Contents lists available at ScienceDirect

Phytochemistry Letters

journal homepage: www.elsevier.com/locate/phytol

Molecular confirmation, constituents and cytotoxicity evaluation of twomedicinal Piper species used by the Manobo tribe of Agusan del Sur,PhilippinesMark Lloyd G. Dapara,d,*, Cesar G. Demayoc, Ulrich Meved, Sigrid Liede-Schumannd,Grecebio Jonathan D. Alejandroa,b,da The Graduate School and Research Center for the Natural and Applied Sciences, University of Santo Tomas, España Boulevard, 1015 Manila, Philippinesb College of Science, University of Santo Tomas, España Boulevard, 1015 Manila, Philippinesc Department of Biological Sciences, College of Science and Mathematics, Mindanao State University - Iligan Institute of Technology, 9200 Iligan City, PhilippinesdDepartment of Plant Systematics, University of Bayreuth, Universitätsstr. 30, D-95447 Bayreuth, Germany

A R T I C L E I N F O

Keywords:BLASTnCytotoxicityITSLunasiaPhytochemicalsPiper aduncumPiper decumanum

A B S T R A C T

The Manobo tribe in the Philippines is culturally rich in ethnomedicinal practices and known to use popularplants locally named as “Lunas” (meaning “cure”). One of these is “Lunas-bagon tapol” which was previouslyidentified as Lunasia sp. (Rutaceae) based on vegetative morphology. The other species is “Lunas-buyo” which issimilar in ethnomedicinal use and morphology. This broadly-based study verifies these two “Lunas”-namedspecimens and evaluates the phytochemicals present and cytotoxic properties. The sequences of nuclear ITS, andplastid rbcL, and matK were investigated for species identification. Molecular confirmation using BLASTn nu-cleotide database query revealed that the two confused “Lunas”-named specimens were members of Piperaceaeand not Rutaceae. Phylogeny of Asian Piper using ITS sequences revealed “Lunas-bagon tapol” as Piper decu-manum L. and “Lunas-buyo” as Piper aduncum L. with strong support (BS = 100 %). Both Piper species similarlyshowed the presence of alkaloids, flavonoids, steroids, tannins and fatty acids but the absence of cyanogenicglycosides. Also, P. decumanum has moderate amount of saponins while P. aduncum contains moderate amount ofanthraquinones. A cytotoxic activity test using trypan blue exclusion method against normal lymphocytes fromhuman blood showed low toxicity (91.9 % viable cells) for P. decumanum and mild toxicity (88.3 % viable cells)for P. aduncum when subjected to 1000 μg/ml of the stem ethanolic extracts. These results support the practicalapproach of molecular-based taxon identification and provide the biochemical and biological basis as to theconstituents present and cytotoxic properties of these medicinal Piper species for future pharmacological re-search, conservation priorities and ecological management.

1. Introduction

The Philippines is a multi-cultural country with more than a hun-dred ethnolinguistic groups (Philippine Statistics Authority (PSA, 2016)classified according to its own unique identity, language, socio-politicalsystems, and practices (National Commission on Indigenous Peoples(NCIP, 2010). The Manobo indigenous group among others is one of themost numerous Philippine ethnic groups occupying a wide area ofdistribution in Mindanao, Philippines using several medicinal plants fordisease treatment and medication due to poor access of healthcareservices. The Manobo tribe found in Agusan provinces is known as theAgusan Manobo inhabiting communities near Agusan River Valley andhighlands above mountain drainage systems making their cultural

identity firmly rooted in their natural habitat alongside Agusan River.Hence, Manobo was named after the “Mansuba” which means riverpeople, coined from the “man” (people) and the “suba” (river). Giventheir location as a significant habitat for most of their indigenous(medicinal) plants, their traditional medical practice of using medicinalplants has long been practiced until these days for their primary diseasetreatment and healthcare.

Some of their notable medicinal plants are locally known as “Lunas”in Bisaya and Manobo dialects which means “cure” with several speciesassociated under this name. An example of which is the “Lunas-bagontapol” which was previously identified under the genus Lunasia Blanco(Rutaceae) based solely on leaf morphology (Dapar and Demayo, 2017;Dapar et al., 2018). However, this identification was somewhat

https://doi.org/10.1016/j.phytol.2020.01.017Received 23 September 2019; Received in revised form 17 December 2019; Accepted 16 January 2020

⁎ Corresponding author at: The Graduate School, University of Santo Tomas, España Boulevard, 1015 Manila, Philippines.E-mail address: marklloyd.dapar.gs@ust.edu.ph (M.L.G. Dapar).

Phytochemistry Letters 36 (2020) 24–31

1874-3900/ © 2020 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

T

uncertain, because vegetatively, Rutaceae can be confused easily withPiperaceae. Both families comprise woody or non-woody climbers, andalso some trees, shrubs, and herbs, with alternate or opposite andaromatic leaves bearing essential oils (Bramley and Utteridge, 2015).Likewise, both families share similar biomedical properties as anti-bacterial and cytotoxic against cancer cells, as proven for Lunasia spe-cies (Prescott et al., 2007) and for Piper species (Wang et al., 2014).Both families were also reported rich in alkaloid content as potent cy-totoxic constituent namely piperine for Piper nigrum L. (Li et al., 2011;Umadevi et al., 2013), and lunacridine for Lunasia amara Blanco(Prescott et al., 2007). Ethnomedicinally, both species constitute aneffective treatment against cancer, stomach troubles, gastralgia, diar-rhea, vomiting, hypertension, food poisoning and snakebites as re-ported by Macabeo and Aguinaldo (2008) for L. amara, and Salehi et al.(2019) for Piper species. As a result, Dapar and Demayo (2017) andDapar et al. (2018) previously identified the “Lunas-bagon tapol” as onepossible new variety of Lunasia amara in addition to the two varieties L.amara var. amara and L. amara var. babuyanica (Merr.) Hartley in thePhilippines. The Agusan Manobo ethnomedicinally claimed “Lunas-bagon” vine species as traditional source of anti-inflammatory, anti-motility, antihistamine, antiparasitic, antibacterial, antitoxin, and an-tiviral medicines (Dapar and Demayo, 2017).

