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Molecular confirmation, constituents and cytotoxicity evaluation of two medicinal Piper species used by the Manobo tribe of Agusan del Sur, Philippines
Phytochemistry Letters 36 (2020) 24–31
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Molecular confirmation, constituents and cytotoxicity evaluation of two medicinal Piper species used by the Manobo tribe of Agusan del Sur, Philippines
T
Mark Lloyd G. Dapara,d,*, Cesar G. Demayoc, Ulrich Meved, Sigrid Liede-Schumannd, Grecebio Jonathan D. Alejandroa,b,d a
The Graduate School and Research Center for the Natural and Applied Sciences, University of Santo Tomas, España Boulevard, 1015 Manila, Philippines College of Science, University of Santo Tomas, España Boulevard, 1015 Manila, Philippines Department of Biological Sciences, College of Science and Mathematics, Mindanao State University - Iligan Institute of Technology, 9200 Iligan City, Philippines d Department of Plant Systematics, University of Bayreuth, Universitätsstr. 30, D-95447 Bayreuth, Germany b c
ARTICLE INFO
ABSTRACT
Keywords: BLASTn Cytotoxicity ITS Lunasia Phytochemicals Piper aduncum Piper decumanum
The Manobo tribe in the Philippines is culturally rich in ethnomedicinal practices and known to use popular plants locally named as “Lunas” (meaning “cure”). One of these is “Lunas-bagon tapol” which was previously identified as Lunasia sp. (Rutaceae) based on vegetative morphology. The other species is “Lunas-buyo” which is similar in ethnomedicinal use and morphology. This broadly-based study verifies these two “Lunas”-named specimens and evaluates the phytochemicals present and cytotoxic properties. The sequences of nuclear ITS, and plastid rbcL, and matK were investigated for species identification. Molecular confirmation using BLASTn nucleotide database query revealed that the two confused “Lunas”-named specimens were members of Piperaceae and not Rutaceae. Phylogeny of Asian Piper using ITS sequences revealed “Lunas-bagon tapol” as Piper decumanum L. and “Lunas-buyo” as Piper aduncum L. with strong support (BS = 100 %). Both Piper species similarly showed the presence of alkaloids, flavonoids, steroids, tannins and fatty acids but the absence of cyanogenic glycosides. Also, P. decumanum has moderate amount of saponins while P. aduncum contains moderate amount of anthraquinones. A cytotoxic activity test using trypan blue exclusion method against normal lymphocytes from human 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 practical approach of molecular-based taxon identification and provide the biochemical and biological basis as to the constituents present and cytotoxic properties of these medicinal Piper species for future pharmacological research, conservation priorities and ecological management.
1. Introduction The Philippines is a multi-cultural country with more than a hundred ethnolinguistic groups (Philippine Statistics Authority (PSA, 2016) classified according to its own unique identity, language, socio-political systems, and practices (National Commission on Indigenous Peoples (NCIP, 2010). The Manobo indigenous group among others is one of the most numerous Philippine ethnic groups occupying a wide area of distribution in Mindanao, Philippines using several medicinal plants for disease treatment and medication due to poor access of healthcare services. The Manobo tribe found in Agusan provinces is known as the Agusan Manobo inhabiting communities near Agusan River Valley and highlands 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 river people, coined from the “man” (people) and the “suba” (river). Given their location as a significant habitat for most of their indigenous (medicinal) plants, their traditional medical practice of using medicinal plants has long been practiced until these days for their primary disease treatment and healthcare. Some of their notable medicinal plants are locally known as “Lunas” in Bisaya and Manobo dialects which means “cure” with several species associated under this name. An example of which is the “Lunas-bagon tapol” 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
Corresponding author at: The Graduate School, University of Santo Tomas, España Boulevard, 1015 Manila, Philippines. E-mail address: [email protected] (M.L.G. Dapar).
