solutions are superior in catalyst loading was reduced down to 10% solubilizing a polar substrate compared to less (Entry 10). In this condition, addition of brine is polar derivatives. Adding salt to an aqueous favoured and effective than solely employing solution with moderate polarity decreases water as the solvent. solubility. On the other hand, adding a (ionic) salt to solution with high polarity increases Addition of selected organic acids did not solubility provided no common ion effect is improve yield and enantioselectivity (Table 2). observed and a globular, micelle structure is This implies that a Bronsted acid-mediated formed in aqueous solutions. The structure of mechanism may not be involved in the catalytic water near the head group is organized, thus, pathways. Instead, a Bronsted-base catalyzed allowing weak interaction with other ions in generation of enol and H-bonding stabilized

37 Baltazar AMR, Budde S, Faderl C, Verano AB, & Macabeo APGActa Manilana 64 (2016)

intermediate is observed. In addition to these enantiomeric ratio was observed when the acid additive serving as Bronsted co-catalyst, electrophile is dimethoxylated at C-3 and C-4 for the results obtained by Masse and co-workers 4g. (Entry 7) Sluggish reaction period was also [13] agree with our findings that improved observed when the substituent is a methyl group catalytic performance can only be obtained in at C-4 (Entry 4 for 4d). Finally, among the electron polar aprotic solvent (for example DMSO) but withdrawing substituents at C-4, the presence not in polar, protic solvents such as water. In all of a nitrile group (Entry 2) effected a drop in the cases, the additive weak organic acids were yield of the Aldol product 4b compared to the 4- afforded in moderate yield but with high nitro derivative 4a (Entry 1). enantioselectivities up to 98% (Table 2). This indicates that in the course of the reaction Based on the results obtained where a high development, the catalyst additive associates selectivity towards the R enantiomer is favoured, with the reactants similar to that of micelles. a hydrogen-bonding — stabilized transition state is proposed whereby the incipient bond is To determine the scope of the reaction, various formed between the Re face of the benzaldehyde benzaldehyde derivatives were also screened electrophile and the Si face of the enol (Table 3). Aldol reaction of aromatic aldehydes nucleophile. 7 with acetone 8 afforded the -hydroxy derivatives 4 in moderate to excellent yields. The 4-(m-chlorophenyl)-4-hydroxy-2-butanone 4e R was obtained with the high yield, with almost Re face no self-aldolization product and excellent Si face H enantioselectivity (Entry 5). Significant decrease O in yield was noted when the substituent is a O H H bromine atom at C-4 (Entry 6, 4f) while a drop in N N N N H H N O O Table 2. Effect of Bronsted acid additives H

Re face Entry Additive Isolated Yield er (R:S) Si face R 1 L-Tartaric acid 34 98:2 2 L-Citric acid 17 95:5 Figure 1. Proposed transition state model for the 3 Benzoic acid 33 75:25 enantioselective, direct aldol reaction with 1c.

Table 3. Scope of the organocatalyzed Aldol reaction with 1c

OH O R1 CHO 1 O 1b,brine R + rt * R2 R2 7 8 4 Reaction Isolated Entry R1 R2 Product 4 er (R:S) Time (h) Yield

1 H NO2 4a 1 78 99:1 2 H CN 4b 2.5 26 99:1 3 H OMe 4c 3 99 99:1 4 H Me 4d 24 37 99:1 5 H Cl 4e 2 98 >99 6 H Br 4f 2 9 99:1 7 OMe OMe 4g 2.5 50 51:49

38 Enantioselective chiral 2-pyridyl-2-imidazoline

Four chiral picolinylimidazoline organocatalysts [7] Davenport AJ, Davies DL, Fawcetta J, & Russella were successfully synthesized and employed DR. Chiral Pyridylimidazolines: Synthesis, Arene as catalysts for the direct asymmetric Aldol Ruthenium Complexes and Application in Asymmetric Catalysis. Journal of the Chemical reaction of benzaldehydes and acetone. The C - 2 Society, Perkin Transactions 2001; 1:1500– symmetric imidazoline 1c was observed to be 1503. the best organocatalyst with brine as the [8] Xu J, Guan Y, Yang S, Ng Y, Peh G, & Tan CH. reaction solvent and 10 mol% as the best Asymmetric Baylis-Hillman reactions promoted catalyst loading at room temperature. Moderate by chiral imidazolines. Chemistry — An Asian to excellent amounts of the -hydroxy Aldol Journal 2006; 1:724–729. [9] Tydlitát J, Bureš F, Kulhánek J, & Rù•ièkab A. product 4 were obtained in excellent 1,2-Disubstituted Hexahydro-1H-benzo[d] enantioselectivity favoring the R enantiomer. imidazoles: Synthesis, Characterization, and Our study provides a green, easy approach and Stability. Synthesis 2010; 22:3934–3940. straightforward enantioselective access to - [10] Fruhstorfer W & Muller-Calgan H (E. Merck hydroxy ketone motifs. Aktiengesellschaft, Darmstadt, Germany) Imidazoline Derivatives. US Patent 3, 147, 275 REFERENCES (September 1, 1964). [11] Bhor S, Anilkumar G, Tse MK, Klawonn M, Doebler [1] Bhanushali M & Zhao C-G. Developing Novel C, Bitterlich B, Grotevendt A, & Beller M. Synthesis Organocatalyzed Aldol Reactions for the of a New Chiral N,N,N-Tridentate Pyridine Enantioselective Synthesis of Biologically Active bisimidazoline Ligand Library and Its Application Molecules. Synthesis 2011; 12:1815–1830. in Ru-Catalyzed Asymmetric Epoxidation. [2] Liu WJ & Gong LZ. Asymmetric Organocatalysis. Organic Letters 2005; 7:3393–3396. Topics in Organometallic Chemistry 2011; [12] Ishihara M & Togo H. An Efficient Preparation of 36:153–206. 2-Imidazolines and Imidazoles from Aldehydes [3] MacMillan DWC. The Advent and Development with Molecular Iodine and (Diacetoxyiodo) of Organocatalysis. Nature 2008; 455:304–308. benzene. Synlett 2006; 37:227–230. [4] Alemán J & Cabrera S. Applications of Asymmetric [13] Delaney JP & Henderson LC. Investigating Ionic Organocatalysis in Medicinal Chemistry. Effects Applied to Water Based Organocatalysed Chemical Society Reviews 2013; 42:774–793. Aldol Reactions. International Journal of [5] Denmark SE, Heemstra JR, & Beutner GL. Molecular Sciences 2011; 12:9083–9094. Catalytic, Enantioselective, Vinylogous Aldol [14] Mase N, Nakai Y, Ohara N, Yoda H, Takabe K, Reactions. Angewandte Chemie-International Tanaka F, & Barbas CFJ III. Investigating Ionic Edition 2005; 44:4682–4698. Effects Applied to Water Based Organocatalysed [6] Liu H & Du D-M. Recent Advances in the Aldol Reactions. Journal of the American Synthesis of 2-Imidazolines and their Applications Chemical Society 2006; 128:734–735. in Homogeneous Catalysis. Advanced Synthesis & Catalysis 2009; 351:489–519.

39 Acta Manilana 64 (2016), pp. 41–50 Printed in the Philippines ISSN: 0065–1370

Pulse electrodeposited tin/palladium/exfoliated graphene oxide (Sn/Pd/EGO) catalyst for direct ethanol fuel cell applications

Paulo Irvin A. Chang1, Joshua L. Tan2, & Bernard John V. Tongol*2–4

1Department of Mathematics and Physics, College of Science; 2Graduate School; 3Department of Chemistry, College of Science; 4Research Center for the Natural and Applied Sciences University of Santo Tomas, 1015 Manila, Philippines

This study aims to utilize pulse electrodeposition to synthesize tin/palladium/exfoliated graphene oxide (Sn/Pd/EGO) catalyst composite as anode material for ethanol oxidation reaction (EOR) which finds significant application in Direct Ethanol Fuel Cell (DEFC) application. The electrocatalytic activity of the prepared catalysts towards ethanol oxidation was evaluated using cyclic voltammetry in 1 M ethanol in 0.1 M NaOH. Cyclic voltammetry showed that the addition of Sn to Pd improved the catalytic activity of Pd towards EOR in alkaline solution. The bimetallic Sn/Pd catalyst on glassy carbon electrode (Sn/Pd/GCE) showed a higher catalytic activity with a peak current density of 28.1 mA cm-–2, compared to Pd/GCE (23.39 mA cm–2). The electrocatalytic activity of bimetallic Sn/Pd catalyst further improved when EGO was used as a support (Sn/Pd/EGO) with a peak current density of 36.7 mA cm–2. Atomic force microscopy revealed that the catalysts were deposited on the edges of the graphene sheets forming bright clusters with dark fringes on the middle of the structure. X-ray photoelectron spectroscopy confirmed the presence of Pd and Sn metals and their oxides. This study demonstrates that pulse electrodeposition could offer a much simpler and faster approach in the development of high performance catalysts for EOR.

Keywords: direct ethanol fuel cells, graphene, Sn/Pd, AFM, XPS, pulse electrodeposition

INTRODUCTION characteristics, fuel cells have high potentials to be used as a primary energy source. Research on alternative energy source includes the development of fuel cells. Fuel cells are Fuel cells generate electric current through attractive due to their minimal carbon emission electrochemical reactions with the help of metal foot print, high theoretical thermodynamic based catalysts. Platinum has been reported to efficiency (not restricted by Carnot limit), and have a high catalytic activity compared to other zero use of fossil fuels [1]. With these metals and it is often the anode catalyst of choice [2]. However, palladium (Pd) is more preferred because it is cheaper and more abundant than *To whom correspondence should be addressed Pt. In addition, Pd has been reported to have a [email protected] / higher catalytic activity towards ethanol [email protected]

© 2016 UST Research Center for the Natural and Applied Sciences, Manila, Philippines Chang PIA, Tan JL, & TongolBJVActa Manilana 64 (2016)

oxidation reaction than platinum in alkaline methods in the preparation and deposition of media [3, 4]. Moreover, this condition gives a the catalysts. There are few reports [18, 19] on broader option in combining other metal catalyst the use of electrochemical techniques for the with palladium [5]. Thus, Pd is more practical in deposition of Pd-based catalysts on graphene terms of synthesizing catalysts for commercial support. Nagaraju and Suresh [18] use. electrochemically synthesized Pd nanoparticles decorated-graphene using graphene oxide Palladium as catalyst alone is susceptible to solution (1 mg/mL, synthesized via modified poisoning by carbonaceous species in alkaline Hummer’s method) and palladium ions (1 mg medium. To prevent poisoning intermediates to PdCl2 in 1 M KCl) in a single step using cyclic form on the surface of Pd catalyst, it is usually voltammetry from 0 to –1.2 V vs. Ag/AgCl at a combined with promoter metals [6] by providing scan rate of 100 mV/s. The peak current density necessary intermediates for the reaction due to for ethanol oxidation was found to be 50 mA the bi-functional mechanism [7]. Pd separates cm–2 which is higher than chemically ethanol by chemisorption, resulting to an synthesized PdSn/C [15]. adsorbed carbonaceous species (CO). The promoter reacts with water which leads to the Recently, pulse deposition technique has been

formation of -OHads species that is needed to used by Hsieh and co-workers to oxidize CO at a lower potential which results to electrochemically deposit Pd [19] on oxidized its desorption. This frees up more active sites graphene for proton exchange membrane fuel of Pd for the EOR to occur, thus giving higher cells. Star-like Pd clusters consisting of several catalytic activity. nanotips are well deposited over the GO sheets, and the primary size of the cluster ranges from Aside from adding promoters to improve 100 to 150 nm. The presence of GO sheets is catalytic activity, the catalyst can be deposited presumed to render multifunctional roles in on a support material with large surface area facilitating the dispersion of metallic Pd clusters, such as carbon-based materials [8, 9]. Studies ionic diffusion, and charge transfer [19]. Some have shown that Pd catalyst supported by advantages of pulse electrodeposition technique graphene gave enhanced catalytic activity [10, include ease of electrode fabrication and low 11]. Graphene can improve the performance of cost compared to other methods [19]. To the the catalyst by providing deposition sites for best of our knowledge, there is no report on the the (electro)chemical reduction of the catalyst. pulse electrodeposition of bimetallic PdSn This results to an effective dispersion of the catalyst on graphene oxide for EOR. This study catalyst particles, leading to a higher effective aims to prepare and characterize pulse surface area. A higher effective surface area electrodeposited PdSn catalyst on would provide the catalyst more active sites for electrochemically exfoliated graphene oxide. The the oxidation of the fuel to occur, resulting in a parameters for pulse electrodeposition of Pd and higher catalytic activity [12]. Sn were optimized based on the current density registered using cyclic voltammetry (CV) Investigations have suggested that bimetallic towards EOR in basic medium. catalysts of Pd alloyed with other metals can effectively enhance the electrocatalytic activity METHODOLOGY towards EOR in alkaline media. Majority of the past research on Pd based catalysts are alloyed Materials and reagents. Precursor salt with Ni [10, 11, 13], Co [14], Ru [5], Sn [15–17], solutions used for the catalysts such as

among others. These studies use chemical palladium(II) chloride (PdCl2), tin(II) chloride

42 Pulse electrodeposited tin/palladium/exfoliated graphene oxide

dihydrate (SnCl2  2H2O), potassium bromide electrode, and a platinum rod as the counter (KBr), and sodium hydroxide (NaOH) were all electrode. The pulse parameters were varied purchased from Sigma Aldrich. Ethanol (J.T. using the eChem software (v1.6). Baker, USA) and sulfuric acid (RCI Labscan, Thailand) are of analytical grade. All solutions Characterization of the catalyst. The were prepared using ultrapure water electrochemical characterization of the catalyst (TOC<5.00 ppb; Resistivity = 18.2 Mcm, towards ethanol oxidation reaction was Millipore, USA). performed with a potentiostat (eDaq, Australia) at a potential range of –900 mV to 400 mV, and at Preparation of graphene via electrochemical a scan rate of 50 mV s–1. Morphological exfoliation. Electrochemical exfoliation of characterization was done with NX-10 atomic graphite to produce graphene was adapted from force microscope system (Park Systems, South Su et al. [20] with minor modification. Graphite Korea) in non-contact mode. A PPP-NCHR tip plate (Graphtek LLC, USA), 3”  1”, was used with a tip radius of curvature of 10 nm, nominal as the source of graphene. The graphite plate frequency of 330 kHz, and a force constant of was clipped on the positive terminal (anode) of 43 N m–1 was used to probe the samples. This the DC power supply and a platinum plate was done to observe the topography of the (3 cm  1 cm) was clipped to the negative deposited catalyst composites. The surface terminal (cathode). The electrodes were elemental analysis was performed using submerged in a solution of 0.5 M H2SO4 and was ESCALAB 250 XPS system (Thermo Fischer, subjected to a conditioning bias of +1 V for Theta Probe System, USA) with a

30 min and exfoliating bias of +6 V for 5 min. monochromatic Al K radiation at 150 W, in the These biases were repeated until a desired of pass energy (PE) mode (PE = 20 eV). amount of graphene was exfoliated. RESULTS AND DISCUSSION The exfoliated graphite material was filtered and washed with ultrapure water to remove excess In Pulse Electro-Deposition (PED), the potential sulfates. The sulfate-free graphite material was is alternated between two potentials. Each pulse dispersed in N,N-dimethylformamide (DMF) and consists of an ON-time and an OFF-time. ON- sonicated for 30 min. The resulting dispersion time is the time where the reduction potential is was centrifuged and decanted to remove the applied while OFF-time is the time where zero unexfoliated graphite. The electrochemically potential is applied. The size and thickness of exfoliated graphene (EGO) material was washed the deposited catalyst can be controlled by and filtered using 0.45 m membrane filter to varying the pulse parameters, which can have remove the DMF. The EGO was dispersed in an influence in the performance of the catalyst water and was freeze-dried to obtain powdered [21]. Reduction potentials of –625 mV and – EGO [10]. 525 mV were used for the deposition of Pd and Sn, respectively, based on CV measurements of Pulse electrodeposition of Pd and Sn. A the corresponding metallic solutions on glassy carbon electrode. solution of 10 mM PdCl2 and 10 mM

SnCl2 × 2H2O in 1 M HCl was prepared using ultrapure water. The pulse electrodeposition Effect of ON-Time and OFF-Time. The technique was performed with a potentiostat synthesis of Pd catalyst on GCE was controlled (eDaq, Australia) on a three electrode set-up with by varying and optimizing the duration of the the glassy carbon electrode (GCE) as the pulse parameters, i.e. ON-time and OFF-time. The working electrode, Ag/AgCl as the reference optimum parameters were chosen based on the

43 Chang PIA, Tan JL, & TongolBJVActa Manilana 64 (2016)

highest current density (mA  cm–2) of the anodic of ON-time was at 10 ms as it yielded the highest peak at the positive-going potential scan which catalytic activity. is related to electrocatalytic activity towards EOR. This anodic peak, in the potential range The optimized ON-time was then used in the of –0.2 V to 0.2 V in the positive-going scan is optimization of the OFF-time. The CV curve in attributed to the primary oxidation of ethanol Fig. 2 shows the catalytic activity increased mainly as acetate [15]. when the OFF time was increased. This may be attributed to the mechanism of pulse The ON-time was first optimized while holding electrodeposition. During ON-time, Pd2+ ions are the OFF-time constant at 10 ms. The CV curve reduced to the electrode which produces a in Fig. 1 shows that the catalytic activity initially concentration gradient at the electrode surface. increased to 23.3 mA cm–2 with an ON-time of At short OFF-time, there is not enough time for 10 ms. Also, the electrocatalytic activity of Pd the Pd2+ ions to diffuse into the concentration towards EOR continuously decrease as ON-time gradient near the electrode. This would cause increases. Long deposition times would prolong the next cycle to deposit less Pd. Longer duration the growth of the deposited catalyst, resulting of OFF-time would provide more time for the in a larger deposit with less effective surface ions to diffuse through the bulk, providing area for EOR [22]. Thus the optimized duration enough Pd2+ ions to be deposited on the

25 – AA25 – ) ) 2 2 20 – 5 ms 20 – 10 ms

15 – 10 ms 25 ms 15 – 25 ms 50 ms

10 – 50 ms 10 – 75 ms 100 ms 100 ms 5– 5– Current Density (mA/cm Current Density (mA/cm

0– 0– -1 -0.8-0.6 -0.4 -0.2 00.2 0.4 0.6 -1 -0.8-0.6 -0.4 -0.2 00.2 0.4 0.6 Potential (V vs. Ag/AgCl) Potential (V vs. Ag/AgCl)

23.3 25 – 24.6 23.4 – BB 23.1 23.2 – 24 – 23.6

) 23.3 2 )

2 23 – 23 – 22.8 22.8 – 22.6 – 22.5 22 –

22.4 – 21 – 22.2 – 20 – 19.4 22 – 21.8 21.8 Current Density (mA/cm 19 – Current Density (mA/cm 21.8 – 21.6– 18– 0 1020 30 40 5060 70 80 90 100 0 1020 30 40 5060 70 80 90 100 ON Time (ms) OFF Time (ms) Figure 1. Optimization of the ON-time parameter Figure 2. Optimization of the OFF-time parameter for the electrodeposition of Pd. The OFF-time for the electrodeposition of Pd. The optimized parameter was kept constant at 10 ms. (A) CV ON-time of 10 ms was used. (A) CV measurements done using 1 M ethanol in 0.1 M measurements done in 1 M ethanol in 0.1 M NaOH and (B) corresponding graph. NaOH and (B) corresponding graph.