Another medicinal plant, which is classified by the Agusan Manobotribe to be closely related with “Lunas-bagon tapol” in terms of med-icinal uses and morphology, is locally known as “Lunas-buyo”. As aconsequence, the identity of these two “Lunas”-named specimens(“Lunas-bagon tapol” and “Lunas-buyo”) poses uncertainty in terms oftheir true familial and generic affinities.

Piper is diverse in habit and inhabits diverse habitat (Sanderson andDonoghue, 1994), and has a widespread distribution occurring inAmerican tropics with 700 spp. and in Southern Asia with 300 spp.(Jaramillo and Manos, 2001). Gardner (2006) recognized 15 climbingPhilippine Piper species with only one taxon (P. myrmecophilum C.DC.)accepted as endemic while Pelser et al. (2011 onwards) reported 65Piper species occurring in the Philippines. Piper nigrum L. (black pepper)and P. betle L. (betel leaf) are some of the economically importantspecies of Piper (Chaveerach et al., 2006; Scott et al., 2008; Fan et al.,2011) while others are considered to be ecologically significant(Fleming, 1981; Bizerril and Raw, 1998). With the large number ofPiper species, vegetative identification up to the species level is difficult.Meticulous investigation of its reproductive parts like fruits and in-fructescence are very important for species identification(Suwanphakdee and Chantaranothai, 2011).

The unique composition of the genetic material for each speciesmakes DNA-based identification a useful tool for accurate speciesidentification. Therefore, correct identification of a medicinal plantshould be examined using a molecular approach (Sanubol et al., 2014)for consistency of species and pharmacological investigations of naturalproducts (Thomford et al., 2018). Although plant-based drug discoveryin the ethnobotanical approach provides future drug leads,

authentication of the plant material is a great challenge and opportu-nity (Jachac and Saklani, 2007). Chemical composition, morphology,and molecular data of some species pairs must be studied for accuratespecies identification (Sanubol et al., 2014). Moreover, the use of in-ternal transcribed spacer (ITS) of nuclear ribosomal DNA is a powerfulphylogenetic marker showing high interspecific divergence and greaterdiscriminatory power over the plastid regions (Hollingsworth, 2011; Liet al., 2014) like matK and rbcL.

It was therefore important that the true identity of the two “Lunas”-named specimens be verified using the molecular sequences of thenuclear ITS, plastid matK and rbcL. The evaluation of the phytochemicalconstituents and cytotoxic activities of the extracts from the two specieswere included to further evaluate their similarities and differences. Theinformation generated in this study not only resolve true species iden-tity of the two species but also provide the basis for their ethnome-dicinal use and as a potential source of medicine. This study can alsopave the way for the future conservation of these two ethnomedicinalplant species as one of the critical genetic resources of the AgusanManobo in Mindanao, Philippines.

2. Materials and methods

2.1. Sample collection

Fieldwork started with consultation and meeting of the tribalcommunity with the council of elders to discuss research intent aspurely academic. As agreed, a certification from the tribal council ofelders was given following ritual observation with the community as acultural tradition to grant permission on the conduct of the study. Freeprior informed consents (FPICs) from the only two community tribalhealers were acquired. When the tribal certification and FPICs weresecured, the national regulators duly provided certification and permitfollowing the by-laws on the protection of the indigenous peoples andthe permission to collect wild plant samples as issued from the regionalgovernment administration. The National Commission on IndigenousPeoples of CARAGA Administrative Region (NCIP-CARAGA) issuedcertification (no. R13-2019-01), and the Department of Environmentand Natural Resources of CARAGA Administrative Region (DENR-CARAGA) granted wildlife gratuitous permit (no. R13-2019-12) for thisstudy. All necessary permit and certifications were reviewed by theUniversity of Santo Tomas Graduate School - Ethics Review Board(USTGS-ERC) and granted ethics approval (protocol no. GS-2019-PN007) before plant collection. The collection of the plant samples wasfollowed through guided field walks with the two tribal healers who areknowledgeable of their medicinal plants. Both tribal healers verified thecollected plant materials and similarly classified both collected plantsamples with the initial word “Lunas” but different second names de-noting different plant species accordingly and locally named one as“Lunas-bagon tapol” and the other as “Lunas-buyo”. Samples of “Lunas-bagon tapol” (Fig. 1A) was collected in Mt. Ararat, Bayugan City,

Fig. 1. Field photographs of “Lunas”-named specimens. (A) “Lunas-bagon tapol” (Piper decumanum L.) leaves, (B) “Lunas-buyo” (Piper aduncum L.) leaves and (C)“Lunas-buyo” (Piper aduncum L.) inflorescence. Photos taken by N.K.G. Alfeche (A) and M.LG. Dapar (B&C).