https://doi.org/10.1016/j.phytol.2020.01.017 Received 23 September 2019; Received in revised form 17 December 2019; Accepted 16 January 2020 1874-3900/ © 2020 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
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uncertain, because vegetatively, Rutaceae can be confused easily with Piperaceae. Both families comprise woody or non-woody climbers, and also some trees, shrubs, and herbs, with alternate or opposite and aromatic leaves bearing essential oils (Bramley and Utteridge, 2015). Likewise, both families share similar biomedical properties as antibacterial and cytotoxic against cancer cells, as proven for Lunasia species (Prescott et al., 2007) and for Piper species (Wang et al., 2014). Both families were also reported rich in alkaloid content as potent cytotoxic 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 an effective treatment against cancer, stomach troubles, gastralgia, diarrhea, vomiting, hypertension, food poisoning and snakebites as reported by Macabeo and Aguinaldo (2008) for L. amara, and Salehi et al. (2019) for Piper species. As a result, Dapar and Demayo (2017) and Dapar et al. (2018) previously identified the “Lunas-bagon tapol” as one possible new variety of Lunasia amara in addition to the two varieties L. amara var. amara and L. amara var. babuyanica (Merr.) Hartley in the Philippines. The Agusan Manobo ethnomedicinally claimed “Lunasbagon” vine species as traditional source of anti-inflammatory, antimotility, antihistamine, antiparasitic, antibacterial, antitoxin, and antiviral medicines (Dapar and Demayo, 2017). Another medicinal plant, which is classified by the Agusan Manobo tribe to be closely related with “Lunas-bagon tapol” in terms of medicinal uses and morphology, is locally known as “Lunas-buyo”. As a consequence, the identity of these two “Lunas”-named specimens (“Lunas-bagon tapol” and “Lunas-buyo”) poses uncertainty in terms of their true familial and generic affinities. Piper is diverse in habit and inhabits diverse habitat (Sanderson and Donoghue, 1994), and has a widespread distribution occurring in American tropics with 700 spp. and in Southern Asia with 300 spp. (Jaramillo and Manos, 2001). Gardner (2006) recognized 15 climbing Philippine Piper species with only one taxon (P. myrmecophilum C.DC.) accepted as endemic while Pelser et al. (2011 onwards) reported 65 Piper species occurring in the Philippines. Piper nigrum L. (black pepper) and P. betle L. (betel leaf) are some of the economically important species 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 of Piper species, vegetative identification up to the species level is difficult. Meticulous investigation of its reproductive parts like fruits and infructescence are very important for species identification (Suwanphakdee and Chantaranothai, 2011). The unique composition of the genetic material for each species makes DNA-based identification a useful tool for accurate species identification. Therefore, correct identification of a medicinal plant should be examined using a molecular approach (Sanubol et al., 2014) for consistency of species and pharmacological investigations of natural products (Thomford et al., 2018). Although plant-based drug discovery in the ethnobotanical approach provides future drug leads,
authentication of the plant material is a great challenge and opportunity (Jachac and Saklani, 2007). Chemical composition, morphology, and molecular data of some species pairs must be studied for accurate species identification (Sanubol et al., 2014). Moreover, the use of internal transcribed spacer (ITS) of nuclear ribosomal DNA is a powerful phylogenetic marker showing high interspecific divergence and greater discriminatory power over the plastid regions (Hollingsworth, 2011; Li et 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 the nuclear ITS, plastid matK and rbcL. The evaluation of the phytochemical constituents and cytotoxic activities of the extracts from the two species were included to further evaluate their similarities and differences. The information generated in this study not only resolve true species identity of the two species but also provide the basis for their ethnomedicinal use and as a potential source of medicine. This study can also pave the way for the future conservation of these two ethnomedicinal plant species as one of the critical genetic resources of the Agusan Manobo in Mindanao, Philippines. 