44 Pulse electrodeposited tin/palladium/exfoliated graphene oxide

electrode [23] . The highest catalytic activity of time parameters have an effect towards the 24.6 mA m–2, was obtained with an OFF-time of electrocatalytic activity of Pd towards EOR. 50 ms. Effect of Pd:Sn ratio and deposition sequence. Based on the results, the optimized ON-time and As previously stated, the promotional effect of OFF-time were 10 ms and 50 ms, respectively. Sn in Pd-Sn catalysts in the EOR can be These results show that the ON time and OFF explained by the bifunctional effect, in which

Sn and/or SnOx have stronger interactions with

hydroxyl group (OHads) while Pd has excellent properties in the adsorption and dissociation of Table 1. Different Ratios of the Number of Pulses for Pd and Sn ethanol [15]. However, higher Sn content will also decrease the occupancy of active Pd atoms Ratio of the Number Number number of of pulses of pulses on the surface, and consequently impair the pulses for Pd for Sn overall performance of dissociation of adsorbed 4:1 100 25 ethanol. Therefore, an optimal Sn content will 2:1 100 50 be observed as a result of such rival effects of 1:1 100 100 1:2 50 100 Sn when alloying with Pd [15]. 1:4 25 100

AA30 – 25 – 25 – ) ) 2 2 20 – 1:4 1:4 20 – 1:2 1:2 15 – 1:1 15 – 1:1

10 – 2:1 2:1 10 – 4:1 4:1 5– 5– Current Density (mA/cm Current Density (mA/cm

0– 0– -1 -0.8-0.6 -0.4 -0.2 00.2 0.4 0.6 -1 -0.8-0.6 -0.4 -0.2 00.2 0.4 0.6 Potential (V vs. Ag/AgCl) Potential (V vs. Ag/AgCl)

30 – BB30 – 28.2 26.1 24.3 ) ) 25 – 25 – 2 2 22.3 20.8 20.4 19.8 20 – 20 – 17.3

15 – 15 – 12.3

10 – 10 – Current Density (mA/cm Current Density (mA/cm 5– 3.45 5–

0 – 0 – 1:4 1:21:1 2:1 4:1 1:4 1:21:1 2:1 4:1 Sn to Pd Ratio of the Number of Pulses Sn to Pd Ratio of the Number of Pulses Figure 3. Optimization of Pd and Sn ratios with Sn Figure 4. Optimization of Pd and Sn ratios with Pd deposited first on GCE before Pd (i.e. Pd/Sn/ deposited first on GCE before Sn (i.e. Sn/Pd/ GCE). The ON-time was optimized at 10 ms and GCE). The ON-time was optimized at 10 ms and OFF-time was optimized at 50 ms. (A) CV OFF-time was optimized at 50 ms. (A) CV measurements done in 1 M ethanol in 0.1 M measurements done in 1 M ethanol in 0.1 M NaOH and (B) corresponding bar graphs. NaOH and (B) corresponding bar graphs.

45 Chang PIA, Tan JL, & TongolBJVActa Manilana 64 (2016)

Table 1 shows the different ratios of the number As shown in Fig. 5A, the peak current density of pulses used in depositing the Pd-Sn bimetallic is dependent on the sequence of deposition of catalyst. The CV data in Fig. 3 and Fig. 4 show the alloy. The CV profile shows that depositing the performance of catalysts prepared with Pd first then Sn (Sn/Pd) gave a higher peak different ratios of number of pulses for current density of 28.2 mA cm–2 (Fig. 4) palladium and tin. The CV results revealed that compared to 24.3 mA cm–2 when depositing Sn the bimetallic catalyst synthesized with a ratio before Pd (Pd/Sn) (Fig. 3) and 25.4 mA cm–2 when of 4:1 (Pd:Sn) gave the highest current density co-depositing Pd and Sn (PdSn) (Fig. 5). This while the catalyst deposited with a ratio of 1:2 may indicate that when Sn is deposited first and 1:4 yielded even lower current densities. before Pd, Sn may have been covered by Pd, This suggests that, as the number of pulses for and could not provide OH– species that can Pd was decreased, there were less active Pd sites oxidize adsorbed carbonaceous species, which and more Sn particles deposit which decrease can improve the catalytic activity of the Pd the electrocatalytic activity towards EOR. catalyst.

30 – AA40 – 35 – 25 – ) ) 2 2 Sn/Pd/GCE 30 – With EGO 20 – Pd/Sn/GCE 25 – Without EGO

15 – PdSn/GCE 20 –

Pd/GCE 15 – 10 – 10 – 5– Current Density (mA/cm Current Density (mA/cm 5–

0– 0– -1 -0.8-0.6 -0.4 -0.2 00.2 0.4 0.6 -1 -0.8-0.6 -0.4 -0.2 00.2 0.4 0.6 Potential vs. Ag/AgCl (V) Potential vs. Ag/AgCl (V )

30 – 45 – 27.9 BB 24.83 40 – 35.65 23.39 23.83 ) 25 – ) 2 2 35 –

20 – 30 – 27.9 25 – 15 – 20 –

10 – 15 – 10 – Current Density (mA/cm Current Density (mA/cm 5– 5–

0 – 0– Pd/GCE Pd/Sn/GCE Sn/Pd/GCE PdSn/GCE Sn/Pd/GCE Sn/Pd/EGO Figure 5. Comparison of sequential and co- Figure 6. (A) CV profiles of Sn/Pd on glassy carbon deposition of Pd and Sn on glassy carbon electrode and Sn/Pd on EGO with Pd deposited electrode (GCE) and Pd on GCE. The ratio was first before Sn with a ratio of 4:1. The ON-time optimized at 4:1 Pd:Sn. The ON-time and OFF- was optimized at 10 ms and OFF-time was time were optimized at 10 ms and 50 ms, optimized at 50 ms. CV was done in 1 M ethanol respectively. (A) CV measurements done in 1 M in 0.1 M NaOH. (B) Bar graphs showing the ethanol in 0.1 M NaOH and (B) corresponding error bars at the range of +/–1 SD of the bar graphs showing precision of CV experiment. measurements.

46 Pulse electrodeposited tin/palladium/exfoliated graphene oxide

Table 2. Repeatability Test of the Optimized Pd/GCE, Pd/Sn/GCE, Sn/Pd/GCE, and PdSn/GCE in Three Trials Relative Standard Trial 1 Trial 2 Trial 3 Mean Standard Catalyst Deviation (mA/cm2) (mA/cm2) (mA/cm2) (mA/cm2) Deviation (mA/cm2) (%) Pd/GCE 24.18 22.34 23.65 0.947 23.39 4.04 Pd/Sn/GCE 24.31 24.83 22.34 1.31 23.83 5.50 Sn/Pd/GCE 27.73 27.86 28.12 0.20 27.90 0.71 PdSn/GCE 25.10 25.36 24.05 0.69 24.83 2.77

Table 3. Repeatability Test of the Optimized Sn/Pd/GCE and Sn/Pd/EGO for Three Trials Relative Standard Trial 1 Trial 2 Trial 3 Mean Standard Catalyst Deviation (mA/cm2) (mA/cm2) (mA/cm2) (mA/cm2) Deviation (mA/cm2) (%) Sn/Pd/GCE 28.12 27.86 27.73 0.20 27.90 0.71 Sn/Pd/EGO 36.66 32.85 37.45 2.45 35.65 6.87

To test for precision, CV was done in three separate trials (i.e. three samples of each catalyst system were prepared and the CV towards EOR was measured for each sample) on the optimized Pd/GCE, Pd/Sn/GCE, Sn/Pd/GCE, and PdSn/ GCE. Table 2 shows the current densities of the three trials, the standard deviation, and the relative standard deviation (RSD) for each catalyst. Figure 5B displays the average peak current density of the catalysts together with its corresponding error bars. The RSD values for the samples are low, with Sn/Pd/GCE having the lowest RSD of 0.71%. This suggests that the CV measurements towards EOR for the Sn/ Figure 7. AFM image (10 mm  10 mm) of Sn/Pd/ Pd/GCE catalyst composites synthesized EGO at a ratio of 1:4. For the synthesis of the catalyst composite, the ON-time and OFF-time through pulse electrodeposition are repeatable. were optimized at 10 ms and 50 ms, respectively. Electrodepositing the metallic particles on EGO allows proper dispersion of the catalyst by providing nucleation sites for the density of 35.65 mA/cm2. Repeatability test was electrochemical reduction of the catalyst [12]; done by performing the experiment three times. this could also lessen the use of the catalyst Table 3 shows the results of each of the three metal without sacrificing its performance. The trials, the standard deviations for Sn/Pd/GCE voltammogram in Fig. 6A shows an and Sn/Pd/EGO. Figure 6B displays the average improvement on the performance of the catalyst peak current density of the catalysts together when deposited on EGO, giving a peak current with its corresponding error bars. Both catalysts

47 Chang PIA, Tan JL, & TongolBJVActa Manilana 64 (2016)

2.00E+04 AB3.00E+05

1.80E+04

2.00E+05 1.60E+04

Counts /s 1.40E+04 Counts /s 1.00E+05

1.20E+04

1.00E+04 0.00E+00 348 346344 342 340 338336 334 332 330 498 496494 492 490 488486 484 482 480 Binding Energy (eV) Binding Energy (eV) Figure 8. XPS analysis of pulse electrodeposited PdSn on EGO support: (A) Pd 3d and (B) Sn 3d core level spectra.

show RSD of less than 10%. This suggests that chemical reduction [17]. Meanwhile, the binding

fabricating Sn/Pd/EGO through pulse energies of Sn 3d5/2 (487.3 eV) and 3d3/2 (496.0 eV) electrodeposition is repeatable. for the electrochemically prepared PdSn/EGO composite (Fig. 8B) are higher than those of Morphological characterization. Figure 7 metallic Sn (485.0 eV and 493.4 eV). This shows bright spherical clusters deposited on indicates the presence of ionic Sn as SnO or graphene surface. This shows the successful SnO2 on the surface of Pd-Sn catalyst. The deposition and the growth of the Sn/Pd predominance of ionic Sn over metallic Sn has catalysts. Figure 7 shows that the metallic been observed by several studies [15, 17], particles appear as bright clusters in wire-like indicating that the catalyst is PdSn alloy, while arrangement. This may suggest that graphene Sn atoms on the surface are partially oxidized did provide deposition sites for Pd, as the into SnO or SnO2. [PdCl]2– ions were first adsorbed on the edges of the oxidized graphene. Under pulsed potential CONCLUSION condition, the ionic concentration is sufficiently high to deposit the Pd clusters. With increasing This study has utilized pulse electrodeposition number of potential pulses, the average size of for the fabrication of Sn/Pd/EGO catalyst. The the Pd cluster gradually enlarges, presumably, use of electrochemical techniques has provided the bright clusters in wire-like arrangement [19]. a simple and fast method of fabricating high performance catalyst for ethanol oxidation X-ray photoelectron spectroscopy analysis. reaction. Cyclic voltammetric studies revealed Figure 8A shows the Pd 3d core level spectra of that pulse parameters such as ON-time and OFF- pulse electrodeposited PdSn on EGO support. time, ratio of the binary catalyst, mode and The two distinct peaks located at 335.6 eV and sequence of deposition, among others, have an effect on the performance of the catalyst 340.8 eV can be assigned to the Pd 3d5/2 (high- towards ethanol oxidation reaction. The Sn/Pd/ energy band) and Pd 3d3/2 (low-energy band) spin orbit states of metallic Pd, while the less EGO catalyst (i.e. Pd deposited first before Sn, intense doublets around 337.1 eV nd 342.5 eV with 10 ms ON-time and 50 ms OFF time, and a ratio of 4:1 [Pd:Sn]) gave the highest catalytic are attributable to 3d3/2 and 3d5/2 peaks of PdO. The peak assignments are in agreement with a activity with an average current density of previous study for PdSn catalyst prepared by 35.65 mA/cm2. AFM image showed that the

48 Pulse electrodeposited tin/palladium/exfoliated graphene oxide

catalysts were deposited as bright clusters on [9] Wang Y, Wang X, & Li C. Electrocatalysts of Pd- graphene surface. XPS analysis confirmed the Co supported on carbon black or ball-milled presence of Pd and Sn which exist in metallic carbon nanotubes towards methanol oxidation in alkaline media. Applied Catalysis B: and oxide forms. Environmental 2010; 99:229–234. [10] Tan JL, Villar PG, De Jesus AM, & Tongol BJV. A ACKNOWLEDGMENT green approach to the synthesis of Pd-Ni/ Graphene via electrochemical exfoliation of The research funding from the Philippine graphite from used battery for the Council for Industry, Energy, and Emerging electrocatalysis of ethanol. Journal of Chinese Technology Research and Development of the Chemical Society 2014; 61:774–777. Department of Science and Technology [11] Krishna R, Fernandez DM, Ventura J, Freire C, & (PCIEERD-DOST) is gratefully acknowledged. Titus E. Facile synthesis of reduced graphene oxide supported Pd@Ni B/RGO nanocomposite: The authors are grateful as well to the Center x Novel electrocatalyst for ethanol oxidation in for Core Research Facilities, Daegu Gyeongbuk alkaline media. International Journal Hydrogen Institute of Science and Technology (DGIST), Energy 2016; 41:11811–11822. South Korea, for the XPS analysis. [12] Dong L, Gari RRS, Li Z, Craig MM, & Hou S. Graphene-supported platinum and platinum- REFERENCES ruthenium nanoparticles with high electrocatalytic activity for methanol and ethanol. [1] Modibedi R, Masombuka T, & Mathe M. Carbon Carbon 2010; 48(3):781–787. supported Pd-Sn and Pd-Ru-Sn nanocatalysts [13] Zhang Z, Xin L, Sun K, & Li W. Pd-Ni for ethanol electro-oxidation in alkaline medium. electrocatalysts for efficient ethanol oxidation International Journal of Hydrogen Energy 2011; reaction in alkaline electrolyte. International 36:4664–4672. Journal of Hydrogen Energy 2011; 36(20):12686– [2] Lamy C, Lima A, LeRhun V, Delime F, Countanceau 12697. C, & Leger JM. Recent advance in the [14] Wang Y, Zhao Y, Yin J, Liu M, Dong Q, & Su Y. development of direct alcohol fuel cells (DAFC). Synthesis and electrocatalytic alcohol oxidation Journal of Power Sources 2002; 105:283–296. performance of Pd-Co bimetallic nanoparticles [3] Xu C, Shen P, & Liu Y. Ethanol electrooxidation supported on graphene. International Journal on Pt/C and Pd/C catalysts promoted with oxide. of Hydrogen Energy 2014; 39:1325–1335. Journal of Power Sources 2007; 164:527–531. [15] Du W, Mackenzie KE, Milano DF, Deskins NA, Su [4] Xu C, Cheng L, Shen P, & Liu Y. Methanol and D, & Teng X. Palladium-Tin alloyed catalysts for ethanol electrooxidation on Pt and Pd supported the ethanol oxidation reaction in an alkaline on carbon microspheres in alkaline media. medium. ACS Catalysis 2012; 2:287–297. Electrochemistry Communications 2007; 9:997– [16] Mao H, Wang L, Zhu P, Xiu Q, & Li Q. Carbon-

1001. supported PdSn-SnO2 catalyst for ethanol [5] Antolini E. Catalysts for direct ethanol fuel cells. electro-oxidation in alkaline media. International Journal of Power Sources 2007; 170:1–12. Journal of Hydrogen Energy 2014; 39:17583– [6] Hayre R, Cha SW, Colella W, & Prinz F. Fuel Cell 17588. Fundamentals, 2nd ed. (New York: John Wiley & [17] Feng Y, Bin D, Zhang K, Ren F, Wang J, & Du Y. Sons, 2008). One-step synthesis of nitrogen-doped [7] Watanabe M & Motoo S. Electrocatalysis by ad- graphene-supported PdSn bimetallic catalysts for atoms: Part II. Enhancement of the oxidation of ethanol oxidation in alkaline media. RSC methanol on platinum by ruthenium ad-atoms. Advances 2016; 6:19314–19321. Journal of Electroanalytical Chemistry and [18] Nagaraju DH & Suresh GS. Green chemistry route Interfacial Electrochemistry 1975; 60(3):267– to the synthesis of palladium nanoparticles on 273. reduced graphene oxide for ethanol fuel cell [8] Hsieh C, Wei J, Lin J, & Yang B. Preparation of application. ECS Electrochemistry Letters 2012; Pt-Co nanocatalysts on carbon nanotube 1(2):F1-F3. electrodes for direct methanol fuel cells. Diamond & Related Materials 2011; 20:1065–1071.

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[19] Hsieh T, Liu Y, & Roy A. Pulse electrodeposited [22] Jagdeep S, Peiris N, & Wijayantha U. Rapid and Pd nanoclusters on graphene-based electrodes simple potentiostatic deposition of copper(II) for proton exchange membrane fuel cells. oxide thin films. Electrochemistry Electrochimica Acta 2012; 64:205–210. Communications 2014; 42:68–71. [20] Su CY, Lu AY, Xu Y, Chen FR, Khlobystov AN, & [23] Hsieh C, Wei J, Hsiao H, & Chen W. Fabrication Li LJ. High-quality thin graphene films from fast of flower-like platinum clusters onto graphene electrochemical exfoliation. ACS Nano 2011; sheets by pulse electrochemical deposition. 5:2332–2339. Electrochimica Acta 2012; 64:177–182. [21] Chandrasekar MS & Pushpavanam M. Pulse and pulse reverse plating – conceptual, advantages, and applications. Electrochimica Acta 2008; 53:3313–3322.