M.L.G. Dapar, et al. Phytochemistry Letters 36 (2020) 24–31

25

Agusan del Sur (coded MGD-002) while “Lunas-buyo” (Fig. 1B&C) wascollected in Mt. Pinagalaan, Bayugan City, Agusan del Sur (coded18AG-002). Voucher specimens were deposited in the University ofSanto Tomas Herbarium (USTH). Leaf samples were placed in zip-locked bags containing silica gel for molecular analysis (Chase andHills, 1991). Mature stems were collected from each “Lunas”-namedspecimens for phytochemical and cytotoxicity analyses since this is theplant part used for cure by the tribal community. Collected stems werewashed with water, air-dried, and pulverized into a fine powder using ablender. The preparation and extraction procedure followed the pro-tocol used by Uy et al. (2019) to obtain the viscous crude extracts whichwere finally stored in vials for phytochemical screening and cytotoxi-city testing.

2.2. DNA extraction, amplification, and sequencing

The total genomic DNA was extracted from silica gel-dried leaftissues of “Lunas-bagon tapol” and “Lunas-buyo” samples following theprotocols of DNeasy Plant Minikit (Qiagen, Germany). The ITS(nrDNA), matK, and rbcL (cpDNA) markers were used for this study. Thegene regions, primer pairs, sequences, and PCR parameters, as shown inTable 1 were used for amplification reactions using Biometra Thermo-cycler T-Gradient. Amplicons were purified using Qia-Quick PCR Pur-ification Kit (Qiagen, Germany) following the manufacturer’s protocoland sent to MACROGEN Inc., Seoul, South Korea, for bidirectional se-quencing.

2.3. Sequence and phylogenetic analysis

The newly generated sequences of each sample were assembled andedited using Codon Code Aligner v8.0.2, trimmed and checked forambiguous nucleotides. Species identity was evaluated using BLASTnsearch algorithm available in the GenBank (www.ncbi.nim.nih.gov).Available sequences of all reported Philippine and other Asian Piperspecies using the three markers (ITS, matK and rbcL) were initiallychecked for inclusion in the phylogenetic analysis. Of the three mar-kers, only ITS was subjected to phylogenetic analysis since it has suf-ficient number of sequences as compared to the chloroplast markers(matK and rbcL) with only 16 each available sequences in the GenBank.Out of the 65 Piper species occurring in the Philippines (Pelser et al.,2011 onwards), only 23 species are with ITS sequences in the GenBank.Additionally, 22 Asian Piper species from the study of Jaramillo andManos (2001) and Jaramillo et al. (2008) were also included in theanalysis. A total of forty-seven accessions of Piper species including tworecognized non-native Philippine species (Piper aduncum L. and Piperumbellatum L.) and two outgroups (Peperomia elongata Kunth and Sar-corhachis naranjoana (C.DC.) Trel.) were included in parsimony analysisof the ITS marker (Table 2).

Multiple alignment of sequences used MUSCLE (Edgar, 2004) im-plemented in MEGA v.7.0.18 (Kumar et al., 2016) and was finallyedited by visual inspection. Parsimony analysis was done using TBRsearch method with 100 random addition of initial trees and examinedusing 1000 bootstrap replicates. Consistency index (CI; Kluge andFarris, 1969) and retention index (RI; Farris, 1989) were calculated toestimate homoplasy.

2.4. Phytochemical screening and cytotoxicity test

The phytochemical screening was based on the protocol describedby Aguinaldo et al. (2005) with slight modification. Qualitative as-sessment was done on the stem ethanolic extracts to screen for phyto-chemicals present such as alkaloids, saponins, flavonoids, steroids,

Table 1Gene regions, primers and amplification protocols used for Polymerase Chain Reaction.

Gene Region Primer Name Reference Primer Sequence (5’-3’) PCR Protocol

ITS (ITS1, 5.8S gene, andITS2)

P17F Alejandro et al.(2005)

5'-CTACCGATTGAATGGTCCGGTGAA-3ʹ 97 °C 90 s; 35 cycles of 97 °C 20 s, 72 °C 90 s, 72 °C 30 s; 72°C 7 min26S-82R 5ʹ-TCCCGGTTCGCTCGCCGTTACTA-3ʹ

rbcL rbcL_aF Bafeel et al. (2012) 5ʹ-ATGTCACCACAAACAGAGACTAAAGC-3ʹ 95 °C 1 min; 35 cycles of 95 °C 30 s, 51 °C 30 s, 68 °C 1 min;68 °C 5 minrbcL_aR 5ʹ-GTAAAATCAAGTCCACCRCG-3ʹ

matK 3F_KimF Costion et al. (2011) 5ʹ-CGTACAGTACTTTTGTGTTTACGAG-3ʹ 94 °C 5 min; 35 cycles of 94 °C 30 s, 52 °C 30 s, 72 °C 50 s; 5min at 72 °CIR_KimR 5ʹ-ACCCAGTCCATCTGGAAATCTTGGTTC-3ʹ

Table 2Nucleotide sequence database accession numbers of taxa used in the ITS phy-logenetic analysis.

Species GenBank accession numbers

OUTGROUPPeperomia elongata Kunth AF275213Sarcorhachis naranjoana (C.DC.) Trel. AF275210