2. Materials and methods 2.1. Sample collection Fieldwork started with consultation and meeting of the tribal community with the council of elders to discuss research intent as purely academic. As agreed, a certification from the tribal council of elders was given following ritual observation with the community as a cultural tradition to grant permission on the conduct of the study. Free prior informed consents (FPICs) from the only two community tribal healers were acquired. When the tribal certification and FPICs were secured, the national regulators duly provided certification and permit following the by-laws on the protection of the indigenous peoples and the permission to collect wild plant samples as issued from the regional government administration. The National Commission on Indigenous Peoples of CARAGA Administrative Region (NCIP-CARAGA) issued certification (no. R13-2019-01), and the Department of Environment and Natural Resources of CARAGA Administrative Region (DENRCARAGA) granted wildlife gratuitous permit (no. R13-2019-12) for this study. All necessary permit and certifications were reviewed by the University of Santo Tomas Graduate School - Ethics Review Board (USTGS-ERC) and granted ethics approval (protocol no. GS-2019PN007) before plant collection. The collection of the plant samples was followed through guided field walks with the two tribal healers who are knowledgeable of their medicinal plants. Both tribal healers verified the collected plant materials and similarly classified both collected plant samples with the initial word “Lunas” but different second names denoting different plant species accordingly and locally named one as “Lunas-bagon tapol” and the other as “Lunas-buyo”. Samples of “Lunasbagon 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). 25
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Table 1 Gene 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, and ITS2) rbcL
P17F 26S-82R rbcL_aF rbcL_aR 3F_KimF IR_KimR
Alejandro et al. (2005) Bafeel et al. (2012)
5'-CTACCGATTGAATGGTCCGGTGAA-3ʹ 5ʹ-TCCCGGTTCGCTCGCCGTTACTA-3ʹ 5ʹ-ATGTCACCACAAACAGAGACTAAAGC-3ʹ 5ʹ-GTAAAATCAAGTCCACCRCG-3ʹ 5ʹ-CGTACAGTACTTTTGTGTTTACGAG-3ʹ 5ʹ-ACCCAGTCCATCTGGAAATCTTGGTTC-3ʹ
97 °C 90 s; 35 cycles of 97 °C 20 s, 72 °C 90 s, 72 °C 30 s; 72 °C 7 min 95 °C 1 min; 35 cycles of 95 °C 30 s, 51 °C 30 s, 68 °C 1 min; 68 °C 5 min 94 °C 5 min; 35 cycles of 94 °C 30 s, 52 °C 30 s, 72 °C 50 s; 5 min at 72 °C
matK
Costion et al. (2011)
Agusan del Sur (coded MGD-002) while “Lunas-buyo” (Fig. 1B&C) was collected in Mt. Pinagalaan, Bayugan City, Agusan del Sur (coded 18AG-002). Voucher specimens were deposited in the University of Santo Tomas Herbarium (USTH). Leaf samples were placed in ziplocked bags containing silica gel for molecular analysis (Chase and Hills, 1991). Mature stems were collected from each “Lunas”-named specimens for phytochemical and cytotoxicity analyses since this is the plant part used for cure by the tribal community. Collected stems were washed with water, air-dried, and pulverized into a fine powder using a blender. The preparation and extraction procedure followed the protocol used by Uy et al. (2019) to obtain the viscous crude extracts which were finally stored in vials for phytochemical screening and cytotoxicity testing.
Table 2 Nucleotide sequence database accession numbers of taxa used in the ITS phylogenetic analysis. Species OUTGROUP Peperomia elongata Kunth Sarcorhachis naranjoana (C.DC.) Trel. INGROUP Piper abbreviatum Opiz P. aduncum L. (1) P. aduncum L. (2) P. albispicum C.DC. P. arborescens Roxb. P. arboricola C.DC. P. austrocaledonicum C.DC. P. baccatum Blume P. bavinum C.DC. P. betle L. P. boehmeriaefolium Wall. P. brevicuspe (Miq.) Merr. P. caninum Blume P. celtidiforme Opiz P. cordatilimbum Quisumb. P. decumanum L. (1) P. decumanum L. (2) P. densum Bl. P. fragile Benth. P. guineense Schumach. & Thonn. P. gymnostachyum C.DC. P. hancei Maxim. P. hymenophyllum Miq. P. korthalsii Miq. P. laosanum C.DC. P. lessertianum (Miq.) C.DC. P. lolot C.DC. P. macropiper Pennant P. majusculum Blume P. medinillifolium Quisumb. P. muricatum Blume P. mutabile C.DC. P. myrmecophilum C.DC. P. nigrum L. P. pendulispicum C.DC. P. penninerve C.DC. P. pierrei C.DC. P. porphyrophyllum N.E.Br. P. quinqueangulatum Miq. P. retrofractum Vahl P. sarmentosum Roxb. P. sorsogonum C.DC. P. thomsonii (C.DC.) Hook.f. P. toppingii C.DC. P. umbellatum L. P. urdanetanum C.DC. P. wallichii (Miq.) Hand.-Mazz.