50 Acta Manilana 64 (2016), pp. 51–57 Printed in the Philippines ISSN: 0065–1370

Intelligent backpack weight assessment system

Diana Marie M. Bulatao, Precious Mae V. Carolino, Aizel Cate S. Equipaje, Cristine Jin DS. Estrada, Paolo Adrian D. Papa, & Edison A. Roxas*

Department of Electronics Engineering, Faculty of Engineering University of Santo Tomas, 1015 Manila, Philippines

Overloaded backpacks have been shown to be associated with the tendency of having back, neck, shoulder pains, posture problems and even musculoskeletal disorders. A design for a backpack system is presented that will alleviate these negative effects. The backpack system is equipped with sensors and microprocessors that will alert the user when the load exceeds his maximum weight bearing capacity. The system will automatically obtain the anthropometric measurements of the user and determine from these data the acceptable load, which has been suggested to be 10 to 15% of the user’s body weight.

Keywords: backpack load, intelligent system, maximum weight bearing capacity

INTRODUCTION emergent and developing; therefore, they are more vulnerable to injury. With the strongest From a canvas bag in the 1920’s to a limited- muscles in the back and in the abdomen, it is edition backpack featuring solar panels, it is possible to support backpacks with excessive evident how backpacks have technologically weights. However, it is alarming that year after advanced to compensate the additional demands year, U.S. Consumer Product Safety Commission of consumers [1]. Granted that there are cell reports 5,500 Americans suffering from phones and iPod compartments, and padded backpack-related injuries and that 64 percent of sleeves for laptops, as well as air cushioning in those diagnosed are children [2]. the straps, there are other issues which concerns overloading that although recognized and A study led by Manila Doctor’s Hospital have addressed, a serious monitoring and control provided statistics indicating that 54% of the should be implemented for the prevention of grade 5–6 elementary students from selected long term health problems. schools in Metro Manila have been found out to be carrying backpacks of weight more than Several studies have shown a considerable 10% of their body mass. From these group, 23% relationship between backpack weight and the of which are identified as having pain in the occurrence of health-related effects especially back [3]. A noticeable effect of excessive loading to the students since their bodies are still being of backpack is when there is a need for a child to bend forward [4]. Low back pains or degenerative *To whom correspondence should be addressed disorders due to carrying loads with bent back [email protected]

© 2016 UST Research Center for the Natural and Applied Sciences, Manila, Philippines Bulatao DMM at al.Acta Manilana 64 (2016) are brought about by bearing loadings over circumference, and the weight of the backpack. extended time [5]. This innovation will provide solution to the negative effects of backpack overloading by As a result, experts suggest that the suitable implementing a technology that could be weight of backpacks should be, at maximum, 10– designed and implemented to diminish the 15% of a person’s body weight. It is scientifically negative effects of excessive weight of upper proven that going over this value will induce extremity load and thus adapt ways to be able the occurrence of neck and back pain. Cases of to alert the user with his maximum weight bearing such discomforts have been the complaint of capacity. 10–30% of healthy children who have experienced these in their teenage years [6]. EXPERIMENTAL

Efforts to prevent backpack injuries emerge to System hardware reduce the number of affected individuals. First, the solution is basically about eliminating the A lay-out of the intelligent backpack system is need to carry the backpack such as by adding shown in Fig. 1. The prototype was made from rollers or wheels so that it can be pulled rather an existing frame of a hiking backpack which than to be carried. It is certainly helpful but in was then reinforced to fit the desired prototype. real practice, this method has been less than It was composed of a three-division effective because of reluctance of many, compartment with two wide padded shoulder particularly the adolescents, to use this straps. It also had a devised waist belt, which wheeled-feature backpack. Furthermore, the had a limit switch and a series of resistors to backpack usually has to be lifted at some point measure the waist circumference of the user. such as when the student will ride to a vehicle Weight sensor. The weight sensor that was used or when the pathway of the student is not in the study is a Flexiforce A201 force sensor as appropriate for the wheels to be used. Then, displayed on Fig. 2. In a circuit, it acts as a force there comes the additional structures of the sensing resistor. The application of the force to backpack such as wide, padded shoulder straps, and belt to be fastened at the abdomen and/or hip.

As these attempts are unsuccessful, researchers 1 2 1 Microcontroller 2 SD/MMC Shield continue to conduct study with regard to the 3 DS 1307 RTC 4 Ultrasonic Sensors (x2) prevention of backpack injuries until the use of 5 FlexiForce technology arise. The present innovation 6 9v Battery Pack includes studies about equipping the backpack with a weight assessment system using mechanical or electrical devices [7]. 3 6 This paper describes a design for a backpack 4 5 system equipped with sensors and microprocessors that will alert the user when Figure 1. Backpack layout the load exceeds his maximum weight bearing capacity. The system will automatically obtain the anthropometric measurements of the user such as the height, weight, and waist Figure 2. Flexiforce A201 sensor [8]

52 Intelligent backpack weight assessment system

the active sensing area would cause to a change concentrate the load on the Flexiforce itself and in resistance of the sensing element in inverse to allow precise measurement proportion to the force applied. Figure 3 shows the variation of the sensor resistance with the Ultrasonic sensor. A US-100 ultrasonic sensor weight load. A puck, or shim, was placed on the was incorporated in the system to measure the sensing area to concentrate and ensure that it distance from the bottom of the backpack to the captures the applied load whether its surface is ground as, as a requirement to be used in larger than the sensor diameter. estimation of the height of the user. The sensor assesses features of a target by interpreting the The force sensor into an application was echoes from transmitted sound waves. It incorporated to a force-to-voltage circuit (Fig. establishes the distance of the object through 4). A 5.1 k gave the most precise sensitivity of the time interval as the signal was sent as to its the sensor. A fixed resistor of 100 k was also echo had received. Figure 6 presents the location used to ample the voltage divider circuit. of the ultrasonic sensor, in actual.

Figure 5 displays the actual setup of the Devised waist belt system. A waist belt system Flexiforce sensor on a devised base. The base was devised to measure the waist circumference is designed such that its size is fitted at the of the user. This data is necessary to approximate bottom of the backpack. The base is also the weight of the user [9]. The system is based equipped with soft flexible springs to on the ability of the system to generate several

30

25 )

 20

15

10 Resistance ( 5

0 0246810 Weight (kg) Figure 5. Actual setup of the Flexiforce A201 sensor Figure 3. Calibration curve of weight sensor on the base of the backpack (Flexiforce A201)

Aceduino Nano ATMega328

5V Gnd A0

R1

5.1k? R2 100k?

Figure 4. Schematic circuit for the Flexiforce sensor Figure 6. Location of the ultrasonic sensor

53 Bulatao DMM at al.Acta Manilana 64 (2016) predetermined waist measurements using an remote location which can cause the sensor array of resistors. being actuated by an object other that human operation. It involves a series of 1K ohm resistors placed on the belt as shown in Fig. 7. Every resistor Microcontroller. All of the sensors were was positioned adjacent to each other with a interfaced into a microcontroller which is constant displacement of 20 mm in between. The programmed to store the acquired data to the gaps correspond to a fixed adjustment that fits memory card for the database system. It is also the waist circumference of the user. exploited to compute all of the needed values as the algorithm to be followed were also etched Once the movable/adjustable strap is engaged into it. The microcontroller that was used in this to a certain position, the corresponding resistor study is Aceduino ATmega328 as displayed on is neutralized and sends open circuit signal to Fig. 8. the microprocessor. Each resistor corresponds to a waist measurement. System operation scheme

Limit switch. A limit switch was integrated in The schema of the system operation is presented the buckle of the belt system for the on Fig. 9. The initialization of the variables to be measurement of the waist circumference of the used on all of the consequent procedures are user. It is a type of sensor that cut off power obtained under the input pre-processing stage. automatically at or near the limit of travel of a Gathered data from the sensors were assessed moving object controlled by any means. This to equip in the preparation for processing. sensor is operational when there is a contact on an actuator of the switch that is mounted on a Calculation of the adequate weight of the backpack will be executed subsequent to the

Figure 7. Series of resistors with limit switch for the belt system

Figure 9. General overview of how the overall Figure 8. Aceduino ATmega328 microcontroller system works

54 Intelligent backpack weight assessment system preparation of the variables to be utilized in the Load = 0.467819849 × Flexiforce + 2.006886307 process. Given that the approximation of the (1) body weight of the user can be determined, the equivalent 12.5% of its body weight can be where the variable Flexiforce represents the attained. This will search out the maximum voltage measured in accordance to the load capacity of the body of an individual that he inside the backpack. ought to carry. Moreover, it represents the utmost value under a tolerance of ±2.5% that is The linear regression model derived from the still acceptable for the user as the weight of the voltage measured by the Flexiforce sensor load. yielded a predicted backpack load for every weight applied with a range of 1.5–9 kg. Evaluation whether the backpack is at acceptable Interpreting the equation, for each unit increase weight was implemented through the comparison in the Flexiforce sensor value, the voltage between the actual weight of the backpack increases with 0.467819849 units. This equation detected by the sensor and the calculated is then used to do a forecast in the backpack acceptable load for the user consequently. In load using sensor value measured by the this process, it will determine the safe load Flexiforce. carriage for an individual and set it out directly to the output through a feedback. Model for user’s height. Equation 2 expressed the linear regression model of height on the If the load is unsafe or has exceeded the distance measured by the ultrasonic sensor in threshold for maximum capacity that an the following form: individual should carry, it has a buzz like sound that has a continuous increasing frequency to Height = 1.495935171 × Ultrasonic + convey awareness to the user. Furthermore, 32.94391256 (2) there is a database that will serve as the where the variable Ultrasonic represents the monitoring means for the weight of the backpack distance measured from the bottom of the and the user’s height, weight and waist backpack to ground. circumference, through a memory card. There will only be a transmission of data onto the The linear regression model derived from the database only if the weight sensor will detect a distance measured by the ultrasonic sensor substantial change in load. yielded a predicted user’s height for every individual respondent. Interpreting the equation, Modelling. To process both the linear and for each unit increase in the ultrasonic distance, multiple regressions in this study, the Analysis the user’s height increases with 1.495935171 ToolPak add-in in Excel is utilized. There are three units. This equation is then used to do a forecast models developed corresponding to the in the user’s height using whatever distance prediction of the height of the user, weight of measured by the ultrasonic sensor. the user, and the weight of the backpack load. Model for user’s weight. Equation 3 expressed Model for backpack load. Equation 1 expressed the multiple regression model of the user’s the linear regression model of Load on the weight on the height of the user and on the voltage measured by the Flexiforce sensor in waist circumference of the user in the following the following form: form:

55 Bulatao DMM at al.Acta Manilana 64 (2016)

Weight = 1.540038776 × Height + 1.279653512 for the different backpack loads such as the test × Waist – 225.3635219 (3) weight, and the corresponding 10%, 12.5%, and 15% of the user’s body weight. In general, the where the variable Height represents the height ranges of percent difference are from around of the user and the waist represents the waist 0% up to 10%. circumference of the user. Table 3 shows the ranges of percent differences The multiple regression model derived from the for the anthropometric measurements such as height and waist circumference yielded a the weight, height, and waist circumference of predicted weight for every individual the user. In general, the ranges of percent respondent with age between 10–24 years old. difference are again from around 0–10%. Interpreting the equation, for each unit increase in the height, the user’s weight increases with Percent differences for the different backpack 1.540038776 units, and for each unit increase in loads and for the anthropometric measurements waist circumference, the user’s weight increases are also computed. The measured values coming with 1.279653512 units. This equation is then from the sensors and the theoretical values are used to do a forecast in the user’s weight using compared in this part. It is observed that only whatever height and waist circumference measured by the ultrasonic sensor and belt system, respectively. Table 1. Audio Response DATA Alert System RESULTS AND DISCUSSION Alert goes Alert goes Alert goes off at off at 10% off at 15% In order to check the accuracy of the weight 12.5% of of user’s of user’s user’s alert backpack, measurements were carried out body body body weight weight on a total of 120 respondents ranging from 10 to weight 24 years old; 62 were males and 58 were females. Hit 101 102 97 Their anthropometric measurements such as Miss 19 18 23 their weight, height, and waist circumference are Total 120 120 120 Percentage Hit 84.17% 85% 80.83% first obtained. After which, they are asked to wear the backpack and the respondents carried a test weight of 3 kg, and their corresponding Table 2. Ranges of Percent Differences for the Different Backpack Loads 10%, 12.5%, and 15% of their body weight. The proponents monitored whether the alert system Ranges of Percent Differences for the Backpack Loads goes off whenever the 10% of the weight of the Minimum Maximum user or a weight more than that is reached. A Test Weight (3 kg) 0.33% 10.67% “hit” is achieved when the alarm triggered, 10% of Body Weight 0% 8.33% otherwise, it is “miss”. 12.5% of Body Weight 0.06% 10.51% 15% of Body Weight 0.09% 10.10% Table 1 shows the summary of the audio response wherein 84.17%, 85%, and 80.83% of Table 3. Ranges of Percent Differences for the the tests indicate that the system was able to Anthropometric Measurements give off an alarm at the stage where the load of Ranges of Percent Difference for the the backpack is at 10%, 12.5% or 15% of the Anthropometric Measurements Minimum Maximum user’s body weight, respectively. Weight 0.03% 9.70% Height 0.01% 10.20% Table 2 shows the ranges of percent differences Waist Circumference 0% 9.52%

56 Intelligent backpack weight assessment system minimal values of percentage error are present measurement is obtained, and the instantaneous on both cases. anthropometric measurements of the user and the corresponding bag weight he is carrying. For the test weight of 3 kg, the percent difference ranges from 0.33% to 10.67%. For a backpack CONCLUSION load corresponding to 10% of the user’s body weight, the percent difference ranges from 0% A backpack that can automatically approximate to 8.33%. For a backpack load corresponding to the weight of the user in order to determine the 12.5% of the user’s body weight, the percent precise weight of the load appropriate for the difference ranges from 0.06% to 10.51%. And body by establishing the maximum weight for a backpack load corresponding to 15% of bearing capacity has been successfully the user’s body weight, the percent difference designed and implemented. ranges from 0.09% to 10.10%. REFERENCES For the user’s weight, the percent difference [1] Horovitz B. August 2007. [Online]. Available: http:/ ranges from 0.03% to 9.70%. For the user’s /abcnews.go.com/. height, the percent differences ranges from [2] Zielonka J. Why Your Child Needs a “Backpack 0.01% to 10.20%. And for the user’s waist Buddy” 2011. circumference, the percent difference ranges [3] P.P.Society. Backpacks and Children. Philippine from 0% to 9.52%. Having minimal values of Pediatric Society Policy Statements 2006; 1(3):11–16. percentage error implies that the system [4] Don’t Let Backpacks Get You Down. Children’s embedded on the backpack will be able to Healthcare of Atlanta 2009. provide weights close to the actual load being [5] Luttman A, Jager M, & Griefahn B. Preventing carried by the user. In addition, the weight, Muscoloskeletal Disorders in the Workplace. height, and waist circumference will be closely Protecting Workers’ Health Series 2003; 5:1– approximated with the actual measurements of 32. the user. [6] Ramprasad M, Alias J, & Raghuveer A. Effect of Backpack Weight on Postural Angles in Preadolescent Children. Indian Pediatrics 2010 For the database monitoring part, Fig. 10 July; 47:575–580. displays a sample of a content of a .txt file saved [7] Ross A. System and Related Methods for in an SD memory card. The database stores Preventing Back Injury. (United States of America details involving the date and time the Patent 7404506, 2008 July 29). [8] Tekscan. Flexiforce Sensors User Manual. (South Boston Massachusetts: Tekscan, Inc., 2010). 2/7/2015 11:26:19 [9] Training Manual No. 1: Limit Switches – 101. height:152.18 cm 1206 Hatton Road. waist circumference: 73 cm bagweight:8.32 kg weight46.55 kg

2/7/2015 11:26:22 height:152.34 cm waist circumference: 73 cm bagweight:8.50 kg weight46.66 kg

2/7/2015 11:26:25 height:152.21 cm waist circumference: 74 cm bagweight:8.29 kg weight47.15 kg Figure 10. Sample content of the database

57 Acta Manilana 64 (2016), pp. 59–66 Printed in the Philippines ISSN: 0065–1370

Mathematical modeling of thin-layer drying kinetics of carabao mango in a hot-air dryer

Fidel Ivan T. Labutong, Janet Stephanie F. Pastores, Angelyn C. Yeung & Lola Domnina B. Pestaño*

Department of Chemical Engineering, Faculty of Engineering University of Santo Tomas, 1015 Manila, Philippines

Carabao mango, also known as the Philippine mango, is one of the most important varieties of mango cultivated in the Philippines. Like any other fruit, carabao mango is susceptible to various diseases making it highly perishable and has a very short span of shelf life. One way of extending the shelf life of carabao mango is through drying. Drying mango meat slices until moisture content (MC) of the mango pulp at 12% inhibits the growth of microorganisms and the action of enzymes that causes spoilage. This research aims to develop a drying method to determine the suitable thin-layer mathematical model that describes the drying kinetics of thinly sliced carabao mangoes and subsequently to predict the optimum drying temperature and drying time to attain 12% MC in the mango pulp. Three mathematical models, namely, Laplace Transform Model, Page Model, and Non-linear Decomposition Model were examined to describe the drying behavior of thin mango slices at 60, 70, and 80°C using a hot-air batch dryer. Laplace Transform model gave the best fit with the least total error. The optimum temperature was observed to be at 60°C being more efficient to produce quality dried carabao mangoes at the shortest drying time of 209.51 min.

Keywords: carabao mango, drying kinetics, hot-air batch dryer, mathematical modeling

INTRODUCTION varieties of mango cultivated in the Philippines. The variety is reputed internationally due to its Mango (Mangifera indica) is one of the top sweetness and exotic taste [2]. Carabao mango important fruit crops grown in the Philippines. like any other fruit is susceptible to various It ranks third next to banana and pineapple diseases making it highly perishable and has a based on volume export and value. Fresh and very short span of shelf life. As a result, food processed mangoes have already established preservation methods were developed to extend inside and out of the Philippines where Hong its storage span. One way of extending the shelf Kong and Japan absorb 89% of the country’s life of carabao mango is through drying. Removal export [1]. Carabao mango, also known as the of water is necessary so that growth of Philippine mango, is one of the most important microorganisms will not manifest.