INGROUPPiper abbreviatum Opiz MH493098P. aduncum L. (1) AF275157P. aduncum L. (2) EF060061P. albispicum C.DC. AY572317P. arborescens Roxb. AF275202P. arboricola C.DC. AY572319P. austrocaledonicum C.DC. MH493124P. baccatum Blume MH493135P. bavinum C.DC. AF275199P. betle L. AF275201P. boehmeriaefolium Wall. AF275204P. brevicuspe (Miq.) Merr. AY572321P. caninum Blume AF275195P. celtidiforme Opiz AF275205P. cordatilimbum Quisumb. AY572323P. decumanum L. (1) AF275203P. decumanum L. (2) MH493337P. densum Bl. AY615963P. fragile Benth. MH493373P. guineense Schumach. & Thonn. MH493383P. gymnostachyum C.DC. AY572325P. hancei Maxim. EF450272P. hymenophyllum Miq. AY572327P. korthalsii Miq. AF275208P. laosanum C.DC. AY572326P. lessertianum (Miq.) C.DC. MH493410P. lolot C.DC. AY326208P. macropiper Pennant MH493418P. majusculum Blume MH493434P. medinillifolium Quisumb. AY667455P. muricatum Blume EF060076P. mutabile C.DC. DQ868737P. myrmecophilum C.DC. AY572328P. nigrum L. AF275198P. pendulispicum C.DC. DQ868742P. penninerve C.DC. AF275206P. pierrei C.DC. AF275200P. porphyrophyllum N.E.Br. MH493522P. quinqueangulatum Miq. MH493537P. retrofractum Vahl AF275196P. sarmentosum Roxb. MH493595P. sorsogonum C.DC. AY572320P. thomsonii (C.DC.) Hook.f. DQ868749P. toppingii C.DC. AY572322P. umbellatum L. AF275174P. urdanetanum C.DC. AF275207P. wallichii (Miq.) Hand.-Mazz. EF450289

M.L.G. Dapar, et al. Phytochemistry Letters 36 (2020) 24–31

26

tannins, fatty acids, anthraquinones, and cyanogenic glycosides. Posi-tive results were qualitatively observed as indicated in each of themethods of phytochemical screening used. The findings were recordedusing a 3-point scale [+ turbid, ++ moderate and +++ heavy] inscoring based on the Handbook of Philippine Medicinal Plants by DePadua et al. (2005). Cell viability testing using trypan blue exclusionassay following the protocol used by Dela Peña et al. (2019) was per-formed to determine the number of viable normal lymphocytes sub-jected to each of the plant's ethanolic extracts at a maximum con-centration (1000 μg/ml). The trypan blue dye exclusion assay is aquantitative method to determine the percentage viability of viablecells and damaged nonviable cells. Viable cells have an intact cellularmembrane with a clear appearance for not taking up the blue dye,whereas nonviable cells are stained blue for taking up the dye as cellmembranes are damaged. The lymphocyte culture was prepared fol-lowing the procedure of GE Healthcare (2007) from the BiologicalResearch and Services Laboratory of the Natural Sciences ResearchInstitute of the University of the Philippines in Diliman. The supple-mented RPMI medium (RPMI with fetal bovine serum, Penicillin-Streptomycin, Amphotericin B), Triton X-100 (0.1 %), and DMSO inPBS (2 %) were used as the negative, positive and vehicle control intriplicates, respectively. The number of live and dead lymphocytes werecounted, and the cell density is computed.

3. Results

3.1. Nucleotide analysis

The BLASTn results of successfully sequenced ITS, matK, and rbcL ofthe two “Lunas”-named specimens identified these as members of thegenus Piper (Piperaceae) with e-values = 0 and %identity of ≥95.98 %(Table 3). Multiple sequence alignment of Piper species using ITS se-quences contained 781 positions, of which 266 were parsimony in-formative (Table 4). In the ITS consensus phylogenetic tree (Fig. 2),sample MGD-002 (“Lunas-bagon tapol”) grouped with Piper decumanumL. while sample 18AG-002 (“Lunas-buyo”) grouped with Piper aduncumL. with strong support (BS = 100 %). The CI and RI are 0.53 and 0.73,respectively (Table 4).

3.2. Distribution and morphological comparisons

The two identified Piper species were compared with the distribu-tion and morphology as indicated in the Co's Digital Flora of thePhilippines (Pelser et al., 2011 onwards) and with the respective typespecimens housed in B and P. Piper aduncum is a widely naturalized andinvasive non-native species in the Philippines, contrary to P. decu-manum which is a native species of Mt. Hilong-hilong (previouslyknown as Mt. Urdaneta), a medium elevated forest in Agusan del Norte,Philippines (Pelser and Barcelona, 2017). The Royal Society Expeditionof Chew (1972) to the Solomon Islands in 1965 recorded the distribu-tion of P. decumanum in Philippine Islands, Celebes, Moluccas, and NewGuinea while P. aduncum is distributed in Mexico, Central America,northern South America, West Indies, and naturalized in many places ofMalesian floristic regions including the Philippines. The two neotropicsPiper (P. aduncum and P. umbellatum) grouped together (BS = 95 %)

which conforms with the only two reported non-native Philippine Piperspecies (Pelser et al., 2011 onwards).

The two molecularly confirmed Piper species were concurrent withtheir morphology as compared to the diagnostic characters conciselydescribed by Gardner (2003, 2006, 2013) and from comprehensiverevision of Chew (1972). The identified P. decumanum is a dioecious,completely glabrous climber; it possesses an ovate, often rugose laminawith acute to bluntly acuminate apex, asymmetrically cordate to aur-iculate base, and often shorter than the auricular base, sheathing at thelower half petioles. On the other hand, the identified P. aduncum, co-inciding with its diagnostic characters, constitutes monoecious shrubswith ovate, broad lamina with long acuminate apex, lightly asymme-trical cordate base, and slightly longer than the sinus of the lamina basebut shorter than the peduncular stalk petioles; and often bisexual,usually curved, as long as leaves, with flowers borne in dense spiralsinflorescences.

3.3. Phytochemical constituents and cytotoxic activities

The results of the phytochemical screening of the ethanolic extractsof the two “Lunas”-named specimens revealed the presence of variableamounts of constituents like alkaloids, flavonoids, steroids, tannins, andfatty acids but none of cyanogenic glycosides (Table 5). Piper decu-manum has moderate to heavy amounts of these phytochemicals whencompared to P. aduncum which showed turbid to moderate quantities.Also, Piper decumanum lacks anthraquinones while saponins are missingin P. aduncum.