2.2. DNA extraction, amplification, and sequencing The total genomic DNA was extracted from silica gel-dried leaf tissues of “Lunas-bagon tapol” and “Lunas-buyo” samples following the protocols of DNeasy Plant Minikit (Qiagen, Germany). The ITS (nrDNA), matK, and rbcL (cpDNA) markers were used for this study. The gene regions, primer pairs, sequences, and PCR parameters, as shown in Table 1 were used for amplification reactions using Biometra Thermocycler T-Gradient. Amplicons were purified using Qia-Quick PCR Purification Kit (Qiagen, Germany) following the manufacturer’s protocol and sent to MACROGEN Inc., Seoul, South Korea, for bidirectional sequencing. 2.3. Sequence and phylogenetic analysis The newly generated sequences of each sample were assembled and edited using Codon Code Aligner v8.0.2, trimmed and checked for ambiguous nucleotides. Species identity was evaluated using BLASTn search algorithm available in the GenBank (www.ncbi.nim.nih.gov). Available sequences of all reported Philippine and other Asian Piper species using the three markers (ITS, matK and rbcL) were initially checked for inclusion in the phylogenetic analysis. Of the three markers, only ITS was subjected to phylogenetic analysis since it has sufficient 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 and Manos (2001) and Jaramillo et al. (2008) were also included in the analysis. A total of forty-seven accessions of Piper species including two recognized non-native Philippine species (Piper aduncum L. and Piper umbellatum L.) and two outgroups (Peperomia elongata Kunth and Sarcorhachis naranjoana (C.DC.) Trel.) were included in parsimony analysis of the ITS marker (Table 2). Multiple alignment of sequences used MUSCLE (Edgar, 2004) implemented in MEGA v.7.0.18 (Kumar et al., 2016) and was finally edited by visual inspection. Parsimony analysis was done using TBR search method with 100 random addition of initial trees and examined using 1000 bootstrap replicates. Consistency index (CI; Kluge and Farris, 1969) and retention index (RI; Farris, 1989) were calculated to estimate homoplasy.
GenBank accession numbers AF275213 AF275210 MH493098 AF275157 EF060061 AY572317 AF275202 AY572319 MH493124 MH493135 AF275199 AF275201 AF275204 AY572321 AF275195 AF275205 AY572323 AF275203 MH493337 AY615963 MH493373 MH493383 AY572325 EF450272 AY572327 AF275208 AY572326 MH493410 AY326208 MH493418 MH493434 AY667455 EF060076 DQ868737 AY572328 AF275198 DQ868742 AF275206 AF275200 MH493522 MH493537 AF275196 MH493595 AY572320 DQ868749 AY572322 AF275174 AF275207 EF450289
2.4. Phytochemical screening and cytotoxicity test The phytochemical screening was based on the protocol described by Aguinaldo et al. (2005) with slight modification. Qualitative assessment was done on the stem ethanolic extracts to screen for phytochemicals present such as alkaloids, saponins, flavonoids, steroids, 26
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Table 3 BLASTn results of newly sequenced samples for markers ITS, matK, and rbcL. Local Name
“Lunas-bagon tapol” “Lunas-buyo”
Sample code/ Voucher
Place of Collection
MGD-002/ USTH 015544 18AG-002/ USTH 015181
Mt. Ararat, Bayugan City Agusan del Sur Mt. Pinagalaan, Bayugan City, Agusan del Sur
(nrDNA) ITS
(cpDNA) matK
(cpDNA) rbcL
BLAST ID
e-value
% identity
BLAST ID
e-value
% identity
BLAST ID
e-value
% identity
Piper decumanum Piper aduncum
0.0
99.68 %
0.0
97.79 %
99.85 %
99.69 %
0.0
98.67 %
Piper macropiper Piper aduncum
0.0
0.0
Piper decumanum Piper aduncum
0.0
95.98 %
tannins, fatty acids, anthraquinones, and cyanogenic glycosides. Positive results were qualitatively observed as indicated in each of the methods of phytochemical screening used. The findings were recorded using a 3-point scale [+ turbid, ++ moderate and +++ heavy] in scoring based on the Handbook of Philippine Medicinal Plants by De Padua et al. (2005). Cell viability testing using trypan blue exclusion assay following the protocol used by Dela Peña et al. (2019) was performed to determine the number of viable normal lymphocytes subjected to each of the plant's ethanolic extracts at a maximum concentration (1000 μg/ml). The trypan blue dye exclusion assay is a quantitative method to determine the percentage viability of viable cells and damaged nonviable cells. Viable cells have an intact cellular membrane with a clear appearance for not taking up the blue dye, whereas nonviable cells are stained blue for taking up the dye as cell membranes are damaged. The lymphocyte culture was prepared following the procedure of GE Healthcare (2007) from the Biological Research and Services Laboratory of the Natural Sciences Research Institute of the University of the Philippines in Diliman. The supplemented RPMI medium (RPMI with fetal bovine serum, PenicillinStreptomycin, Amphotericin B), Triton X-100 (0.1 %), and DMSO in PBS (2 %) were used as the negative, positive and vehicle control in triplicates, respectively. The number of live and dead lymphocytes were counted, and the cell density is computed.