Drying, a unit operation involving the removal *To whom correspondence should be addressed of moisture content (MC) from a wet solid [3], of [email protected]

© 2016 UST Research Center for the Natural and Applied Sciences, Manila, Philippines Labutong FIT, Pastores JSF, Yeung AC, & Pestaño LDBActa Manilana 64 (2016)

agricultural products is of great importance for MATERIALS AND METHODS the preservation of food by human beings [4]. Drying inhibits the growth of bacteria and has Materials. Fresh, healthy, yellow carabao been practiced worldwide since ancient times mangoes from Davao, Philippines with exact to preserve food. ripeness aging from 10–15 days were carefully chosen for this study. The fruits were tapped Drying mango meat slices until MC of the mango and patted to feel the exact ripeness. The pulp at 12–15% inhibits the growth of mangoes were washed, wiped with a clean piece microorganisms and the action of enzymes that of cloth, peeled manually and sliced into causes spoilage [5]. In the food industry, mango rectangular shape with varied length and width is not only dried for the purpose of preservation to fill the spaces on the tray but keeping the but also for modification of taste, flavor and thickness constant at 5-mm thickness for thin- texture needed to meet consumer preferences. layer drying using a sterilized stainless steel Color is one of the factors that influence the knife. The procedure was done in an enclosed impact of dried mango in the market. As the room with a stainless steel work table and sink mango is being dried, browning occurs. This is for washing up. The whole procedure was due to the oxidation of an enzyme naturally carried out hygienically to preserve the occurring in plants or the operating temperature. freshness of the mangoes free from Oxidation is a reaction that cannot be prevented contaminants. after mango has already been peeled while high operating temperatures can lead to deterioration Hot-air dryer. A hot-air batch dryer was of the nutrients, create off-flavors and induce fabricated with dimensions 20 cm × 20 cm × 20 textural changes [6]. cm. It was made of aluminum sheets, with its inner part insulated with aluminum laminated air The drying kinetics of food is a complex bubble insulation material. It consisted of the phenomenon and requires simple following parts: (1) a drying chamber, which representations to predict the drying behavior, housed, (2) a static square tray, (3) a heating and for optimizing the drying parameters. Many element made of an electric coil that is enclosed investigators have carried out mathematical in a tubular duct, (4) an axial blower that modeling and experimental studies on the thin- facilitates the distribution of air along the tubular layer drying of various vegetables and fruits duct towards the drying chamber, (5) a [7]. Studies on thin-layer drying were done on temperature control system that controls and potato slices [8], onion slices [9], sweet cherry [10], and banana [11]. However, there is limited information and research on drying kinetics of mango slices in literature [7]. This paper is carried out to develop a drying method to determine the suitable thin-layer mathematical 1 2 model that describes the drying kinetics of thinly sliced carabao mangoes and subsequently to 4 5 predict the optimum drying temperature and 3 drying time to attain 12% MC. 6

Figure 1. Diagram of the hot-air dryer: (1) drying chamber, (2) tray, (3) heating element, (4) axial blower, (5) temperature control system, (6) air channeling sheet, and (7) exhaust hood.

60 Mathematical modeling of thin-layer drying kinetics of carabao mango in a hot-air dryer displays the temperature in the drying chamber, respectively. The R was calculated from the (6) a perforated air channeling sheet that directs experimental data using Equation 2: the heated air perpendicularly through the tray, W Xd ' and (7) an air exhaust hood. A diagram of the R  S (2) hot-air dryer is shown in Fig. 1. A dt where W is the weight in g, of the dry solid, A is Drying experiment. The hot-air dryer was s the surface drying area in m2, X’ is the bulk MC installed in an environment with 30% relative air and t is time. Equation 2 can be rearranged and humidity and an ambient air temperature of integrated. The integral form Equation 3 was about 28°C. The air velocity through the dryer used to obtain the drying time. was controlled by the speed of the blower. A portable handheld multifunction anemometer t W X '2 Xd ' dt  S (3) TA465 was used to measure the temperature and 0 A X '1 R air velocity inside the hot-air dryer and the relative humidity of the environment. The drying data (MR vs. t) at 60, 70 and 80°C were fitted into the three mathematical models: Single layers of thinly sliced carabao mango Page Model, Non-linear Decomposition Model, were arranged on the tray and was heated at and Laplace Transform Model. Drying is a temperatures of 60–80°C at 10°C intervals every complex process and as a means to simplify the 15 min until constant weight is obtained. The analysis of the drying kinetics of mangoes, spaces in between the three rows of the mango empirical expressions are used [12]. slices ensure that drying air circulates in the Mathematical modeling using thin-layer drying drying chamber and comes in contact with both equations are used to evaluate drying time of sides of the mango slices that conform to the products based from experimental data. Models conditions of thin-layer drying. Before beginning are often used to study the variables involved the experiments, the air dryer system was in the process, predict drying kinetics of the preheated in order to achieve a desirable steady product and optimize the operating parameters state condition of temperature at constant air and circumstances [13]. Values of the correlation velocity of 0.80 m s–1. coefficient, R2, for Page Model and Non-linear Decomposition Model, the time constant, tau,  Weighing of air-dried mango slices was done for the Laplace Transform Model and the total using a precision balance with an accuracy of error for the three models were computed. The 0.01 g. Drying experiments for each temperature selection of the best model to describe the were conducted in triplicate. drying behavior of mango slices will be based on the least total error and the goodness of fit Mathematical modeling. The parameters that of a model. will be used in the modeling are the moisture ratio (MR) and the drying rate (R). The MR The Page Model was successfully used to during drying was calculated using Equation 1: describe the drying characteristics of some M  M agricultural products [10, 14, 15]. The Page Model MR  e [16, 17] that was used in this study was derived M  M (1) i e by linearizing the R equation shown in Equation 4. where M, Mi and Me are the MC, in g, at any n time t, the initial MC and the equilibrium MC, MR aekt (4)

61 Labutong FIT, Pastores JSF, Yeung AC, & Pestaño LDBActa Manilana 64 (2016)

Equation 5 is the linearized form of Equation 4. The determination of the MR is represented by (5) the concentration [20] and is represented below MR  ln a  kt n in Equation 9. The Laplace Transform Model and Non-Linear n1 MRo Decomposition Model are used for prediction n1 MR  n1 (9) of the behavior of a system as reduction of 1 (n  )1 ktMRo moisture proceeds until it reaches equilibrium [18]. The Laplace Transform Model was derived Determination of drying time to attain 12% from a material balance of the system in the MC. The drying time needed to attain the experiment. The general material balance states needed 12% MC was determined through that the input minus the output of the system is Lagrange interpolation. This method generates equal to the rate of accumulation as shown in nth-order polynomials that pass through (n + 1) Equation 6. points. These polynomials attempt to produce dM interpolation values of increased accuracy by M  M  (6) assuming a curvature in the relationship of the i o dt data. Its general equation is represented by Equation 6 is then formulated to its Laplace Equation 10. The three versions are Equations Transform Model [19], integrated and simplified 11–13. forming Equation 7 used in modeling the drying rate characteristics of the carabao mango slices. The Lagrange interpolation: t t n     (7) fn x)(  Li xfx i )()( M  M f  M f e  ieM  (10) i0

The Non-linear Decomposition Model is based First-Order version: on the differential equation for batch x  x x  x decomposition as given in Equation 8. xf )(  1 xf )(  0 xf )( 1 x  x 0 x  x 1 (11) 1 1 0 1 1 0   (n  )1 kt n1 n1 (8) C Co

Second-Order version:

(x  x1)(x  x2 ) (x  x0 )(x  x2 ) 1 xf )(  xf 0 )(  xf 1)( (x0  x1)(x0  x2 ) (x1  x0 )(x1  x2 )

(x  x0 )(x  x1) (12)  (xf 2 ) (x2  x0 )(x2  x1)

Third-Order version:

x(  x1 x)(  x2 x)(  3 )x x(  x0 x)(  x2 x)(  3 )x 3 )x(f  0 )x(f  1 )x(f x( 0  x1 x)( 0  x2 x)( 0  3 )x x( 1  x0 x)( 1  x2 x)( 1  3 )x

x(  x0 x)(  x1 x)(  3 )x x(  x0 x)(  x1 x)(  2 )x (13)  2 )x(f  3 )x(f x( 2  x0 x)( 2  x1 x)( 2  3 )x x( 3  x0 x)( 3  x1 x)( 3  2 )x

62 Mathematical modeling of thin-layer drying kinetics of carabao mango in a hot-air dryer

Quality of dried carabao mango. The air-dried Temperature is one of the main factors that carabao mango slices were stored in sealed influence the drying kinetics of carabao mango. containers made from low density polyethylene Drying rate has a direct proportional relationship at an ambient temperature of 28°C. The air-dried with temperature making the R faster at elevated samples were set aside for five weeks to check if temperature. The shortest drying time to reach browning of the air-dried samples occurs. equilibrium moisture was obtained using 80°C as drying temperature, which only took 570 min., RESULTS AND DISCUSSION followed by 70°C, which took 660 min. and finally, 60°C which took 735 min. The color of dried Drying behavior. The drying curves (Fig. 2) mango slices was observed to vary depending show the changes in the experimental data-MR on the temperature. As the temperature of the mango slices with drying time at indicated increased, the product became darker in color. temperatures (60, 70 and 80°C). The drying of the thin mango slices exhibited the characteristic Mathematical modeling. The MR data were moisture desorption behavior. An initial high rate fitted into the three models. The results, namely, of moisture removal was followed by slower values of the correlation coefficient, R2, for the moisture removal in the latter stages. The Page Model and Non-Linear Decomposition characteristic behavior is due to the various Model, the time constant, tau,  for the Laplace forms in which water is present in food products Transform Model and the total error for the [21]. This is a general trend reported for other three models were recorded and are shown in food products e.g. mulberry, eggplant, tomatoes, Table 1. The best results based on the least total sweet pepper, and peach slices [22–26]. error were shown by the Laplace Transform Model at the three temperatures studied. The Laplace Transform Model yielded notable accuracy, precisely modeling the data series of 6– Drying Characteristics of Air-Dried Carabao Mango carabao mango slices and predicting its future 5– behavior. Using visual inspection, the Laplace Transform Model also gave the best fit as 4– 60°C shown in Fig. 3. Hence, the Laplace Transform 70°C 3– Model may be assumed to represent the thin- 80°C layer drying behavior of carabao mango slices. 2–

Moisture Ratio Similar findings were reported for the air-drying 1– kinetics of thin slices of coconut meat [19]. Earlier research works reported that the Page 0– 0 100 200 300 400 500 600 700 800 Model represent the drying kinetics of raw Drying Time (min) mango slices [7, 27]. Figure 3 shows the Figure 2. The mean drying characteristics of air- experimental drying kinetic data as they are fitted dried mango slices at different temperatures

Table 1. Summary of Results

Non-linear Laplace Page Model Temp. Decomposition Model Transform Model n R2 Total Error n R2 Total Error  Total Error 60C 0.75 0.9572 5.7400 1.14 0.9353 2.0536 59.0 0.3058 70C 0.64 0.9570 6.0859 1.19 0.9067 1.3676 41.0 0.4843 80C 0.66 0.9528 6.6387 1.20 0.9469 3.7151 37.2 1.6976

63 Labutong FIT, Pastores JSF, Yeung AC, & Pestaño LDBActa Manilana 64 (2016)

6– Page Model, 60°C with the simulated models at 60°C drying 5– temperature.

4– Drying time analysis. The drying time for the 3– carabao mango slices to reach the desired MC 2– Expt’l of 12% at which the growth of microorganisms Predicted 1– is unlikely to occur was significantly affected Moisture Ratio 0– by the drying temperature. The Laplace 0 100 200 300 400 500 600 700 800 Transform Model was used to predict the –1 – Time (min) optimum drying time at which the MC of the

6– Non-Linear Decomposition Model, 60°C mango slices will be 12%. The drying time was computed using Lagrange interpolation. The 5– average drying time to reach 12% MC from the 4– initial range of 42–50% was 213.43, 149.44 and 3– 139.22 min at drying temperatures of 60, 70 and 2– Expt’l 80°C, respectively. Table 2 shows the calculated Predicted 1– heating time as predicted by the Laplace Moisture Ratio Transform Model. A shorter heating time at 80°C 0– 0 100 200 300 400 500 600 700 800 was utilized to attain a 12% MC in the mango –1 – Time (min) slices except for 70°C at Trial 3 due to changes in the surrounding conditions, which affected 5– Laplace Transform Model, 60°C the ability of the air to absorb moisture. 4– Observation of the color of the air-dried mango 3– pulp. The appearances of dark spots that 2– indicate the growth of microorganism were Expt’l monitored by visual inspection from the air-dried 1– Predicted

Moisture Ratio mangoes. With 1-week interval of monitoring, it 0– was observed that after 5 weeks, browning of 0 100 200 300 400 500 600 700 800 –1 – Time (min) the mango pulp was not evident for all air-dried carabao mango samples. The retention of the Figure 3. Comparison of the experimental and predicted values of MR vs. time at 60oC using yellow color of the mango pulp is due to the the three models removal of water using hot-air drying that inhibits the growth of microorganisms.

CONCLUSION Table 2. Calculated drying time at different treatment temperatures as A drying method was developed using a hot-air predicted by the Laplace Transform dryer to determine the drying kinetics of carabao Model mango. The Laplace Transform Model best fits the drying kinetics of thinly sliced carabao Temperature Trial No. 60C 70C 80C mango with the least total error and good visual 1 209.51 min 154.67 min 139.28 min inspection. The optimum drying temperature 2 215.24 min 154.28 min 139.21 min was observed to be at 60°C being more efficient 3 215.54 min 139.96 min 139.17 min to produce quality dried carabao mangoes at

64 Mathematical modeling of thin-layer drying kinetics of carabao mango in a hot-air dryer

the shortest drying time of 209.51 min to reach a [3] Levenspiel O. Chemical Reaction Engineering. MC of 12% wet basis. (John Wiley & Sons, 2003). [4] Togul I & Pehlivan D. Modelling of Thin Layer Nomenclature Drying Kinetics of Some Fruits Under Open-Air M — mass of mangoes and water, g at any time, Sun Drying Process. Journal of Food t Engineering 2004. [5] Clary C, Farid M, Fasina O, Hui Y, Noomhorm A, & Me — equilibrium moisture content, g water; Welti-Chanes J. Food Drying Science and Mi — initial moisture content, g water at t = 0 MR — moisture ratio Technology: Microbiology, Chemistry, MC — moisture content at any time, g water at Applications. In: Sablani S & Rahman S, any time t Fundamentals of Food Dehydration, pp. 1–2. a, k — drying constant (Lancaster: DEStech Publications Inc., 2008). n — order of the reaction, positive integer [6] Da Silva WP, Silva CMDPS, Gama FJ, & Gomes R — drying rate, g of liquid evaporated per s- JP. Mathematical models to describe thin-layer m2 of solid surface drying and to determine drying rate of whole bananas. Journal of the Saudi Society of WS — weight of dry solid, g A — Surface drying area of solid, m2 Agricultural Sciences 2014; 13:(1),67–74. X’ — bulk moisture content of the solid [7] Akoy EOM. Experimental characterization and modeling of thin-layer drying of mango slices. X’1 — bulk moisture content at time 0, g of liquid/ g of dry solid at t=0 International Food Research Journal 2014; 21(5):1911–1917. X’2 — bulk moisture content at time t, g of liquid/ g of dry solid at time t [8] Aghbashlo M, Kianmehr MH, & Arabhosseini A. Modeling of thin-layer drying of potato slices in Greek Letters length of continuous band dryer. Energy t — residence time, min Conservation and Management 2009; 50:1348– 1355. Subscripts [9] Arslan D & Özcan MM. Study the effect of sun, f — final oven and microwave drying on quality of onion i, o — initial slices. LWT-Food Science and Technology e — equilibrium 2010; 43:1121–1227. [10] Doymaz I & Ismail O. Drying characteristics of ACKNOWLEDGMENTS sweet cherry. Food and Bioproducts Processing 2011; 89:31–38. The authors gratefully acknowledge the helpful [11] da Siva WP, Silva C, Gama F, & Gomes J. comments and suggestions of Engr. Beatriz A. Mathematical models to describe thin- layer drying Belmonte, Engr. Mark Emile H. Punzalan and Ms. and to determine drying rate of whole bananas. Essence Jean P. Logan. Journal of the Saudi Society of Agricultural Sciences 2013. htt:// dx.doi.org/10.1016/ j.jssas.2013.01.003. REFERENCES [12] Clary C, Farid M, Fasina O, Hui Y, Noomhorm A, & Welti-Chanes J. Food Drying Science and [1] FAO, 2013 Commodity and Trade Policy Technology: Microbiology, Chemistry, Research Working Paper No. 42. Market Applications. In: Lypez-Malo A & Rios-Casas L Structure and Distribution of Benefits from Solar Assisted Drying of Foods, p. 84. Agricultural Exports: The Case of the Philippine (Lancaster: DEStech Publications Inc., 2008). Mango Industry. http://www.fao.org/3/a- [13] Belessiotis VG & Karathanos VT. Application of ar709e.pdf accessed: 30/01/2017 a thin layer equation to drying data fresh and [2] Delmo G. Carabao mango: Philippine’s sweet semi-dried fruits. Agricultural Engineering pride. Far Eastern Agriculture, 2010. Retrieved Resources 1999; 355–361. 1 August 2014. From http:// [14] Singh S, Raina GS, Bawa AS, & Saxena DG. www.businessmirror.com.ph/en/business/ Effect of pretreatments on drying and entrepreneur/32407-pmsfc-modernizing-mango- rehydration kinetics and colour of sweet potato farming-in-phl slices. Drying Technology 2006; 24:1487–1497.

65 Labutong FIT, Pastores JSF, Yeung AC, & Pestaño LDBActa Manilana 64 (2016)

[15] Hassan-Beygi SR, Aghbashlo M, Kinamehr MH, [21] Tunde-Akintunde TY & Afon AA. Modelling of & Massad J. Drying characteristics of walnut hot-air drying of pretreated cassava chips. (Juglan regia L.) during convection drying. Agricultural Engineering International (CIGR International Agrophysics 2009; 23:129–135. Ejournal, Manuscript 1493) 2009. [16] Agrawal YC & Singh RC. Thin layer drying studies [22] Doymaz I. Pretreatment effect on sun drying of on short grain rough rice (ASAE Paper No. 77– mulberry fruits (Morus alba L.). Journal of Food 3531. (MI, U.S.A.: St. Joseph, 1977). Engineering 2004; 78:591–596. [17] Zhang Q & Litchfield JB. An optimization of [23] Ertekin C & Yaldiz O. Drying of eggplant and Intermittent corn drying in a laboratory scale thin selection of a suitable thin layer drying model. layer dryer. Drying Technology 1991; 9:383–395. Journal of Food Engineering 2004; 63:349–359. [18] Charbonnel C, Ghoul M, Guiga W, & Ioannou I. [24] Doymaz I. Air-drying characteristics of tomatoes. Food and Bioproducts Processing Frozen Journal of Food Engineering 2007; 78:1291– mirabelle plum drying: Kinetics, modeling and 1297. impact on biochemical properties. (2010) [25] Vengaiah PC & Pandey JP. Dehaydration kinetics [19] Pestaño LB. Mathematical Modeling of the drying of sweet pepper. (Capsicum annum L.). Journal process of coconut meat [Online]. In: Third of Food Engineering 2007; 81:282–286. International Conference on Advances in [26] Kingsly RP, Goyal RK, Manikantan MR, and Ilyas Applied Science and Environmental SM. Effects of pretreatments and drying air Engineering (ASEE 2015) (USA: ©Institute of temperature on drying behaviour of peach slice. Research Engineers and Doctors, 2015). ISBN: International Journal of Food Science and 978–1–63248–055–2. Technology 2007; 4:65–69. [20] Jose WI & Pestaño LB. Engineering an Improved [27] Goyal RK, Kingsly ARP, Manikantan MR, & Ilyas Coconut Processing System in the Philippines at SM. Thin-layer drying kinetics of raw mango the Farm-Level. Journal of Advanced slices. Biosystems Enginerring 2006; 95(1):43– Agricultural Technologies 2016; 3(1):58–62. 49.