Evaluation of cytotoxic effect of the stem ethanolic extracts of thetwo “Lunas”-named specimens showed low (91–100 % viable cells; DelaPeña et al., 2019) for P. decumanum and mild toxicity (81–90 % viablecells; Dela Peña et al., 2019) for P. aduncum to normal lymphocytesfrom human blood (Table 6). This cytotoxicity result may be attributedto the absence of cyanogenic glycosides in the extracts from two plants(Dela Peña et al., 2019; Uy et al., 2019).

4. Discussion

The use of molecular sequences to verify species identity of med-icinal plants with species problem due to similar local names andconfusing identity was found to be very useful. The two “Lunas”-namedspecimens were molecularly confirmed as the indigenous Piper decu-manum for “Lunas-bagon tapol”, and the introduced P. aduncum for

Table 3BLASTn results of newly sequenced samples for markers ITS, matK, and rbcL.

Local Name Sample code/Voucher

Place of Collection (nrDNA) ITS (cpDNA) matK (cpDNA) rbcL

BLAST ID e-value % identity BLAST ID e-value % identity BLAST ID e-value % identity

“Lunas-bagontapol”

MGD-002/USTH 015544

Mt. Ararat, Bayugan CityAgusan del Sur

Piperdecumanum

0.0 99.68 % Piperdecumanum

0.0 97.79 % Pipermacropiper

0.0 99.85 %

“Lunas-buyo” 18AG-002/USTH 015181

Mt. Pinagalaan, BayuganCity, Agusan del Sur

Piper aduncum 0.0 99.69 % Piper aduncum 0.0 98.67 % Piperaduncum

0.0 95.98 %

Table 4Characteristics of molecular dataset used in this study.

Parameters ITS

Number of species 47Number of individuals 51Alignment length (bp) 781Variable sites 412Parsimony-informative characters 266Consistency index (CI) 0.53Retention index (RI) 0.73

M.L.G. Dapar, et al. Phytochemistry Letters 36 (2020) 24–31

27

Fig. 2. Strict consensus tree derived from 34 equally parsimonious trees based on the phylogenetic analysis of ITS sequence data. Length = 938 steps; consistencyindex = 0.53; retention index = 0.73. Branch lengths are drawn to scale. Percentage of 1000 bootstrap replicates is given when higher than 50 %. Highlighted cladesin gray showed where the newly included samples written in red (MGD-002 and 18AG-002) are nested. (For interpretation of the references to colour in this figurelegend, the reader is referred to the web version of this article.)

M.L.G. Dapar, et al. Phytochemistry Letters 36 (2020) 24–31

28

“Lunas-buyo” based on the nuclear ITS sequences. The result of mole-cular confirmation of these two Piper species reinforced traditionalknowledge of Agusan Manobo on their ethnoclassification as differentspecies which are only named similarly on its first word due to itshomologous ethnomedicinal uses. This molecular verification is sup-ported by the morphological diagnostic characteristics of Piper speciesin the Philippines by Chew (1972) and Gardner (2003, 2006, 2013).Relatively, P. decumanum is still unresolved species with a low con-fidence level (The Plant List, 2013) but integrative molecular approachcoupled with assessment of available data from morphology and eth-noclassification could provide a more accurate species identification.Vernacular names are essential for preserving traditional knowledgeand recording ethnotaxa (Ghorbani et al., 2017) but reliance on themoften result to erroneous identification resulting to research invalida-tion (Bennett and Balick, 2014; Rivera et al., 2014). This molecularconfirmation study could serve as useful future reference for properidentification of unresolved species and even unidentifiable collectedplant materials. Correct identification of plant species can be verifiedaccurately by means of integrative molecular approach for all phar-macological studies and natural product researches.

Vegetative identification of P. decumanum could be difficult becausethis species is most of the time sterile according to theManobo residentsand upon collection. Most dioecious Piper species like P. decumanumhave resemblances among and within species (Chaveerach et al., 2008).In fact, Sanubol et al. (2014) recommended the use of chemical com-position and molecular data aside from morphology in the identifica-tion of Piper species. Even in P. aduncum when collected with in-florescences, morphology alone is often insufficient for identificationbecause Piper species have small flowers with uniform vegetative andfloral morphology (Suwanphakdee et al., 2016). Within and amongPiper species, controversies exist regarding their true identity basedsolely on morphology; thus, correct identification should be corrobo-rated by molecular data (Sanubol et al., 2014) which is demonstratedby this present work again.

Integration of molecular data with the morphology of plants is alsoneeded to test traditional limits of identification. Different DNA-basedmolecular markers are applied in various fields, and their application istremendously increasing for species characterization of medicinalplants (Shaw et al., 2005; Sucher and Carles, 2008). The BLASTnmethod estimates the reliability of species identification as a sequence

similarity search program to determine the sequence of interest(McGinnis and Madden, 2004) in identifying the statistical relatednessand significance of matches in the genus Piper. The result of this mo-lecular confirmation further supported the use of ITS sequences forphylogenetic analysis of Piper species in Asia, the South Pacific and theNeotropics by Jaramillo and Manos (2001) and Jaramillo et al. (2008)as well as the phylogeny of Malesian-Pacific Piper species byAsmarayani (2018).