which conforms with the only two reported non-native Philippine Piper species (Pelser et al., 2011 onwards). The two molecularly confirmed Piper species were concurrent with their morphology as compared to the diagnostic characters concisely described by Gardner (2003, 2006, 2013) and from comprehensive revision of Chew (1972). The identified P. decumanum is a dioecious, completely glabrous climber; it possesses an ovate, often rugose lamina with acute to bluntly acuminate apex, asymmetrically cordate to auriculate base, and often shorter than the auricular base, sheathing at the lower half petioles. On the other hand, the identified P. aduncum, coinciding with its diagnostic characters, constitutes monoecious shrubs with ovate, broad lamina with long acuminate apex, lightly asymmetrical cordate base, and slightly longer than the sinus of the lamina base but shorter than the peduncular stalk petioles; and often bisexual, usually curved, as long as leaves, with flowers borne in dense spirals inflorescences. 3.3. Phytochemical constituents and cytotoxic activities The results of the phytochemical screening of the ethanolic extracts of the two “Lunas”-named specimens revealed the presence of variable amounts of constituents like alkaloids, flavonoids, steroids, tannins, and fatty acids but none of cyanogenic glycosides (Table 5). Piper decumanum has moderate to heavy amounts of these phytochemicals when compared to P. aduncum which showed turbid to moderate quantities. Also, Piper decumanum lacks anthraquinones while saponins are missing in P. aduncum. Evaluation of cytotoxic effect of the stem ethanolic extracts of the two “Lunas”-named specimens showed low (91–100 % viable cells; Dela Peña et al., 2019) for P. decumanum and mild toxicity (81–90 % viable cells; Dela Peña et al., 2019) for P. aduncum to normal lymphocytes from human blood (Table 6). This cytotoxicity result may be attributed to the absence of cyanogenic glycosides in the extracts from two plants (Dela Peña et al., 2019; Uy et al., 2019).
3. Results 3.1. Nucleotide analysis The BLASTn results of successfully sequenced ITS, matK, and rbcL of the two “Lunas”-named specimens identified these as members of the genus Piper (Piperaceae) with e-values = 0 and %identity of ≥95.98 % (Table 3). Multiple sequence alignment of Piper species using ITS sequences contained 781 positions, of which 266 were parsimony informative (Table 4). In the ITS consensus phylogenetic tree (Fig. 2), sample MGD-002 (“Lunas-bagon tapol”) grouped with Piper decumanum L. while sample 18AG-002 (“Lunas-buyo”) grouped with Piper aduncum L. with strong support (BS = 100 %). The CI and RI are 0.53 and 0.73, respectively (Table 4).
4. Discussion The use of molecular sequences to verify species identity of medicinal plants with species problem due to similar local names and confusing identity was found to be very useful. The two “Lunas”-named specimens were molecularly confirmed as the indigenous Piper decumanum for “Lunas-bagon tapol”, and the introduced P. aduncum for
3.2. Distribution and morphological comparisons The two identified Piper species were compared with the distribution and morphology as indicated in the Co's Digital Flora of the Philippines (Pelser et al., 2011 onwards) and with the respective type specimens housed in B and P. Piper aduncum is a widely naturalized and invasive non-native species in the Philippines, contrary to P. decumanum which is a native species of Mt. Hilong-hilong (previously known as Mt. Urdaneta), a medium elevated forest in Agusan del Norte, Philippines (Pelser and Barcelona, 2017). The Royal Society Expedition of Chew (1972) to the Solomon Islands in 1965 recorded the distribution of P. decumanum in Philippine Islands, Celebes, Moluccas, and New Guinea while P. aduncum is distributed in Mexico, Central America, northern South America, West Indies, and naturalized in many places of Malesian floristic regions including the Philippines. The two neotropics Piper (P. aduncum and P. umbellatum) grouped together (BS = 95 %)
Table 4 Characteristics of molecular dataset used in this study.