66 Acta Manilana 64 (2016), pp. 67–74 Printed in the Philippines ISSN: 0065–1370

A review of recent literature on repairable-item inventory systems

Marilyn C. Mabini

Resarch Center for the Natural and Applied Sciences & Faculty of Engineering University of Santo Tomas, 1015 Manila, Philippines

This review includes papers found in the literature for repairable item inventory systems since 1997. The idea is to complement related earlier reviews until 1997, which are mentioned in the paper for the benefit of the interested reader. The papers are classified into two groups, based broadly on the types of inventory systems: single echelon and multi-echelon systems. A number of modeling approaches found in the papers in both groups are noted.

Keywords: repairable inventories, single echelon system, multi-echelon system

INTRODUCTION repairable-item inventory, as noted by Jung et al. [3], based on the US Defense Department Repairable items are items which, upon failure, supply system inventory report of September are capable of being restored back to working 2000. Meanwhile, Diaz and Fu [2] observe that condition through repair. These items are usually repairable items represent almost 2/3 of the total high valued, infrequently demanded, and highly inventory value, both in the Caracas subway essential to operations. Because of their high station in Venezuela and in the US Air Force. cost, these items are generally cheaper to repair than to replace. The management of repairable inventories has received considerable research attention since Although comprising a small percentage of the the 1960s. Early papers were concerned with number of items stocked, repairable items items in the military sector. Throughout the usually account for a significant part of years, studies in this area have dealt with items investment in inventories. In Nahmias [1], it is in the aviation industry, e.g., aircraft and its reported that repairable items represented components; transportation industry, e.g., approximately $10 billion in the US military sector trains, buses and their components; and certain alone in 1976. This amount went up to $34 billion commercial products such as computers and in 1994, as noted by Diaz and Fu [2] based on a copy machines. The present paper surveys the 1994 private communication from O’Malley. By literature on repairable-item inventory 2000, the US military had $46.4 billion of management from 1997 onwards, thus complementing earlier reviews by Nahmias [1], Mabini and Gelders [4], Diaz and Fu [2], and *To whom correspondence should be addressed [email protected] Guide and Srivastava [5].

© 2016 UST Research Center for the Natural and Applied Sciences, Manila, Philippines Mabini MCActa Manilana 64 (2016)

In Nahmias [1], we find a survey of mathematical SINGLE-ECHELON SYSTEMS models for determining stocking levels of repairable inventories. The models are classified A single-echelon repairable inventory system into three groups, namely, continuous review, usually consists of a repair shop and a periodic review, and models based on cyclic warehouse which holds inventories of queueing systems. The analytical approaches serviceable items and non-serviceable ones employed in developing and solving the models awaiting repair. The system has two possible are outlined in the paper. Meanwhile, the article sources of serviceable inventories, namely, of Mabini and Gelders [4] is a partial review of returns from repair and external purchases. the printed literature on repairable item The single-echelon problem is usually modelled inventory systems either as a deterministic problem with batch Diaz and Fu [2] focus on multi-echelon models procurement and repair, or as a stochastic for repairable inventories, highlighting certain problem with one-for-one replenishment. The key relationships. They review Sherbrooke’s studies of Teunter [7, 8], Koh et al. [9], and Multi-Echelon Technique for Recoverable Item Konstantaras [10] belong to the former group. Control (METRIC) model [6], its extensions and Teunter [7] proposes EOQ formulas for the variations, and discuss a number of general manufacturing and recovery batch quantities of queueing models. Finally, they take up the items that can be recovered, i.e., repaired, elements in the models that must be addressed refurbished, or remanufactured. His model in order for them to be applicable to industrial allows disposal, and differentiates holding costs situations. for manufactured and recovered items. In another paper, Teunter [8] investigates Guide and Srivastava [5] examine various inventory systems in situations where demand repairable inventory models proposed in the may be filled with new or recovered items, where literature, in the light of the major underlying recovered items are considered to be “as good assumptions for those models. The authors use as new.” Assuming deterministic demand rate a three-way classification for the models: and return fraction, he presents formulas for solution methodology, single versus multi- determining optimal lot sizes for the production echelon, and exact versus approximate solutions. or purchase of new items and for the recovery of returned items. The objective is to minimize In this paper, we present the material in the total of lot ordering costs for production repairable-item inventory literature since 1997, and for recovery, and inventory holding costs in two general groups: single echelon and multi- for recoverable items and new/recovered items. echelon inventory systems. The modelling The formulas derived are valid for finite and approaches used in the papers in both groups infinite production and recovery rates. fall in one or a combination of the following classifications: (1) continuous review, (2) The contribution of Koh et al. [9] is a joint EOQ periodic review, (3) queueing-based models, (4) and EPQ model for the situation where stationary Markov models, (5) heuristics, and (6) simulation. demand can be met from recycles and newly It is worthy to mention that, where work prior to purchased products. The assumption is that a 1997 is relevant to models included in the present given proportion of used products are collected survey, such work is given reference here. from customers and subsequently recovered to “good as new” condition. The model finds the EOQ for newly purchased products and the optimal inventory level of recoverable items that

68 A review of recent literature on repairable-item inventory systems

would initiate the recovery process. Extending Wong et al. [13] consider a single-item repairable the work of Koh et al. [9], Konstantaras [10] inventory system involving multiple hubs and presents a formula for determining the optimal multiple companies. They present a model for inventory level of recoverable items that would finding stocking levels of spare parts for the mark the start of the inspection and recovery system where complete pooling of stock is processes, and the economic order quantity for allowed among the hubs and companies. The procurement. In this study, Konstantaras model minimizes the total of holding, downtime, considers that some recovered items are and transshipment cost, for which the authors perceived to be of secondary quality and can propose a two-stage solution. In Wong et al. be sold at a reduced price. [14], non-zero lateral transshipment time and delayed lateral transhipments are considered for A related work by Omar and Yeo [11] introduces estimating several performance measures in a an inventory model for the production of new single-item repairable inventory system items and repair of used ones. The model involving multiple companies. Allowing assumes a known and finite planning horizon, complete pooling of stock among the deterministic demand, and constant repair rates. companies, the authors present a multi- A numerical solution procedure is proposed and dimensional Markovian model for the problem, numerical examples are provided in the paper. and a two-stage solution method for the same.

Stochastic models for the single-echelon The multi-item case is studied by Wong et al. problem are found in Zijm and Avsar [12], Wong [15] for a two-location repairable spare parts et al. [13], Wong et al. [14], Wong et al. [15], inventory system, which supports expensive Chakravarty [16], Mirzahosseinian [17], Selçuk technical systems with high target availability [18], and Tracht [19], among others. Zijm and levels. A continuous review model is proposed Avsar [12] study a two-indenture maintenance for the inventory system, where lateral and system for a number of identical installations emergency shipments are allowed to absorb which are in use at a single site. The installations stockout situations. The model aims to minimize are assemblies composed of repairable the total cost inclusive of inventory holding, components. Inventories of ready-for-use lateral transhipments and emergency shipments, assemblies and components are maintained at subject to an average waiting time limitation per particular stock points, each one employing a demanded part at each of the two locations. The base-stock policy. Allowing backorders and authors propose a solution procedure based on assuming that an assembly failure is caused by Lagrangian relaxation to find upper and lower only one component at a time, the paper bounds on the optimal total cost; they proposes first a slightly aggregated (but exact) eventually show a narrow average gap between formulation for the system, and subsequently these bounds. presents a near-product-form solution as an approximate steady-state distribution of the The paper of Kilpi [20] specifies three aggregated system. The approximate cooperative strategies for making repairable performance measures are found to be accurate aircraft components available to participating by simulation, and are subsequently used to airlines when needed. Using simulation, the find the optimal base-stock levels, following a strategies ad hoc cooperation, cooperative greedy procedure. pooling and commercial pooling are studied and compared to the alternative of acting alone, i.e. A number of papers in this group consider solo strategy. Findings indicate that generally, pooling and sharing of stocks to meet demand. the benefits from pooling are greater when

69 Mabini MCActa Manilana 64 (2016)

demand for one component type served by one reliability and the efficiency of the repair facility, pool is higher. The author notes, however, that rather than improving the base stock level. The conflicting interests can complicate the latter is said to have little effect on system development of efficient pools. availability.

Several studies on the single-echelon system Alfredsson [23] presents a mathematical pay special attention to the repair aspect. In his framework for finding the quantity of spares and paper, De Haas [21] uses service level as a test equipment needed and for determining measure of performance for a maintenance where repair should take place in a support support system consisting of a group of repair system for a fleet of technical systems. The departments and stock locations intended to author proposes an algorithm to find cost- provide serviceable engines to aircraft. The efficient configurations for the support system. author shows that the service level targets for Tiemessen [24] tackles the repair scheduling the repair departments can be significantly lower problem for a system consisting of a repair shop than those set for the stock locations of the and one stockpoint. The latter is where spare system. The importance of coordinating the parts of multiple critical repairable items are kept performance of the respective repair on stock to support technical equipment. For departments and the stock locations is the objective of minimizing aggregate downtime, emphasized. The paper shows that the well- with backorders allowed, the study evaluates a known METRIC [6] and MOD-METRIC [22] number of dynamic scheduling policies, models can be used to support the deduction of including the myopic allocation rule from the the service level targets. make-to-stock environment. This rule selects the SKU with the largest reduction in expected Chakravarty [16] studies a reliability system with backorders per invested time unit. The myopic identical, repairable components, and at least k allocation rule is shown to outperform the other out of N components are needed for the system heuristic scheduling rules investigated. to operate. Failed components are attended to by a single repairman on a first-come-first-served While many studies assume ample repair basis. The system has K spares which can be capacity, this assumption may not be realistic in tapped to extend its lifetime, following a many situations. Some authors have, therefore, probabilistic rule. The delivery time of a spare is investigated the repairable inventory problem exponentially distributed; and multiple requests with repair capacity limitations. Considering a for spares at any given time are possible. The single-item, single-location problem with limited influence of delivery times on the performance repair capacity, Selçuk [18] proposes an adaptive measures of the system is investigated, using a base stock policy in which the base stock level finite quasi-birth-and-death process to analyze of a repairable item is updated based on the work- the system. in-process inventory level in the repair facility. The update frequency is modeled as a separate Mirzahosseinian [17] investigates a repairable control parameter along with a standard base parts system operating under a Performance- stock level. Emergency shipments are employed Based Logistics (PBL) contract. The closed-loop in stockout situations, whereas priority inventory system is modeled as an M/M/m shipments are used when updating the base queue with Poisson failures for components and stock level. The problem is modelled as a 2- exponential repair times. Analysis of key model dimensional continuous-time Markov chain, and parameters indicated that system availability can solved by matrix geometric methods. Numerical be improved by improving the component

70 A review of recent literature on repairable-item inventory systems results show that, for a given downtime target, operational sites in different areas. The multiple inventory on hand and total cost can supplier inventory system has an internal repair substantially be reduced with the new policy. shop offering several modes of repair with different repair times. Spare parts come from an Meanwhile, the work of Tracht [19] applies to external supplier. The network model, which the repairable item system for expensive parts considers backorders, is shown to efficiently of machines. Here, the impact of varying repair solve the problem for deterministic demands that capacity on the system is investigated. The vary over time. study shows how the stock levels should be adjusted so that a maximum backorder level of Most other multi-echelon models are stochastic, waiting request is guaranteed for the entire year. and follow a one-for-one replenishment policy. Kim et al. [27] address the problem of finding MULTI-ECHELON SYSTEMS the initial spare inventory level for a multi- echelon repairable item inventory system, with A multi-echelon inventory system usually refers a general repair time distribution. He presents to a two-level system composed of a number of an algorithm for determining the inventory level locations, called bases, supported by a central which minimizes the total expected cost while depot. Items fail at the base level, and are repaired satisfying a minimum service rate. there, if possible. If a base has no repair capacity, failed items are sent to the depot for repair and The model by Tracht [28] determines cost- for replacement. The pioneering work of optimal inventory levels for warehouses in a Sherbrooke (1968), known as METRIC, or Multi- two-echelon spare parts supply chain in the Echelon Review Technique for Repairable Item aviation industry. The method takes into Control, is the forerunner of studies on multi- account budget and inventory level limitations. echelon repairable inventory systems. A simulation model is used to validate the calculated inventory levels. Costantino [29] Deterministic models for the multi-echelon presents a spare parts allocation model for the repairable inventory system are introduced in Italian Air Force, whose objective is to minimize Toptal [25] and in Perlman and Levner [26]. Toptal backorders and ensure a 99% system availability. [25] considers a system consisting of a central Solved by marginal analysis, the model depot where failed items are recovered, and a incorporates repair centers with different skills collection center where failed items from retailers in a multi-echelon, multi-item system, where items are temporarily stored until they are brought to have a multi-indentured structure. the central depot for recovery. A mathematical model is proposed for determining the economic A number of authors have also considered the shipment quantities of failed and recovered multi-echelon problem with repair capacity items between the depot and the collection limitations: Díaz and Fu [30], Perlman et al. [31], center. The model strives to minimize the long- Sleptchenko et al. [32], Jung et al. [3], Spanjers run average total cost subject to a service level [33], Lau and Song [34], and Tao [36]. Díaz and constraint. The latter is given by the maximum Fu [30] study a system with limited repair level of failed items in each cycle which are not facilities at the depot, where all failed LRUs are immediately replaced. repaired. They propose approximations based on queuing theory for three cases of queueing Perlman and Levner [26] propose a network at the depot: M/M/s single-class model, M/G/s model for an inventory system which covers single-class model and M/G/s multi-class model. repairable equipment located at several Meanwhile, the work of Perlman, et al. [31]

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describes three models to investigate the effect is allowed on both levels. A near-product-form of congestion in a multi-echelon inventory solution is proposed for determining excellent system employing two modes of repair, each approximations of relevant performance with limited repair capacity. An expanding repair measures. These approximations are then used policy employed at the bases for choosing which to find stock levels which maximize system mode of repair to use is compared with various availability subject to a budget constraint. expediting policies related to congestion externalities. The expanding repair policy which Lau and Song [34] address the corrective considers congestion externalities is shown to maintenance problem in military logistics and result in better system performance present an analytical model for evaluating measurement than an expanding policy with no system performance under limited repair capacity congestion. and nonstationary demands. They develop a METRIC-based optimization algorithm to solve Meanwhile, Sleptchenko et al. [32] investigate the model. In Tao [36], we find a discrete-event the capacitated multi-echelon problem for stochastic simulation model for a closed loop repairable service parts with a hierarchical (multi- multi-echelon repairable inventory system. indentured) structure. They show that if the Using examples, the paper shows how the model repair shop utilization is relatively high, the can be used to study the effect of depot repair frequently used infinite capacity assumption may capacity on expected backorders and to affect system performance and stock allocation determine spares allocation. The results indicate decisions badly. The paper presents a modified that, compared to METRIC, the model is more VARI-METRIC [35] method to allocate service accurate and efficient. part stocks in the network for both cases of item- dedicated and shared repair shops. The repair CONCLUSION shops are modelled as multi-server queueing The management of repairable inventories has systems. Validated by simulation, the technique become increasingly important is the last few is found to be more accurate than the VARI- decades, primarily because of the significant METRIC technique. share of repairable items in the inventory The model by Jung et al. [3], developed in the investment of an organization. This paper is context of a system with finite repair channels, aimed to help present and future researchers in allows the lateral transshipment of a spare from this area, in terms of assessing the work already another base, when needed and where possible, done and determining which important issues based on the availability of stocks there. If no remain unresolved. It is hoped that this paper such spare is available from any of the bases, will be a useful guide to the researcher on the item is backordered. The authors propose repairable inventories in determining future an algorithm to find the spare inventory level at research directions in this area. each base, which minimizes the total expected REFERENCES cost. [1] Nahmias S. Managing repairable item inventory The work of Spanjers [33] presents a slightly systems: A review. In: Schwarz LB (Ed.) Multi- aggregated model for finding the steady state level Production/Inventory Control Systems: probabilities for a closed loop two-echelon Theory and Practice - TIMS Studies in the system with finite repair capacities at the bases Management Sciences,Vol. 16, pp. 253–277. and at the depot. Ready-for-use machines are (North-Holland, Amsterdam: 1981) held in stock at these locations; and backlogging

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74 Acta Manilana 64 (2016), pp. 75–86 Printed in the Philippines ISSN: 0065–1370

Reviving the Philippine shrimp industry: molecular diagnostics and therapeutics

Maria Violeta R. Tare1,2,3, Pocholo Mari T. Arabit1,2, Mark Anthony G. Fran3, David Angelo V. Guanzon1,2, Jalizah Jaira E. Lim1,2, Sharlaine Joi Ann B. Orense1,2, Joselito A. Tabardillo Jr.3, & Mary Beth B. Maningas*1,2,3

1Graduate School; 2Department of Biological Science, College of Science; 3Research Center for the Natural and Applied Sciences, University of Santo Tomas, 1015 Manila, Philippines

In 1994, the Philippines was one of the top three producers in shrimp aquaculture, reaching a peak of 91MT. However, diseases caused a rapid decline in shrimp production, down to less than 50MT in 1997, and the Philippine status in shrimp aquaculture production has dwindled to the 13th spot. Production has not yet recovered and stabilized, as these diseases still plague the industry up to this day. The strict implementation of importation laws for broodstock and better management practices helped the industry to make up for losses, and is now starting to revive. The scientific community is doing its part to hasten this process, shrimp research is of consistent interest to several research institutions providing deeper understanding regarding viral and bacterial diseases and their interactions with the host. However, the challenge remains as to how research can be translated and applied in the field, to provide the shrimp industry with science-based management strategies. This review provides a balance of both basic and applied research with the ultimate goal of reaching out to the industry through useful and beneficial knowledge together with simpler and cost- effective detection methods and alternative, effective immunostimulants.