Moreover, the presence of constituents such as alkaloids, flavonoids,steroids, tannins, fatty acids and anthraquinones are medically im-portant bioactive compounds which scientifically validated the bio-chemical and biological basis of the ethnomedicinal use of Piper speciesamong the indigenous group of Agusan Manobo against cancer, stomachtroubles, gastralgia, diarrhea, vomiting, liver and urological problems,fever, hypertension, food poisoning, skin diseases, snakebites and forwound healing. The comprehensive review of Salehi et al. (2019) on thephytochemistry, biological activities and applications of Piper speciespresented several phytochemicals and essential oils present that arestrongly antioxidant and potentially antibacterial or antifungal agentsagainst human pathogens. Furthermore, Piper species were observed tohave a significant amount of Piplartine, an amide alkaloid which is apotent anticancer agent (Raja Mazlan et al., 2018).

While the characterization of specific phytochemicals in the two“Lunas”-named specimens were not done, variations in the amounts inthe extracts further differentiated the two plant species. Differences inthe level of toxicity were also observed where “Lunas-bagon tapol” haslow toxicity (91–100 %) when compared to mild toxicity (81–90 %) of“Lunas-buyo”. Although trypan blue exclusion assay has its limitationssince trypan blue emits fluorescence when complexed with proteinswhich could be hard to distinguish unstained living cells from fluor-escent dead cells, combining this cell viability analysis with flow cy-tometry technique could be an alternative tool for a quick and reliableresult (Avelar-Freitas et al., 2014). However, this cytotoxicity result issimilar to studies done on nine Piper species with betel-like scents crudeextracts showing significant genotoxicity in HeLa cells but generallynon-toxic in leukocytes (Sanubol et al., 2017). This result may alsovalidate the effective and safe ethnomedicinal use of medicinal Piperspecies used by the Agusan Manobo lending credence on their folkmedicine.

Table 5Results of phytochemical screening of P. decumanum and P. aduncum stem ethanolic extracts.

Phytochemicals Screened Methods Used Indication of Positive Results Piper decumanum Piper aduncum

Alkaloids Mayer’s Test Whitish or cream colored precipitate ++ +Saponins Froth Test Honeycomb froth ++ –Flavonoids Bate-Smith and Metcalf Test Shades of red +++ ++Steroids Keller-Kiliani Test Reddish brown ring obtained at the interface +++ ++Tannins Gelatin Test Precipitate +++ ++Fatty acids Fatty acids test Transparency on filter paper +++ ++Anthraquinones Borntrager’s Test Pink violet or red color in the ammoniacal layer – ++Cyanogenic Glycosides Guignard Test Shades of red – –

(+) indicates present: +turbid, ++moderate, +++heavy; (-) indicates absent.

Table 6Cytotoxic activity of P. decumanum and P. aduncum stem ethanolic extract to normal human lymphocytes.

Treatment Average No. of Live Cells Average No. of Dead Cells Average No. of Total Cells Average Percent of Live Cells

Supplemented RPMIa 61.56 4.00 65.56 93.9Triton X-100b (0.1%) 0.00 56.56 56.56 0.00DMSOc (2%) 49.67 6.67 56.33 88.2P. decumanum (1000 μg/ml) 60.33 5.33 65.67 91.9P. aduncum (1000 μg/ml) 57.67 7.67 65.33 88.3

a Negative control.b Positive control.c Vehicle control.

M.L.G. Dapar, et al. Phytochemistry Letters 36 (2020) 24–31

29

5. Conclusion

Molecular sequences inferred from the nuclear ITS with compara-tive morphology confirmed the identity of “Lunas-bagon tapol” as Piperdecumanum while “Lunas-buyo” as Piper aduncum. Results also showedthat the two confused “Lunas”-named specimens do not only differ onspecies identity but also in the number and amount of phytochemicalconstituents present and cytotoxic properties. It can be concluded thatspecies identities can be confirmed using molecular data when mor-phological data is limited and inconclusive while constituent and cy-totoxic evaluation can be useful in determining variations betweenplant species used in ethnomedicine. These scientific results also vali-dated the tribal claims of Agusan Manobo on the ethnomedicinal usesand application of their medicinal Piper species to further conserve,protect, and investigate these important genetic resources in line withthe government programs and initiatives.

Declaration of Competing Interest

The authors declare that there is no conflict of interest.

Acknowledgements

The first author would like to thank his scholarship grant from theDepartment of Science and Technology - Accelerated Science andTechnology Human Resource Development Program - National ScienceConsortium (DOST-ASTHRDP-NSC) and Alexander von HumboldtFoundation as a Junior Researcher. The last author thanks theDepartment of Health - Philippine Institute of Traditional andAlternative Health Care (DOH-PITAHC) for the funding, and Alexandervon Humboldt Foundation for a renewed research stay at the Universityof Bayreuth (Germany) in 2019.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in theonline version, at doi:https://doi.org/10.1016/j.phytol.2020.01.017.

References

Aguinaldo, A.M., Espeso, E.I., Guevara, B.Q., Nonato, M.G., 2005. Phytochemistry. In:Guevara, B.Q. (Ed.), A Guidebook to Plant Screening: Phytochemical and Biological.University of Santo Tomas, Manila, Philippines.

Alejandro, G.D., Razafimandimbison, S.G., Liede-Schumann, S., 2005. Polyphyly ofMussaenda inferred from ITS and trnT-F data and its implication for generic limits inMussaendeae (Rubiaceae). Am. J. Bot. 92 (3), 544–557.

Asmarayani, R., 2018. Phylogenetic relationships in Malesian-Pacific Piper (Piperaceae)and their implications for systematics. Taxon 67 (4), 693–724.

Avelar-Freitas, B.A., Almeida, V.G., Pinto, M.C., Mourão, F.A., Massensini, A.R., Martins-Filho, O.A., Rocha-Vieira, E., Brito-Melo, G.E., 2014. Trypan blue exclusion assay byflow cytometry. Braz. J. Med. Biol. Res. 47 (4), 307–315.