27
Parameters
ITS
Number of species Number of individuals Alignment length (bp) Variable sites Parsimony-informative characters Consistency index (CI) Retention index (RI)
47 51 781 412 266 0.53 0.73
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Fig. 2. Strict consensus tree derived from 34 equally parsimonious trees based on the phylogenetic analysis of ITS sequence data. Length = 938 steps; consistency index = 0.53; retention index = 0.73. Branch lengths are drawn to scale. Percentage of 1000 bootstrap replicates is given when higher than 50 %. Highlighted clades in 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 figure legend, the reader is referred to the web version of this article.)
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Table 5 Results 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 Saponins Flavonoids Steroids Tannins Fatty acids Anthraquinones Cyanogenic Glycosides
Mayer’s Test Froth Test Bate-Smith and Metcalf Test Keller-Kiliani Test Gelatin Test Fatty acids test Borntrager’s Test Guignard Test
Whitish or cream colored precipitate Honeycomb froth Shades of red Reddish brown ring obtained at the interface Precipitate Transparency on filter paper Pink violet or red color in the ammoniacal layer Shades of red
++ ++ +++ +++ +++ +++ – –
+ – ++ ++ ++ ++ ++ –
(+) indicates present: +turbid, ++moderate, +++heavy; (-) indicates absent.
“Lunas-buyo” based on the nuclear ITS sequences. The result of molecular confirmation of these two Piper species reinforced traditional knowledge of Agusan Manobo on their ethnoclassification as different species which are only named similarly on its first word due to its homologous ethnomedicinal uses. This molecular verification is supported by the morphological diagnostic characteristics of Piper species in the Philippines by Chew (1972) and Gardner (2003, 2006, 2013). Relatively, P. decumanum is still unresolved species with a low confidence level (The Plant List, 2013) but integrative molecular approach coupled with assessment of available data from morphology and ethnoclassification could provide a more accurate species identification. Vernacular names are essential for preserving traditional knowledge and recording ethnotaxa (Ghorbani et al., 2017) but reliance on them often result to erroneous identification resulting to research invalidation (Bennett and Balick, 2014; Rivera et al., 2014). This molecular confirmation study could serve as useful future reference for proper identification of unresolved species and even unidentifiable collected plant materials. Correct identification of plant species can be verified accurately by means of integrative molecular approach for all pharmacological studies and natural product researches. Vegetative identification of P. decumanum could be difficult because this species is most of the time sterile according to the Manobo residents and upon collection. Most dioecious Piper species like P. decumanum have resemblances among and within species (Chaveerach et al., 2008). In fact, Sanubol et al. (2014) recommended the use of chemical composition and molecular data aside from morphology in the identification of Piper species. Even in P. aduncum when collected with inflorescences, morphology alone is often insufficient for identification because Piper species have small flowers with uniform vegetative and floral morphology (Suwanphakdee et al., 2016). Within and among Piper species, controversies exist regarding their true identity based solely on morphology; thus, correct identification should be corroborated by molecular data (Sanubol et al., 2014) which is demonstrated by this present work again. Integration of molecular data with the morphology of plants is also needed to test traditional limits of identification. Different DNA-based molecular markers are applied in various fields, and their application is tremendously increasing for species characterization of medicinal plants (Shaw et al., 2005; Sucher and Carles, 2008). The BLASTn method 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 relatedness and significance of matches in the genus Piper. The result of this molecular confirmation further supported the use of ITS sequences for phylogenetic analysis of Piper species in Asia, the South Pacific and the Neotropics by Jaramillo and Manos (2001) and Jaramillo et al. (2008) as well as the phylogeny of Malesian-Pacific Piper species by Asmarayani (2018). Moreover, the presence of constituents such as alkaloids, flavonoids, steroids, tannins, fatty acids and anthraquinones are medically important bioactive compounds which scientifically validated the biochemical and biological basis of the ethnomedicinal use of Piper species among the indigenous group of Agusan Manobo against cancer, stomach