Keywords: shrimp biotechnology, shrimp immunity, molecular detection

INTRODUCTION the industry has generated profits, foreign exchange earnings and an important source of Marine and freshwater shellfish, including livelihood. However, the expansion of the shrimp shrimps, are a key source of protein in a industry was accompanied by an increased continuously growing population. The incidence of economically significant diseases, increased consumption of shrimps and the particularly of viral and bacterial origin [1, 2]. limited supply from capture fisheries have led to a demand for farmed shrimps and an expansion Among the viruses affecting farmed shrimps in of the shrimp industry in the Philippines. Due to the Philippines are the white spot syndrome the demand for high-value seafood products, virus (WSSV), the taura syndrome virus (TSV) and the Laem-Singh virus (LSNV). WSSV has been used for challenge tests due to its *To whom correspondence should be addressed devastating impact in shrimp populations upon [email protected]

© 2016 UST Research Center for the Natural and Applied Sciences, Manila, Philippines Tare MVR, et al.Acta Manilana 64 (2016)

infection, whereas TSV and LSNV have been the elucidation of gene function in relation to detected in the country for the first time. The WSSV infection and the use of clinical signs, transmission, distribution and immunostimulants for shrimps. incidence of diseases due to these viral pathogens have been reviewed by Andrino- DETECTION OF SHRIMP PATHOGENS Felarca et al. [3] Viral and bacterial pathogens are the most Bacterial agents from the genus Vibrio have also common causes of the shrimp diseases, caused significant morbidity and mortality in accounting for 60% of the economic losses in shrimps in the Philippines. The major species aquaculture for viral diseases and 20% for causing Vibriosis are V. harveyi, V. fluvialis, V. bacterial infections [8]. Early detection and parahaemolyticus, V. damsel, and V. vulnificus constant surveillance of these pathogens remain [4]. It is worth noting that a toxic plasmid of V. to be one of the most effective counter- parahaemolyticus has been found to be the measures against shrimp diseases because it causative agent for an emerging disease in provides ample time for officials to make break- shrimp, acute hepatopancreatic degenerative even strategies and proper aquaculture practices necrotic disease (AHPND) or commonly known to mitigate production losses [9]. Because of as early mortality syndrome (EMS) due to mass their economic importance, rapid and simple mortality of infected shrimp within days of diagnostics for the WSSV, TSV, LSNV, and stocking [5]. species of Vibrio were developed and are reviewed in this paper. These pathogens have posed a significant threat to the shrimp industry, causing millions of Polymerase chain reaction-based methods. In dollars of losses in production. The detection, recent years, molecular detection has become control and prevention of shrimp diseases the most widely accepted technique in disease caused by these pathogenic agents are essential diagnosis because it is faster and more accurate in supporting the ailing shrimp farming industry. than the traditional procedures [10]. Among Efficient detection methods have been provided these, PCR detection and its derivatives are the by molecular diagnostics technology, and most reliable in detecting aquaculture pathogens contribute significantly to the mitigation of [9]. production losses. A therapeutic tool for the White spot disease, caused by WSSV, remains management of shrimp diseases is gene to be one of the most serious viral disease, due silencing, which is based on the elucidation of to its high mortality rate that can take place 3– the interaction between the shrimp and the viral 10 days after post-infection [11]. Infected agents. Immunostimulants have provided an shrimps exhibit loss of appetite, lethargy and effective means for increasing the characteristic white spots located on the immunocompetency and disease resistance in carapace from calcified deposits, symptoms that shrimps, and have been widely accepted by the eventually lead to mass mortality [12]. The virus shrimp culture industry [6, 7]. is viable to a wide range of economically- This review paper intends to provide an insight important shrimp species in the Philippines, and understanding of several aspects of shrimp Peneaus monodon [13], Peneaus vannamei [14], research. It covers loop-mediated isothermal and Macrobrachium rosenbergii [15]. amplification (LAMP)-based and polymerase In 2011, WSSV was detected from P. monodon chain reaction (PCR)-based detection of shrimp samples in Bulacan [13]. The authors also diseases, the utilization of RNA interference for

76 Reviving the Philippine shrimp industry: molecular diagnostics and therapeutics reported that incidental shrimps such as Aside from viral pathogens, bacteria are also Metapenaeus dalli all tested negative in one- major causes of mortalities in shrimp ponds. step and nested PCR, a possible display of Vibriosis is a common example, caused by several resistance to the virus. On the same year, a partial species of Vibrio such as V. alginolyticus [18], sequence of the WSSV isolate from the V. anguillarum [19], V. harveyi [20], V. vulfinicus Philippines was generated [14] and was [20], and V. parahaemolyticus [21], among subsequently used to design PCR primers. PCR others. Initial efforts for the rapid detection of detection of the disease using the designed species of Vibrio were done using PCR and primers showed a low incidence rate of 15% for microbiological methods [22]. A virulent strain P. vannamei. Continued surveillance of WSSV of V. parahaemolyticus was associated with an from June 2014 to July 2015 for both P. monodon emerging disease that causes massive and P. vannamei was conducted in other sites mortalities around Southeast Asia and Mexico in the Philippines and showed a higher total called acute hepatopancreatic necrosis disease prevalence rate of 25% [unpublished data]. (AHPND) [23]. The disease creates lesions in the hepatopancreas in shrimp when the V. The successful detection of WSSV indicated parahaemolyticus plasmid with the toxin gene that other shrimp pathogens may be present but was present in the species [5]. In 2015, the are left undetected and unreported in the presence of the virulent strain of V. country, which may result to the rapid spread of parahaemolyticus was first recorded from across diseases. To prevent this, LSNV became the the country and confirmed by PCR amplification target for possible detection. Initial screening and histopathology, together with studies for the RNA-based virus, which causes microbiological and biochemical techniques. Monodon slow growth syndrome (MSGS), was Alarmingly, the prevalence of AHPND was conducted between the years 1998–2007. The found to be 24% in the Philippines [24]. studies revealed that there have been no carriers of the disease in the country [16]. Early detection is important for the mitigation and the control of the spread of these diseases. However, a recent study [unpublished data] But it remains to be a problem for local shrimp showed the presence of LSNV in P. monodon farms especially for those located far from samples from the province of Bulacan. Detection accredited testing laboratories and do not have was done by observing the external symptoms the capital to invest in self-test detection kits. of the sampled shrimps. LSNV was further The delay in action from time lost in detected in the shrimp samples via PCR transportation and testing of samples can amplification, and the sequenced the target gene greatly reduce shrimp harvest when diseases exhibited a 99% homology to LSNV. are present. It is also expensive because PCR- based assays require expensive reagents, Another RNA-based virus, TSV, was also specialized equipment and technical personnel detected in shrimp farms across the country. TSV to perform. It is of utmost importance that the is capable of causing significant mortalities in diagnosis be rapid, reliable, and can be done reared P. vannamei three days after the onset of on-site. disease [17] and was also previously unreported in the country. In 2014 [unpublished data], TSV Loop-mediated isothermal amplification. A was detected via PCR and the resulting novel and emerging technology called LAMP sequences indicated close homology to the TSV [25] was used as the platform for the developed Taiwan isolate. diagnostic kit for WSSV. LAMP gained popularity in the detection of several pathogens

77 Tare MVR, et al.Acta Manilana 64 (2016)

for the aquaculture sector such as Edwardsiella across the Philippines. A prevalence rate of 88% tarda in Japanese flounder [26], fish iridovirus was observed in Luzon, 63% in Visayas and 65% [27], TSV [28] and WSSV [29]. The LAMP in Mindanao [unpublished data]. reaction uses a specialized polymerase, the Bst polymerase which has a strand displacement The alarming number of pathogens present in activity, and four sets of primers that target six our country remains to be a constant threat to specific regions in the gene of interest, making the Philippine shrimp industry. A summary of LAMP more specific and sensitive to the target the pathogens detected in the Philippines is sequence. The amplification proceeds in presented in Table 1, and the areas covered by isothermal conditions for one hour. The the pathogens are shown in Fig.1, together with visualization of the result is straightforward and their annual prevalence during the period of can be performed by the naked eye. The 2010–2016. Developing rapid and farm-ready simplicity and rapidity of LAMP makes it diagnostic kits for these pathogens are essential desirable as a diagnostic platform for farm in aiding the industry and the government in applications. shrimp health management. Early detection and rapid diagnosis of the disease is only the first Four sets of primers (patented) highly specific step in creating a sustainable and productive for the Philippine WSSV isolates were aquaculture industry, its breakthroughs will go developed [30]. The different sets of primers a long way for the mitigation and prevention of were tested, and the most consistent and diseases, as well as understanding the nature sensitive was used for the diagnostic kit. The of the relationship of the pathogen to its host. designed primers were further optimized [22], In the future lies a rapid, simple and cost- and compared to the conventional PCR for its effective diagnostic platform that can be used analytical sensitivity and specificity. by farm and hatchery operators for timely and accurate on-site detection of pathogens. Utilizing the optimized LAMP assay, a prevalence rate of 48% for WSSV from selected GENE SILENCING THERAPY sites in the Philippines was recorded. Additionally, the assay provided faster and ten The development of strategies for the times more sensitive results than PCR. It was prevention of shrimp disease outbreak could be also noted that the formation of LAMP products facilitated by an understanding of host-virus is possible at temperatures lower than 60°C, and interactions. The suppression of gene the incubation period can be reduced to 45 min expression based on sequence homology with consistent results. between the dsRNA trigger and the target gene comprises RNAi together with a set of related Despite the improvements made for the LAMP cellular processes. It is an approach with protocol, the DNA extraction remains to be time incredible potential for therapeutic purposes, consuming and costly. To resolve this difficulty, using dsRNA that targets viral sequences or our group formulated and developed a rapid, homologous viral sequences in vivo [31]. RNAi simple and cost-effective diagnostic kit that can applications for shrimp diseases has been used for shrimp farm monitoring of WSSV. The reviewed by Maningas and Tare [32], and the developed diagnostic kit includes a simple DNA review is supplemental to the current paper. From extraction kit and protocol (patent pending), the functional analysis of transglutaminase LAMP premix buffers and a fabricated heat block (TGase) and clotting protein (CP) in the shrimp tailor-made for LAMP applications. Using the clotting system using gene silencing as the developed diagnostic kit, WSSV was tested determining assay for the role of the two genes

78 Reviving the Philippine shrimp industry: molecular diagnostics and therapeutics

Table 1. Pathogens of Interest Detected in Shrimp Via Molecular Diagnostics Infected shrimp Molecular detection Sampling sites in the Pathogen Reference species platform Philippines Bacterial

Nicolasora et al. (2014) Vibrio spp. P. vannamei PCR and LAMP Pangasinan

Vibrio parahaemolyticus Bataan, Bulacan, causing Acute P. vannamei PCR Pampanga, Cebu, Bohol, Dabu et al. (2015) Hepatopancreatic General Santos, Saranggani Necrosis Disease Viruses Alenton and Maningas P. monodon PCR and Nested PCR Bulacan (2011) Batangas, Zambales, Capiz P. vannamei PCR Maralit et al. (2011) and General Santos city Batangas, Zambales, Capiz P. vannamei LAMP Maralit et al. (2012) WSSV and General Santos city Bulacan, Batangas, Laoag, P. vannamei PCR and LAMP Nicolasora et al. (2014) Iloilo, andLeyte Bulacan, Bataan, Cebu, P. monodon and PCR and LAMP Davao and General Santos (Unpublished) P .vannamei city Bulacan, Batangas, Bohol TSV P. vannamei PCR (Unpublished) and Cebu Bulacan, Batangas, LSNV P. monodon PCR (Unpublished) Pangasinan and Pampanga

Vibrio Spp. harnessing these relationships for the WSSV 100 90 prevention of disease outbreak [35]. TSV 80 LSNV 70 60 50

Percent 40 WSSV-shrimp homologs were found to be 30 20 10 involved in viral infectivity in kuruma shrimp, 0 2010 2011 2012 2013 2014 2015 2016 Year Marsupenaeus japonicus [36]. This finding was A B instrumental in several studies discussed in this review, reporting the discovered contigs involved in WSSV infection in the freshwater shrimp, Macrobrachium rosenbergii and the black tiger prawn, Penaeus monodon.

Immune defense responses are affected upon Figure 1. (A) Detected shrimp pathogen distribution in the Philippines; (B) Prevalence of shrimp invasion of WSSV, as shown by the pathogens per year. characterization of hemocyanin-like subunits of M. rosenbergii, initially known as contigs 13 and 37. Both genes were found to be [33] to the proposed molecular mechanism of upregulated during the early stages of WSSV the shrimp clotting system [34], succeeding infection [37]. studies followed a trend of uncovering host- virus interactions towards understanding and Contig 23 is one of the most promising candidates for therapeutic development for

79 Tare MVR, et al.Acta Manilana 64 (2016)

having rendered 100% survival rate at seven protecting the shrimps against infection thereby days post-infection when injected with c23- stabilizing shrimp production. dsRNA and infected with WSSV, signifying its interaction with the virus for rendering its IMMUNOSTIMULATION FOR DISEASE protective effect upon silencing both in M. RESISTANCE rosenbergii and P. monodon [38]. The rest of the contigs, 20, 31 and 34 were found to be Another approach that our group has adopted involved in the shrimp’s metabolic processes, towards the prevention of shrimp pathogenesis which also responds to viral activity and triggers is based on the natural process of host responses upon infection [our unpublished immunostimulation. Immunostimulation is the data]. In addition to host genes, the knockdown introduction of molecules into the system of an of a viral gene VP9 has significantly increased organism with the intention of improving its shrimp survival even upon re-infection and is response against infection from pathogenic suspected to play a key role in viral replication causes [41–43]. An update on the use of [39]. immunostimulants in the Philippines has been published [44]. Thus, the coverage of this review A transcriptome database of genes from tiger focuses on the multiple studies engrossed on shrimp that survived WSSV challenged was immunostimulation and done at the generated by Maralit [40], who also unveiled a aforementioned laboratory; all these exploring ubiquitin conjugating enzyme-like gene that the potential of recycled natural products and renders protective effect to WSSV challenged bacterial species found in shrimp gut (our M. rosenbergii [unpublished data]. Table 2 unpublished data) as immunostimulants (Table summarizes the genes utilized by RNA 3). interference discussed in this review. Natural immunostimulants are safe for the An exciting platform has been provided by the environment and cost-effective. In addition, gene silencing technology by showing the effect these can be sourced from bacteria, plant of knocking down a target gene on the organism. products, such as herbs and fruits, and even Interests on its application in shrimp is at a animal scraps, such as shells [42, 45–47]. What steady rate, Fig. 2 summarizes the authors marks these products as viable candidates for intention to continuously join this effort, with immunostimulants is the throng of minerals and the main goal of identifying genes suitable for

RNA interference in vitro

Table 2. Target Genes Used in RNAi Studies Discussed in This Review Functional elucidation of Silencing of viral host genes involved in genes Target Gene Source Reference WSSV infectivity Hemocyanin-like Host Tare et al. (2015) subunits Contig 20 Host Unpublished Mass production Survival of shrimps Development of Contig 23 Host Unpublished of dsRNA upon infection RNAi therapeutics Contig 31 Host Unpublished Figure 2. The conceptual framework of RNAi studies Contig 34 Host Unpublished starting from in vitro production of dsRNA, the Ubiquitin goal is to develop a more efficient, cost- conjugating enzyme- Host Unpublished effective, mass production of dsRNA and the like gene eventual development of RNAi therapeutics for VP9 Viral Alenton et al. (2016) application in the field.

80 Reviving the Philippine shrimp industry: molecular diagnostics and therapeutics

compounds stored within their varying indica crude extract and powdered treatments components. Waste plant products provide rich against WSSV was found to yield 80% survival sources of metabolites such as carbohydrates, rates and elevated immune parameter values [our proteins and mineral constituents. It is also unpublished data]. Mangifera indica kernels abundant in phytochemicals such as flavonoids, have been used as immunostimulant for Penaeus alkaloids, tannins and saponins which act as indicus against WSSV [53]. Ethanolic and hot- antioxidant and defense mechanism against free water extracts from algae, (Fucus, Laminaria, radicals that can cause debilitating disease in and Gracilaria) exhibited antiviral activity and shrimps [48]. aided in the prevention of the attachment of the virus to host cells [54, 55]. Aside from algae, Several natural products derived from plant, fruit berries also exhibit possible algae, animal and bacterial sources have been immunomodulatory properties. A crude explored in our laboratory for their ethanolic extract of a species of raspberry Rubus immunostimulation potential (Table 3). To make coreanus exhibited significantly increased use of the specific compounds, the raw materials immunity and expression of antioxidant enzyme were either subjected to further extraction, activities in P. vannamei after challenged with through either aqueous or ethanolic solutions Vibrio alginolyticus [56]. [15, 49, 50], or introduced in their crude powdered forms [18, 51, 52]. Extracted treatments were more The method of administering immunostimulants widely used since the crude extracts are to the shrimps revolve around three methods: comprised of more purified compounds such as feeding, immersion and injection. Of all the three phenols, tannins and flavonoids [48]. methods, injection is the most invasive and has been devised in a study involving agar- Herbal medicine like Cyanodon dactylon, Aegle carotenoid complex [unpublished data], chitin marmelos, Tinospora cordifolia, Picrorhiza and chitosan [57], hot-water extract of Gelidium kurooa, and Eclipta resulted to better amansi, [58] and hot-water extract of S. performance in the haematological, biochemical duplicatum [59]. Apart from injection, and immunological parameters from the introduction through feeding was also employed immunostimulant-incorporated diet fed to for eregosan [47], Sargassum fusiforme [60] and WSSV-challenged P. monodon [49]. Mangifera

Table 3. A Summary of the Immunomodulatory Studies Done by the Authors in This Paper Extraction Method of Source Components Pathogen Reference Method Introduction Plant Products Citrus microcarpa Vitamin C Ethanol Feeding V. alginolyticus Unpublished Curcuma longa Curcumin Powdered Feeding V. alginolyticus Alambra et al. (2012) Mangifera indica Mangiferin Powdered Feeding WSSV Unpublished Mangifera indica Mangiferin Ethanol Feeding WSSV Unpublished Musa paradisiaca Polyphenols Powdered Feeding V. alginolyticus Unpublished Marine Algae Gracilaria edulis Betaglucan Aqueous Immersion V. alginolyticus Maningas et al. (2013) Gracilaria edulis Betaglucan Aqueous Immersion WSSV Unpublished Sargassum polycystum Fucoidan Ethanol Feeding WSSV Arizo et al. (2015) Animal Sources Shrimp Shells Carotenoids TOSS Injection V. alginolyticus Unpublished Bacterial Sources Exiguobacteria sp. LPS None Feeding V. alginolyticus Unpublished

81 Tare MVR, et al.Acta Manilana 64 (2016)

on fucoidan [61]. Immersion strategies have also damage prevention to host cells, since the been devised and utilized [50, 58]. Between enzyme reactions produced by the humoral feeding and immersion, the former is more widely immune response tend to release free radicals employed since it required a smaller amount of that destroy foreign cells [67]. extracted compounds [50, 59, 62, our unpublished data], as little as 500 mg of an extract Aside from conferring protection against was enough to fuel the whole study [63, 49, 15, infection, growth was also found to be affected our unpublished data]. As far as efficacy and by the administration of immunostimulants. complexity is concerned, all three treatment Crude fucoidan was able to increase the weekly strategies generate lessened mortalities and weight gain of shrimp [15, 68]. Modified feeds higher immune parameter values; however, enhanced the feed utilization and the growth feeding is the simplest mode of administration performance of treated shrimps [69–71]. In due to the minimal volume of extracts required summary, materials containing these and the hassle-free preparation of experimental compounds prove to aid in improving the shrimp treatments [58, our unpublished data]. growth performance and immune response against pathogens through their effective Compounds of immunostimulatory functions are actions in regulating enzyme reactions (Fig. 3). often exploited for their antioxidant properties or their structural similarity to pathogenic Developing immunomodulatory feed additives components [46, 64]. Compounds with molecular from scrap materials paves way to providing structure similar to that of pathogens are termed cost-effective substitutes for commercial as pathogen-associated molecular patterns immunostimulants such as MacroguardTM. A (PAMPs), and are rooted to play a crucial role in U.S. patent 6440466 has been granted to the the activation of the key enzymatic reaction of composite of plant extracts of Lantena camera, the crustacean immune system, such as the Aegle marmelos, Ocimum sanctum, Mimosa proPhenoloxidase (PO) system [65]. The use of pudica, Cynodon dactylon, Curcuma longa, PAMPs tend to produce inverse correlation and Allium sativum which was found useful as between survival and compound prophylactic and therapeutic agents against concentrations [our unpublished data, 15]. viral (WSSV) and bacterial disease [72]. Higher treatment concentrations cause an increase in free radical production that induce irreversible damage to both host and foreign

cells and lead to weaker survival rates despite ORGANIC WASTE MATERIALS GENE EXRESSION the increased immune parameter values. AQUEOUS OR ETHANOLIC Similarly, since more PO reactions are produced, EXTRACTION IMMUNE PARAMETERS METHOD OF an exhaustion of the immune system can be ADMINISTRATION GROWTH PARAMETERS observed as indicated by the drop in immune SURVIVAL ANALYSIS parameter values [68]. INJECTION FEEDS IMMERSION

CHALLENGE TEST Antioxidants have also been employed as an (PROPHYLACTICS / THERAPEUTICS) immunostimulant [our unpublished data, 49, 53]. Antioxidant key compounds are expected to promote increased survivability, heightened Figure 3. Immunostimulation experiments begin with the treatment preparation from organic hemocyte count and increased superoxide materials and continue on to the challenge dismutase levels. This can be related to the experiments wherein immune parameters, growth rate, survival analysis and even gene expression is evaluated.