Bafeel, S., Arif, I., Bakir, M., Al-Homaidan, A., 2012. DNA barcoding of arid wild plantsusing rbcL gene sequences. Genet. Mol. Res. 11 (3), 1934–1941.

Bennett, B.C., Balick, M.J., 2014. Does the name really matter? The importance of bo-tanical nomenclature and plant taxonomy in biomedical research. J.Ethnopharmacol. 152, 387–392.

Bizerril, M.X.A., Raw, A., 1998. Feeding behaviour of bats and dispersal of Piper arboreumseeds in Brazil. J. Trop. Ecol. 14, 109–114.

Bramley, G., Utteridge, T., 2015. The Kew Tropical Plant Families IdentificationHandbook, 2nd ed. Royal Botanic Gardens, Kew.

Chase, M.W., Hills, H.H., 1991. Silica gel: an ideal material for preservation of leafsamples for DNA studies. Taxon 40, 215–220.

Chaveerach, A., Mokkamul, P., Sudmoon, R., Tanee, T., 2006. Ethnobotany of the genusPiper (Piperaceae) in Thailand. Ethnobot. Res. Appl. 4, 223–231.

Chaveerach, A., Sudmoon, R., Tanee, T., Mokkamul, P., 2008. The species diversity of thegenus Piper from Thailand. Acta Phytotaxonomica et Geobotanica 59, 105–163.

Chew, W.L., 1972. The genus Piper (Piperaceae) in New Guinea, Solomon Islands andAustralia. J. Arnold Arbor. 53 (1), 1–25.

Costion, C., Ford, A., Cross, H., Crayn, D., Harrington, M., Lowe, A., 2011. Plant DNAbarcodes can accurately estimate species richness in poorly known floras. PLoS One6 (11).

Dapar, M.L.D., Demayo, C.G., 2017. Folk medical uses of Lunas Lunasia amara Blanco bythe Manobo people, traditional healers and residents of Agusan del Sur, Philippines.

Sci. Int. (Lahore) 29 (4), 823–826.Dapar, M.L.D., Demayo, C.G.D., Senarath, W.T.P.S.K., 2018. Antimicrobial and cellular

metabolic inhibitory properties of the ethanolic extract from the bark of ‘Lunas-Bagon’ (Lunasia sp.). Int. J. Pharm. Sci. Res. 9 (1), 88–97.

De Padua, L.S., Lugod, G.C., Pancho, J.V., 2005. Handbook of Philippine MedicinalPlants. University of the Philippines, Los Baños, pp. 66 1981 (1–4).

Dela Peña, J.F., Dapar, M.L.G., Aranas, A.T., Mindo, R.A.R., Cabrido, C.K., Torres, M.A.J.,Manting, M.M.E., Demayo, C.G., 2019. Assessment of antimicrobial, antioxidant andcytotoxic properties of the ethanolic extract from Dracontomelon Dao (Blanco) Merr.& Rolfe. Pharmacophore 10 (2), 18–29.

Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and highthroughput. Nucleic Acids Res. 32, 1792–1797.

Fan, L.S., Muhamad, R., Omar, D., Rahmani, M., 2011. Insecticidal properties of Pipernigrum fruit extracts and essential oils against Spodoptera litura. Int. J. Agric. Biol. 13,517–522.

Farris, J.S., 1989. The retention index and the rescaled consistency index. Cladistics 5,417–419.

Fleming, T.H., 1981. Fecundity, fruiting pattern, and seed dispersal in Piper amalago(Piperaceae), a bat dispersed tropical shrub. Oecologia 51, 42–46.

Gardner, R.O., 2003. Piper (Piperaceae) in New Guinea: the non-climbing species. Blumea57 (48), 47–68.

Gardner, R.O., 2006. Piper in the Philippine Islands: the climbing species. Blumea 51 (3),569–586.

Gardner, R.O., 2013. Piper (Piperaceae) in New Guinea: the climbing species. Blumea 57,275–294.

GE Healthcare, 2007. Ficoll-Paque PLUS Instructions. GE Healthcare Bio-Sciences, AB,Uppsala, Sweden.

Ghorbani, A., Saeedi, Y., de Boer, H.J., 2017. Unidentifiable by morphology: DNA bar-coding of plant material in local markets in Iran. PLoS One 12 (4).

Hollingsworth, P., 2011. Refining the DNA barcode for plants. Proc. Natl. Acad. Sci. U. S.A. 108 (49), 19451–19452.

Jachac, S.M., Saklani, A., 2007. Challenges and opportunities in drug discovery fromplants. Curr. Sci. 92 (9).

Jaramillo, M.A., Manos, P.S., 2001. Phylogeny and patterns of floral diversity in the genusPiper (Piperaceae). Am. J. Bot. 88 (4), 706–716.

Jaramillo, M.A., Callejas, R., Davidson, C., Smith, J.F., Stevens, A.C., Tepe, E.J., 2008. Aphylogeny of the tropical genus Piper using ITS and the chloroplast intron psbJ–petA.Syst. Bot. 33, 647–660.

Kluge, A.G., Farris, J.S., 1969. Quantitative phyletics and the evolution of anurans. Syst.Zool. 18, 1–32.

Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary geneticsanalysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874.

Li, S., Lei, Y., Jia, Y., Li, N., Wink, M., Ma, Y., 2011. Piperine, a piperidine alkaloid fromPiper nigrum re-sensitizes P-gp, MRP1 and BCRP dependent multidrug resistant cancercells. Phytomedicine 19 (1), 83–87.