82 Reviving the Philippine shrimp industry: molecular diagnostics and therapeutics

Despite the advances made in the use of [4] Manilal A, Selvin J, & George S. In vivo therapeutic immunostimulants, the immune mechanism of potentiality of red seaweed, Asparagopsis crustaceans against viruses is yet to be defined, (Bonnemaisoniales. Rhodophyta) in the treatment of Vibriosis in Penaeus monodon. Saudi Journal and more efficient natural therapeutic strategies of Biological Sciences 2012; 19:165–175. can be developed. Endemic Philippine fruits and [5] Tran L, Nunan L, Redman R, Mohney R, Pantoja plants can be explored for possible efficacy as L, Fitzsimmons C, Kevin & Lightner D. bactericidal and antiviral treatments. Determination of the infectious nature of the agent of acute hepatopancreatic necrosis CONCLUSION syndrome affecting penaeid shrimp. Diseases of Aquatic Organisms 2013; 105:45–55. The emergence of pathogens has yet to cease [6] Pholdaeng K & Pangsamart S. Studies on the and thus, the struggle to develop mitigation immunomodulatory effect of polysaccharide gel extracted from Durio zibethinus in Penaeus techniques to hamper economic loss and monodon shrimp against Vibrio harveyi and promote shrimp production is a persistent WSSV. Fish & Shellfish Immunology 2010; challenge worth pursuing. The development of 28:555–561. molecular diagnostics, RNAi technology and [7] Hsieh T, Wang J, Li C, Kuo C, & Hsieh S. Effects immunostimulants play a vital and significant of Rutin from Toona sinensis on the immune and role in improving shrimp health management and physiological responses of white shrimp practices, and in reducing losses in the shrimp (Litopenaeus vannamei) under Vibrio alginolyticus challenge. Fish & Shellfish farming industry. Immunology 2008; 25:581–588. [8] Flegel T. Historic emergence, impact and current ACKNOWLEDGMENT status of shrimp pathogens in Asia. Journal of Invertebrate Pathology 2012:166–173. Our research work was supported by grants from [9] Caipang C. Utilization of loop-mediated isothermal the Commission on Higher Education-Higher amplification (LAMP) for the detection of Education Research Network (CHED-PHERNet) pathogens in shrimp and fish aquaculture in the and the Department of Science and Technology- Philippines. In Caipang MM, CMA Philippine Council for Agriculture, Aquatic and Biotechnological advances in shrimp health managament in the Philippines, pp. 149–163. Natural Resources Research and Development (Kerala, India: Research Signpost, 2015). (DOST-PCAARRD). [10] Biswas G & SM. Loop-mediated isothermal amplification (LAMP) assays for detection and REFERENCES identification of aquaculture pathogens: current state and perspectives. Applied Microbiology & [1] Hauton C. The scope of the crustacean immune Biotechnology 2014. system for disease control. Journal of [11] Lightner D. Handbook of Shrimp Pathology and Invertebrate Pathology 2012; 110:251–260. Diagnostic Procedures for Diseases of Penaeid [2] Muegue M, Caipang , & Geduspan J. Current Shrimp. (Baton Rouge: World Aquaculture status of shrimp aquaculture in the Philippines. Society, 1996). Biotechnological advances in shrimp health [12] Pradeep B. Biology, Host Range, Pathogenesis management in the Philippines. (Kerala, India: and Diagnosis of White spot syndrome virus. Research signpost, 2015). ISBN: 978–81–308– Indian Journal of Virology 2012. 0558–0. [13] Alenton R & Maningas MBB. Detection of white [3] Andrino-Felarca K, Estante E, & Lazado C. Viral spot syndrome virus on Penaeus monodon and diseases of shrimp in the Philippines. Metapenaeus dali From Bulacan, Philippines. Biotechnological advances in shrimp health Acta Manilana 2011. management in the Philippines. (Kerala, India: Research signpost, 2015). ISBN: 978–81–308– 0558–0.

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[55] Rudtanatip T, Asuvapongpatana S, [64] Vazquez L, Alpuche J, Maldonado G, Agundis C, Withyachumnarnkul B, & Wongprasert K. Sulfated Pereyra-Morales A, & Zenteno E. Review: galactans isolated from the red seaweed immunity mechanisms in crustaceans. Innate Gracilaria fisheri target the envelope proteins Immunity 2009; 15(3):179–188. of white spot syndrome virus and protect against [65] Cerenius L, Lee B, & Söderhäll K. The proPO- viral infection in shrimp haemocytes. Journal of system: pros and cons for its role in invertebrate General Virology 2014; 95(5):1126–1134. immunity. Trends in Immunology 2008; [56] Subramanian V, Jang Y, Kim D, Kang B, & Heo V. 29(6):263–271. Dietary effect of Rubus coreanus ethanolic [66] Sajeevan T, Philip R, & Singh I. Dose frequency: extract on immune gene expression in white leg a critical factor in the administration of glucan as shrimp, Penaeus vannamei. Fish & Shellfish immunostimulant to Indian white shrimp Immunology 2013; 35(3):808–814. Fenneropenaeus indicus. Aquaculture 2009; [57] Wang S & Chen J. The protective effect of chitin 287(3):248–252. and chitosan against Vibrio alginolyticus in [67] Urmi K, Mahboob S, Hossain M, Bhusal P, & Hamid white shrimp Litopenaeus vannamei. Fish & K. Antioxidant Activity and Brine Shrimp Lethality Shellfish Immunology 2005; 19(3):191–204. Bioassay of Different Parts of the Plant [58] Fu Y, Hou W, Yeh S, Li C, & Chen J. The Calotropis gigantea R. Br. (2012). immunostimulatory effects of hot-water extract [68] Immanuel G, Sivagnanavelmurugan M, of Gelidium amansii via immersion, injection and Marudhupandi T, Radhakrishnan S, & Palavesam dietary administrations on white shrimp A. The effect of fucoidan from brown seaweed Litopenaeus vannamei and its resistance against Sargassum wightii on WSSV resistance and Vibrio alginolyticus. Fish & Shellfish immune activity in shrimp Penaeus monodon Immunology 2007; 22(6):673–685. (Fab). Fish & Shellfish Immunology 2012; [59] YehS, Lee C, & Chen J. Administration of hot- 32(4):551–564. water extract of brown seaweed Sargassum [69] Itami T, Asano M, Tokushige K, Kubono K, duplicatum via immersion and injection enhances Nakagawa A, Takeno N, & Takahashi Y. the immune resistance of white shrimp Enhancement of disease resistance of kuruma Litopenaeus vannamei. Fish & Shellfish shrimp, Penaeus japonicus, after oral Immunology 2006; 20(3):332–345 administration of peptidoglycan derived from [60] Huang X, Zhou H, & Zhang H. The effect of Bifidobacterium thermophilum. Aquaculture Sargassum fusiforme polysaccharide extracts 1998; 164(1):277–288. on vibriosis resistance and immune activity of [70] Wang V. Effect of probiotics on growth the shrimp, Fenneropenaeus chinensis. Fish & performance and digestive enzyme activity of Shellfish Immunology 2006; 20(5):750–757. the shrimpPenaeus vannamei. Aquaculture 2007l [61] ChotigeatW, Tongsupa S, Supamataya K, & 269:259–264. Phongdara A. Effect of fucoidan on disease [71] Azad I, Panighari A, Chavali G, Paulpandi S, resistance of black tiger shrimp. Aquaculture Mahima H, & Pitchaiyappan R. Routes of 2004; 233(1). immunostimulation vis-à-vis survival and growth [62] Sung H, Yang Y, & Song Y. Enhancement of of Penaeus monodon postlarvae. Aquaculture microbicidal activity in the tiger shrimp Penaeus 2005; 248(1):227–234. monodon via immunostimulation. Journal of [72] Desai U, Achuthankutty C, & Sreepada R. U.S. Crustacean Biology 1996; 16(2):278–284. Patent No. 6,440,466. (Washington, DC: U.S. [63] Suphantharika M, Khunrae P, Thanardkit P, & Patent and Trademark Office, 2002). Verduyn C. Preparation of spent brewer’s yeast -glucans with a potential application as an immunostimulant for black tiger shrimp, Penaeus monodon. Bioresource Technology 2003; 88(1):55–60.

86 Acta Manilana 64 (2016), pp. 87–98 Printed in the Philippines ISSN: 0065–1370

Natural products-based discovery of antitubercular agents from Philippine medicinal plants — A review

Allan Patrick G. Macabeo1, Oliver B. Villaflores2, Scott G. Franzblau3, & Ma. Alicia M. Aguinaldo2*

1Laboratory for Organic Reactivity Discovery and Synthesis (LORDS) and 2Phytochemistry Laboratory, Research Center for the Natural and Applied Sciences, University of Santo Tomas, Espana, 1015 Manila 3Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago 833 S. Wood Street, Chicago, IL 60612-7231, USA

Tuberculosis is one of the leading causes of death worldwide, more than HIV and malaria. New TB cases are emerging, including multidrug-resistant TB. The development of antimycobacterial assays led to the discovery of secondary metabolites that elicit promising inhibitory activity against Mycobacterium tuberculosis. This review covers literature published from 1999 to 2014 about natural products from Philippine medicinal plants with reported

growth inhibitory activity in vitro against M. tuberculosis H37Rv. The antitubercular compounds were grouped according to plant family and/or chemotype. Some exhibited structural significance but with low inhibitory activity (MICs of >128 g/mL). While other previously reported compounds that were re-isolated exhibited anti-TB activity.

Keywords: antitubercular, anti-Mycobacterium tuberculosis, Philippine medicinal plants, natural products

INTRODUCTION adoption of directly observed treatment short course (DOTS), and overall, the “End TB The World Health Organization listed Strategy” recommended by WHO, to achieve tuberculosis as one of the top ten causes of the global TB target set by 2030 among the newly death worldwide in 2015, and was responsible adopted Sustainable Development Goals. for more deaths than HIV and malaria [1]. It is a However, MDR-TB has been prevalent in all leading killer of people living with HIV, causing countries surveyed. In 2015, an estimated 35% of all deaths. Over 95% of TB deaths occur 480,000 people developed multidrug-resistant TB in low- and middle-income countries. In 2015, (MDR-TB). In the Philippines, an estimated 2.6% an estimated 480,000 people developed of TB cases are with MDR/RR-TB (rifampicin- multidrug-resistant TB (MDR-TB). The TB resistance TB) [1]. Indeed, many are plagued incidence has dropped by an average of 1.5% with the disease, for a variety of reasons, per year since 2000, through TB diagnosis, the foremost of which is poverty.

With the development of inhibitory-based anti- *To whom correspondence should be addressed mycobacterial assays, a number of secondary [email protected]

© 2016 UST Research Center for the Natural and Applied Sciences, Manila, Philippines Macabeo APG, Villaflores OB, Franzblau SG, & Aguinaldo MAMActa Manilana 64 (2016) metabolites from medicinal plants have been inhibitory activity against the acid-fast bacteria, documented to elicit promising inhibitory activity Mycobacterium 607, together with against Mycobacterium tuberculosis [2–4]. representative Gram-positive (S. aureus and B. Natural products and their derivatives have subtilis), Gram-negative bacteria (E. coli and P. been shown to exhibit growth inhibitory activity aeruginosa), and yeast (S. cerevisiae and C. against M. tuberculosis and some have been albicans). This was succeeded by another study selected as inspiration molecules for the creation on 27 plants screened only for possible inhibition of new generation antituberculosis agents [4, of Mycobacterium 607 [10]. As a result of both 5]. Several review papers describing the studies, priority plants were identified for in- discovery of antimycobacterial plant natural depth phytochemical investigation guided by products have been published in the literature bioassays, still using Mycobacterium 607. from 1990 to 2012 [6–8].

Initial studies using M. tuberculosis H37Rv were The Philippines is rich in medicinal plants, much based on the BACTEC 460 radiorespirometric of which remains unexplored, and thus, assay. The results of bioassay guided underutilized. Though more than 800 species of phytochemical studies showed that there was Philippine plants are considered medicinal, there little correlation between the inhibition of is much more to be explored. Taking advantage Mycobacterium 607 and the inhibition of M. of the diversity of our terrestrial plants, the tuberculosis H37Rv. The samples (extracts, sub- Phytochemistry Group of the University of extracts, fractions) which inhibited Santo Tomas Research Center for the Natural Mycobacterium 607 did not inhibit or were Sciences (UST-RCNAS) has committed itself to weakly inhibiting the growth of M. tuberculosis the exploration and utilization of these plants. H37Rv. A later study involving M. tuberculosis

The objective of the group has been to provide H37Rv showed that the results of assays based a scientific rationalization for the use of plants on MABA were highly correlated with the as sources of medicine against TB, either as results obtained from the BACTEC assay [11]. phytopharmaceuticals or as new drugs for As a result, MABA became the method of choice development. for succeeding assays using M. tuberculosis

H37Rv. The in-depth phytochemical investigation is conducted after a random or ethnobotanical This review paper covers studies published from selection of plants followed by screening of the 1999 to 2014 about natural products from crude plant extract against M. tuberculosis Philippine medicinal plants which exhibited

H37Rv for bioactivity. Extraction, isolation and growth inhibitory activity in vitro against slow- purification of constituents is bioassay guided, growing, non-resistant strains of M. wherein the bioassays are the colorimetric tuberculosis H37Rv. Compounds with Microplate Alamar Blue assays (MABA) done antitubercular properties at minimal inhibitory in collaboration with the Institute for TB concentrations (MICs) of less than 128 g/mL Research, University of Illinois, Chicago, U.S.A. were highlighted and grouped according to their The pure compounds are identified through a source of origin (i.e. plant family) and/or combination of spectroscopic techniques. chemotypes. The selection includes natural products isolated mainly from plants, which were Prior to the investigations that used M. classified according to their secondary tuberculosis H37Rv for screening, Chua and co- metabolite type as terpenes (triterpenes), workers [9] conducted a microbiological steroids (sterols, sterones), alkaloids (indole, screening (agar cup method) of 125 plants for quinoline), aromatics (flavonoids, chalcones,

88 Natural products-based discovery of antitubercular agents

xanthones, chromones, etc.), and (1-4) exhibited good inhibitory activity against

phenyldecanoids. In some cases, the collection Mycobacterium tuberculosis H37Rv (MIC up to also covers those structurally significant 16 mg/mL). Liriodenine (4) showed the strongest compounds with low inhibitory activity (MICs antimycobacterial activity (MIC = 16 mg/mL) of >128 g/mL). This review paper also followed by 1 and 5:4 mixture of 1 and 2 [12]. highlights plant compounds that have been previously reported in literature, but which have Uvaria valderramensis Cabuang, Exconde & been re-isolated and observed to exhibit anti- Alejandro. The new Philippine endemic Uvaria TB activity. valderramensis is a small shrub (ca. 3–7 m tall) growing in the lowlands and forests of ANTITUBERCULAR CONSTITUENTS OF Valderrama, Antique, Panay Island, Philippines, SELECTED ANNONACEAE SPECIES and is known locally as usog [13]. Its identification and distinction from other Uvaria Efforts on the discovery of antimycobacterial species were facilitated by morphological and compounds from the custard family, molecular phylogenetic evidences. Two new Annonaceae, have been mainly inspired by a tetrahydroxanthene-1,3(2H)-dione metabolites, number of studies that reported on the structural valderramenols A (5) and B (6), were isolated complexity and potent biological activity of from this plant and were identified. natural products identified to date. Studies Valderramenol A (5) showed antitubercular dating back as early as 2007 have pointed out activity (MIC = 10 g/mL), while grandiuvarone discoveries relating to highly oxygenated (7) and reticuline (8) had weaker activities (MIC cyclohexenes, tetrahydroxanthenoids, styryl- = 32 g/mL) [14]. lactones, and alkaloids as antituberculosis agents. Uvaria rufa Blume. The Philippine medicinal plant Uvaria rufa (susung kalabaw in Filipino) Goniothalamus gitingensis Elmer. is a short climbing shrub found in low- and Phytochemical studies enabled the isolation and medium-altitude forests of Northern Luzon, identification of four secondary metabolites Philippines. In a previous report, the chloroform corresponding to three styryllactones, extract and fractions of U. rufa have been isoaltholactone (1), altholactone (2) and reported to exhibit strong antitubercular activity goniopypyrone (3), and the alkaloid liriodenine [15–16]. However, the major compounds such (4) from the Philippine endemic Annonaceae as (+)–zeylenol (9), ellipeiopsol B (10), ferrudiol species, Goniothalamus gitingensis. The (11), kweichowenol B (12), anabellamide (13), extracts together with the isolated compounds microcarpin A (14), and microcarpin B (15)

OMe OMe O O O O

O O MeO MeO MeO O OH HO O OH O O O O 6 OH OH 5 HO (1) (2) N O MeO Me O O OAc OH O O N O O O HO O HO O OMe 7 (3)(4) 8 Figure 1. Secondary metabolites isolated from G. Figure 2. Compounds isolated from U. gitingensis valderramensis

89 Macabeo APG, Villaflores OB, Franzblau SG, & Aguinaldo MAMActa Manilana 64 (2016)

O

O O O O O O O HO HO HO HO HO OH HO HO OH HO OH O O HO O O O NH HN O O O O O O O

91011 12 13

O OH O OH O O OH OH OH OH HO O HO O OH 14 O OH OH OH O OH O O OH

O O O OH

O OH 16 17 OH OH

15 Figure 3. Compounds identified from U. rufa

CH O N 3 N N N H H H H OH N O O OMe O OMe N N N H OMe OMe H O H O H O 18 19 20 21

CH O 3 CH3 N N HO OMe

N H O N OH N N OMe H H O H

22 23 24 Figure 4. Indole alkaloids from A. scholaris showed weak activity against M. tuberculosis ANTITUBERCULAR ALKALOIDS, (MIC >128 g/mL). In another study, the STEROIDS, AND TERPENOIDS FROM flavonoids quercetin -D-glucoside (16) and  SELECTED APOCYNACEAE SPECIES kaempferol (17) from the n-butanol extract exhibited moderately strong in vitro inhibitory The growing interest in exploring plants of the activity against M. tuberculosis H37Rv (MIC = Apocynaceae family for antitubercular potential 64 g/mL) [16]. mainly spurred from studies reporting indole alkaloids with growth inhibitory effects against a panel of bacterial species. Thus, from 1999 up to the present, five species have been explored

90 Natural products-based discovery of antitubercular agents enabling the identification of alkaloids, steroids, reported indole alkaloids — 19,20E-vallesamine and terpenoids as antimycobacterial (18), a mixture of angustilobine B N4-oxide (19) constituents. and N4-methyl angustilobine B (20), 20S- tubotaiwine (21), 6,7-seco-angustilobine B (22) Alstonia scholaris L. R. Brown. Alstonia and (+)-manilamine (23) from the most bioactive scholaris is a well-known Philippine medicinal alkaloid fractions with 98–99% inhibition — plant, where the root bark is known for its showed weak activities. Among the six antimalarial properties. The crude methanolic compounds, only alkaloid 20S-tubotaiwine (21) extract of A. scholaris collected in Manila demonstrated the highest activity with a demonstrated in vitro antituberculosis activity minimum inhibitory concentration (MIC) of 100 (89% inhibition against M. tuberculosis H37Rv g/mL. Compared with the standard rifampin at 50 g/mL). Gradient pH fractionation of the (MIC = 0.125 g/mL), all alkaloids were alkaloids yielded three alkaloid extracts, AsA, considered inactive [17, 18]. In a separate study AsB, and AsC, which exhibited 69%, 99%, and on A. scholaris collected in Ilocos Sur, fractions 99% inhibition, respectively. Group separation and alkaloid, including the major alkaloid, 19E- by silica gel vacuum liquid chromatography akuammidine (24) showed weak inhibition (VLC) of extracts AsA and AsB afforded against M. tuberculosis H37Rv (MIC = >128 g/ fractions that showed 69–99% inhibition against mL) [19]. the test mycobacterium. The previously Catharanthus roseus L. G. Don. The Madagascar periwinkle, Catharanthus roseus N is a common evergreen sub-shrub growing 1 m OAc tall and occurs worldwide. Our study allowed CO2Me MeO N OH the isolation and identification of the indole Me 25 alkaloid vindoline (25) as the antimycobacterial Figure 5. Structure of vindoline from C. roseus constituent (MIC = 128 g/mL) [20].

N N N MeO MeO MeO H H H N N N HO H O H O H O H H N N H N H O H H O O O O

N N N H OMe OMe OMe O H O H O 26 27 28

N MeO H N HO HO O N H H O O HO

N OMe 30 H O

29 Figure 6. Bisindole alkaloids and a triterpenoid from V. globosa

91 Macabeo APG, Villaflores OB, Franzblau SG, & Aguinaldo MAMActa Manilana 64 (2016)

Voacanga globosa Merr. Voacanga globosa liver disease and as an antidiabetic [24, 25]. The is one of the two known endemic species of crude extract and hexane fractions from M. Voacanga in the Philippines and is used citrifolia showed 89% and 95% inhibition, traditionally to stupefy eels and treat ulcers and respectively, against M. tuberculosis at 100 g/ wounds [21]. Globospiramine (26), a new mL. When tested against M. tuberculosis, E- spirobisindole alkaloid possessing an phytol (35) and cycloartenol (36) exhibited Aspidosperma-Aspidosperma skeleton, MICs of 32 g/mL and 64 g/mL, respectively. together with deoxyvobtusine (27), The sterols stigmasterol (33) and -sitosterol deoxyvobtusine lactone (28), vobtusine lactone (29), and lupeol (30), were isolated and identified through a bioassay-guided purification. Globospiramine (26) showed potent antituberculosis activity against M. O O O O tuberculosis H37Rv as evidenced in MABA (MIC = 4 g/mL) and low-oxygen recovery 31 32 assays (LORA) (MIC = 5.2 mg/mL) [22].

Voacanga megacarpa Merr. Voacanga megacarpa is one of the two endemic species found in the lowland forests of Camarines Sur HO HO 33 34 province. Chromatographic purification of the Figure 7. Sterols and triterpenes from V. megacarpa crude DCM-methanolic extract afforded a 1:1 mixture of lupeol acetate (31) and -amyrin acetate (32), and a 1:1 mixture of stigmasterol (33) and -sitosterol (34). The crude DCM- methanolic and alkaloid extracts, and fractions OH (1 and 2) showed moderate inhibitory activity HO against M. tuberculosis H Rv (MIC = 64 g/ 37 35 36 mL) [23].

ANTITUBERCULAR CONSTITUENTS FROM SELECTED UBIACEAE SPECIES R HO O HO O A family distinguished for its economic and 37 38 medicinal impacts, the Rubiaceae has been tapped to discover antitubercular constituents and other bioactives. Phytochemical efforts thus afforded triterpenes, and sterols as O O bioactive chemical ingredients. 39 40 Figure 8. Sterols and triterpenes from M. citrifolia Morinda citrifolia Linn. Morinda citrifolia is a well-known plant in the Indo-Pacific region and grows everywhere in the Philippines. O CHO O Popularly known as “noni,” the plant has many 14 HO HO uses in traditional medicine. For example, it is 9 used as a treatment for dysentery, heartburn, (41) (42) Figure 9. Compounds from V. odorata

92 Natural products-based discovery of antitubercular agents

(34), and the epidioxysterol (37) (from oxidation family developed from significant findings of 38) exhibited MICs of 128 g/mL, 32 g/mL regarding the inhibitory activity of ginger. and 2.5 g/mL, respectively, while a 2:1 mixture Phytochemical work for antitubercular of the ketosteroids stigmasta-4-en-3-one (39) constituents gave phenyldecanoids, sterols and and stigmasta-4,22-dien-3-one (40) showed an their glycosides and a flavonoid. MIC of 2.0 g/mL. There was insufficient material of 39 and 40 to test these compounds Alpinia purpurata (Vieill.) K. Schum. Alpinia individually [26]. purpurata is locally known in the Philippines as luyang pula or red ginger, and is a native to Villaria odorata (Blanco) Merr. Villaria the Pacific. MABA assay result of its crude odorata is a flowering and fruiting shrub or small ethanolic extract obtained from various plant tree that is distributed in Luzon (Bulacan, parts had shown the leaf extract to possess the Quezon, Albay, Cagayan, Isabela, Camarines, highest activity followed by the rhizome and and Sorsogon Provinces), Visayas (Leyte) and flower extracts. Among the sub-extracts, the Mindanao (Surigao Province). Villarinol (41), a DCM sub-extract exhibited the highest activity new alkenoyloxy alkenol metabolite was isolated followed by hexane and n-butanol sub-extracts. from the dichloromethane extract along with the All fractions obtained from the hexane and DCM known compounds stigmasterol (33) and 4- sub-extracts showed low to moderate activity. hydroxybenzaldehyde (42). The extracts and Fractionation and purification afforded a mixture compounds of V. odorata showed weak of C28 to C32 fatty alcohols, a 3-methoxyflavone inhibition against M. tuberculosis H37Rv and two steroidal glycosides. The two latter (MIC >128 g/mL) [27]. metabolites were spectroscopically identified as kumatakenin (43), sitosteryl-3-O-6-palmitoyl-- ANTITUBERCULAR CONSTITUENTS FROM D-glucoside (44), and -sitosteryl galactoside SELECTED ZINGIBERACEAE SPECIES (45). The fatty alcohols showed an MIC value of 64 g/mL and proved most active compared Better known as the ginger family, the to the flavonoid kumatakenin and the steroidal Zingiberaceae is mainly tropical in distribution, glycosides (MIC >128 g/mL) [28]. and used for its spices, perfume, ornamental and medicinal properties. Interest in species of this

OH O MeO O O O O O O OH 8 OMe HO OH HO OH OH O OH OH 43 44 45 Figure 10. Sterol glycosides and a flavonoid from A. purpurata

O O OH MeO MeO

HO HO 46 47 Figure 11. Phenyldecanoids from Z. officinale

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Zingiber officinale Rosc. The widely occurring with moderately strong inhibitory activity food ingredient, Zingiber officinale has been against the test organism (MIC up to 64 g/mL). traditionally used for arthritis, rheumatism, Chromatographic purification of sub-fraction 1 sprains, muscular aches, pains, sore throats, afforded four compounds which were cramps, constipation, indigestion, vomiting, spectroscopically identified as -amyrin 3- hypertension, dementia, fever, infectious palmitate (48), squalene (49) and a 1:1 mixture diseases, and helminthiasis. In vitro anti-TB of the sterols -sitosterol (33) and stigmasterol susceptibility assay using the radiorespirometric (34). Evaluation of the antimycobacterial method showed that a crude alcoholic extract of activity of 33, 34, 48, and 49 showed the rhizome of Z. officinale inhibited the growth insignificant inhibitory activity against the test of M. tuberculosis H37Rv by 100% at 1000 g/ organism (MIC >128 g/mL) [31]. mL. Bioassay-guided isolation of the constituents present in the bioactive hexane Lunasia amara Blanco. Lunasia amara fraction afforded two phenyldecanoids namely, (Rutaceae) is an erect shrub that grows in 6-shogaol (46) and 6-gingerol (47). Compounds thickets and forests throughout the Philippines 46 and 47 exhibited 100% inhibitory activity at [32]. An ethanolic leaf alcohol extract showed 64 and 32 g/mL, respectively [29]. significant inhibition to M. tuberculosis H37Rv (99% at 1000 g/mL). Bioassay-guided ANTITUBERCULAR CONSTITUENTS FROM chromatographic purification of the hexane MISCELLANEOUS PLANT SPECIES extract (99% at 100 g/mL) and dichloromethane extract (93% at 100 g/mL) afforded two Abutilon indicum Sweet. Abutilon indicum quinoline alkaloids which were identified (Malvaceae) is used in Filipino folk medicine as spectroscopically as kokusagine (7,8- demulcent, diuretic, sedative, aphrodisiac and methylenedioxydictamnine) (51) and antidiabetic remedy [30]. The fractions obtained 4-methoxy-2-phenylquinoline (52). Isolation of after silica gel chromatography of the crude alkaloids from the pH 4 extract of another batch DCM-methanol (1:1) showed the first fraction of ethanolic extract gave graveolinine (4- (MIC = 64 g/mL) to exert the highest inhibition methoxy-2-(3’4’-methylenedioxy)phenylquinoline) against M. tuberculosis H37Rv. Further (53). Alkaloids 51 and 53 demonstrated an MIC separation of this fraction afforded sub-fractions of 16 g/mL while 52 exhibited an MIC of 30 g/ mL [33].

O

O 11 48 49 Figure 12. Triterpenes from A. indicum

OMe OMe OMe

O O O N N N O O

51 52 53 Figure 13. Quinoline alkaloids from L. amara

94 Natural products-based discovery of antitubercular agents

Momordica charantia L. Momordica isolated from the ethanolic (leaf) extract together charantia (Cucurbitaceae) or ampalaya in with the known compound, 2,4-bis(2- Filipino is one of the most popular anti-diabetic phenylpropan-2-yl)phenol (55). Compound 55 plants in Asia, Africa and Latin America and is displayed a moderately strong antitubercular traditionally used as food and medicine [34]. A activity against M. tuberculosis H37Rv (MIC = new lanostane aldehyde, charantal (54), was 14 g/mL) [35].

Pandanus tectorius Soland. var. laevis. HO OH Pandanus tectorius Soland. var. laevis (Pandanaceae) is known for the use of its dried roots as diuretic. During the bioassay-guided purification of the antitubercular chloroform CHO HO OH extract of P. tectorius leaves a new tirucallane- type triterpene, 24,24-dimethyl-5b-tirucall- 54 55 9(11),25-dien-3-one (56) was afforded along with Figure 14. Compounds from M. charantia squalene (49) and a mixture of the phytosterols, -sitosterol (33) and stigmasterol (34). MABA showed that 56 inhibited the growth of M.

tuberculosis H37Rv with an MIC of 64 g/mL, while squalene (49) and the sterol mixture have MICs of 100 and 128 g/mL, respectively [36].

O H Premna odorata Blanco. Premna odorata 56 (Lamiaceae) is a medicinal plant traditionally Figure 15. Structure of 24,24-dimethyl-5b-tirucall- used in Albay Province, Philippines to treat 9(11),25-dien-3-one from P. laevis tuberculosis. The crude methanolic extract and sub-extracts showed poor inhibitory activity

against M. tuberculosis H37Rv (MIC >128 g/ mL). However, increased inhibitory potency was O CN observed for fractions eluted from the DCM sub- H O 5 extract (MIC = 54–120 g/mL). Further 57 58 purification of the most active fraction (MIC = Figure 16. Compounds from P. odorata 54 g/mL) led to the isolation of a 5:3 mixture of

MeOH MeOH HO HO O O HO HO O HO O HO HO OH HO OH O O

HO OH HO OH O O O O OH HO HO OH O O

HO HO OH HO 59 60 Figure 17. Triterpene glycosides from S. odorata

95 Macabeo APG, Villaflores OB, Franzblau SG, & Aguinaldo MAMActa Manilana 64 (2016)

1-heneicosyl formate (57) and 3-(2- (55) are remarkable scaffolds for drug phenylethyl)benzonitrile (58), and 4:1 mixture development, because of their potent of -sitosterol (33) and stigmasterol (44) which antimycobacterial activity. It is also worth noting were identified through GC-MS analysis (with the synergistic antitubercular activity of dereplication) and NMR experiments. The MIC compound mixtures, for example, of stigmasta- of the mixture of compounds 57 and 58 was 8 4-en-3-one (39) and stigmasta-4,22-dien-3-one g/mL [37]. (40) and of 1-heneicosyl formate (57) and 3-(2- phenylethyl)benzonitrile (58). Current efforts are Schefflera luzoniensis Merr. Schefflera centered on investigations of other Philippine luzoniensis (Araliaceae) is an endemic small medicinal plant families. Similar investigations shrub abundant in the lowland to medium forests are also ongoing on antitubercular constituents of Mount Banahaw, Southern Luzon, from microbial sources such as endophytic fungi. Philippines. The antibacterial potential of Schefflera luzoniensis was assessed using the REFERENCES paper disc diffusion and MABA assays. Moderately strong zones of inhibition were [1] WHO (2016b). Global Tuberculosis Report. http:/ /www.who.int/countries/phl/en/ observed for the n-butanol subextract and the [2] Chung GA, Aktar Z, Jackson S, & Duncan K. saponin-rich fractions against the Gram-positive High-throughput screen for detecting bacteria such as Bacillus cereus, antimycobacterial agents. Antimicrobial Agents Staphylococcus epidermidis, and Staphylo- and Chemotherapy 1995; 39:2235–2238. coccus aureus. Also, the saponin-containing [3] Orme I. Search for new drugs for treatment of subfractions showed activity against M. tuberculosis. Antimicrobial Agents and Chemotherapy 2001; 45:1943–1946. tuberculosis H37Rv (MIC=64 g/mL). HPLC-MS  [4] Tripathi RP, Tewari N, Dwivedi N, & Tiwari VK. profiling of the antibacterial fractions revealed Fighting tuberculosis: an old disease with new the presence of two known oleanene glycosides, challenges. Medicinal Research Reviews 2005; namely, schiffoleoside A (59) and F (60) as the 25:93–131. major saponin glycoside constituents. [5] Nayyar A & Jain R. Recent advances in new Angiogenic property of the n-butanol sub- structural classes of antituberculosis agents. extract was revealed by the chicken Current Medicinal Chemistry 2005; 12:1873– 1886. chorio-allantoic membrane assay at a [6] Copp BR. Antimycobacterial natural products. concentration of 10 g/mL while higher Natural Products Reports 2003; 20:535–557. concentrations suppressed neovascularization [7] Okunade AL, Elvin-Lewis MPF, & Lewis WH. (58). Natural antimycobacterial metabolites: current status. Phytochemistry 2004; 65:1017–1032. CONCLUSION [8] Copp BR & Pearce AN. Natural product growth inhibitors of Mycobacterium tuberculosis. The present review compiled 60 natural products Natural Products. Reports 2007; 24:278–297. isolated and identified from a number of [9] Chua NM, Abad RV, Santos PS, Guevara BQ, & Solevilla RC. Microbiological Screening of Some Philippine plants. Those molecules with MICs Philippine Medicinal Plants for Antimicrobial less than 128 mg/mL would be considered as Activities. Acta Manilana 1985; 34:45–90. potential leads for further investigations in the [10] Aguinaldo A & Chua NM. Philippine Medicinal discovery and development of more potent Plants Active Against Mycobacterium 607. Acta antitubercular congeners. For example, Manilana 1988; 37:81–84. valderramenol A (5), globospiramine (26), kokusagine (51), 4-methoxyphenylquinoline (52), and 2,4-bis(2-phenylpropan-2-yl)phenol

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