Li, X., Yang, Y., Henry, R.J., Rossetto, M., Wang, Y., Chen, S., 2014. Plant DNA barcoding:from gene to genome. Biol. Rev. 90 (1), 157–166.

Macabeo, A.P., Aguinaldo, A.M., 2008. Chemical and phytomedicinal investigations inLunasia amara. Pharmacogn. Rev. 2 (4), 317–325.

McGinnis, S., Madden, T., 2004. BLAST: at the core of a powerful and diverse set ofsequence analysis tools. Nucleic Acids Res. 32.

National Commission on Indigenous Peoples (NCIP), 2010. Primer on Census forIndigenous Peoples. National Commission on Indigenous Peoples, Quezon City,Philippines.

Pelser, P.B., Barcelona, J.F., 2017. Co’s Digital Flora of the Philippines. Retrieved fromwww.philippineplants.org on 08 July 2019. .

Pelser, P.B., Barcelona, J.F., Nickrent, D.L., 2011 onwards. Co’s Digital Flora of thePhilippines Retrieved from www.philippineplants.org on 08 July 2019. CDFP.

Philippine Statistics Authority (PSA), 2016. 2010 Census of Population and Housing:Definition of Terms and Concepts. Philippine Statistics Authority, Quezon City,Philippines.

Prescott, T.A.K., Sadler, I.H., Kiapranis, R., Maciver, S.K., 2007. Lunacridine from Lunasiaamara is a DNA intercalating topoisomerase II inhibitor. J. Ethnopharmacol. 109,289–294.

Raja Mazlan, R., Rukayadi, Y., Maulidiani, M., Ismail, I.S., 2018. Solvent extraction andidentification of active anticariogenic metabolites in Piper cubeba L. through 1H-NMR-based metabolomics approach. Molecules 23 (7), 1730.

Rivera, D., Allkin, R., Obo ́n, C., Alcaraz, F., Verpoorte, R., Heinrich, M., 2014. What is ina name? The need for accurate scientific nomenclature for plants. J. Ethnopharmacol.152, 393–402.

Salehi, B., Zakaria, Z.A., Gyawali, R., Ibrahim, S.A., Rajkovic, J., Shinwari, Z.K., Khan, T.,Sharifi-Rad, J., Ozleyen, A., Turkdonmez, E., Valussi, M., Tumer, T.B., Fidalgo, L.M.,Martorell, M., Setzer, W.N., 2019. Piper species: a comprehensive review on theirphytochemistry, biological activities and applications. Molecules 24 (7), 1364.

Sanderson, M.J., Donoghue, M.J., 1994. Shifts in diversification rate with the origin ofangiosperms. Science 265, 1590–1593.

Sanubol, A., Chaveerach, A., Sudmoon, R., Tanee, T., Liehr, T., 2014. Verification of se-lected Piper species (Piperaceae) using morphological characters, molecular data, andchemical constituents. Malay. Nat. J. 66 (3), 60–81.

Sanubol, A., Chaveerach, A., Tanee, T., Sudmoon, R., 2017. Pre-clinical evaluation ofextracts and essential oils from betel-like scent Piper species identified potentialcancer treatment. Afr. J. Tradit. Complement. Altern. Med. 14 (1), 89–102.

Scott, I.M., Jensen, H.R., Philogène, B.J.R., Arnason, J.T., 2008. A review of Piper spp.(Piperaceae) phytochemistry, insecticidal activity and mode of action. Phytochem.Rev. 7, 65–75.

Shaw, J., Lickey, E.B., Beck, J.T., Farmer, S.B., Liu, W., Miller, J., Small, R.L., 2005. The

M.L.G. Dapar, et al. Phytochemistry Letters 36 (2020) 24–31

30

tortoise and the hare II: relative utility of 21 noncoding chloroplast DNA sequencesfor phylogenetic analysis. Am. J. Bot. 92 (1), 142–166.

Sucher, N., Carles, M., 2008. Genome-based approaches to the authentication of medic-inal plants. Planta Med. 74, 603–623.

Suwanphakdee, C., Chantaranothai, P., 2011. A new species and three taxonomic changesin Piper (Piperaceae) from Thailand. Blumea 56, 235–239.

Suwanphakdee, C., Simpson, D.A., Hodkinson, T.R., Chantaranothai, P., 2016. Taxonomicnotes on the genus Piper (Piperaceae). Nord. J. Bot. 34, 605–618.

The Plant List, 2013. Retrieved from http://www.theplantlist.org/ on 08 July 2019.Thomford, N.E., Senthebane, D.A., Rowe, A., Munro, D., Seele, P., Maroyi, A., Dzobo, K.,

2018. Natural products for drug discovery in the 21st century: innovations for novel

drug discovery. Int. J. Mol. Sci. 19 (6).Umadevi, P., Deepti, K., Venugopal, D.V., 2013. Synthesis, anticancer and antibacterial

activities of piperine analogs. Med. Chem. Res. 22, 5466–5471.Uy, I.A., Dapar, M.L.G., Aranas, A.T., Mindo, R.A.R., Cabrido, C.K., Torres, M.A.J.,

Manting, M.M.E., Demayo, C.G., 2019. Qualitative assessment of the antimicrobial,antioxidant, phytochemical properties of the ethanolic extracts of the roots of Cocosnucifera L. Pharmacophore 10 (2), 63–75.

Wang, Y.H., Morris-Natschke, S.L., Yang, J., Niu, H.M., Long, C.L., Lee, K.H., 2014.Anticancer principles from medicinal Piper (胡椒 Hú Jiāo) plants. J. Tradit.Complement. Med. 4 (1), 8–16.

M.L.G. Dapar, et al. Phytochemistry Letters 36 (2020) 24–31

31