troubles, gastralgia, diarrhea, vomiting, liver and urological problems, fever, hypertension, food poisoning, skin diseases, snakebites and for wound healing. The comprehensive review of Salehi et al. (2019) on the phytochemistry, biological activities and applications of Piper species presented several phytochemicals and essential oils present that are strongly antioxidant and potentially antibacterial or antifungal agents against human pathogens. Furthermore, Piper species were observed to have a significant amount of Piplartine, an amide alkaloid which is a potent 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 in the extracts further differentiated the two plant species. Differences in the level of toxicity were also observed where “Lunas-bagon tapol” has low toxicity (91–100 %) when compared to mild toxicity (81–90 %) of “Lunas-buyo”. Although trypan blue exclusion assay has its limitations since trypan blue emits fluorescence when complexed with proteins which could be hard to distinguish unstained living cells from fluorescent dead cells, combining this cell viability analysis with flow cytometry technique could be an alternative tool for a quick and reliable result (Avelar-Freitas et al., 2014). However, this cytotoxicity result is similar to studies done on nine Piper species with betel-like scents crude extracts showing significant genotoxicity in HeLa cells but generally non-toxic in leukocytes (Sanubol et al., 2017). This result may also validate the effective and safe ethnomedicinal use of medicinal Piper species used by the Agusan Manobo lending credence on their folk medicine.
Table 6 Cytotoxic activity of P. decumanum and P. aduncum stem ethanolic extract to normal human lymphocytes. Treatment a
Supplemented RPMI Triton X-100b (0.1%) DMSOc (2%) P. decumanum (1000 μg/ml) P. aduncum (1000 μg/ml) a b c
Average No. of Live Cells
Average No. of Dead Cells
Average No. of Total Cells
Average Percent of Live Cells
61.56 0.00 49.67 60.33 57.67
4.00 56.56 6.67 5.33 7.67
65.56 56.56 56.33 65.67 65.33
93.9 0.00 88.2 91.9 88.3
Negative control. Positive control. Vehicle control. 29
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5. Conclusion
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Shifts in diversification rate with the origin of angiosperms. Science 265, 1590–1593. Sanubol, A., Chaveerach, A., Sudmoon, R., Tanee, T., Liehr, T., 2014. Verification of selected Piper species (Piperaceae) using morphological characters, molecular data, and chemical constituents. Malay. Nat. J. 66 (3), 60–81. Sanubol, A., Chaveerach, A., Tanee, T., Sudmoon, R., 2017. Pre-clinical evaluation of extracts and essential oils from betel-like scent Piper species identified potential cancer 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
Molecular sequences inferred from the nuclear ITS with comparative morphology confirmed the identity of “Lunas-bagon tapol” as Piper decumanum while “Lunas-buyo” as Piper aduncum. Results also showed that the two confused “Lunas”-named specimens do not only differ on species identity but also in the number and amount of phytochemical constituents present and cytotoxic properties. It can be concluded that species identities can be confirmed using molecular data when morphological data is limited and inconclusive while constituent and cytotoxic evaluation can be useful in determining variations between plant species used in ethnomedicine. These scientific results also validated the tribal claims of Agusan Manobo on the ethnomedicinal uses and application of their medicinal Piper species to further conserve, protect, and investigate these important genetic resources in line with the 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 the Department of Science and Technology - Accelerated Science and Technology Human Resource Development Program - National Science Consortium (DOST-ASTHRDP-NSC) and Alexander von Humboldt Foundation as a Junior Researcher. The last author thanks the Department of Health - Philippine Institute of Traditional and Alternative Health Care (DOH-PITAHC) for the funding, and Alexander von Humboldt Foundation for a renewed research stay at the University of Bayreuth (Germany) in 2019. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online 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 of Mussaenda inferred from ITS and trnT-F data and its implication for generic limits in Mussaendeae (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., MartinsFilho, O.A., Rocha-Vieira, E., Brito-Melo, G.E., 2014. Trypan blue exclusion assay by flow 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 plants using 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 botanical 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 arboreum seeds in Brazil. J. Trop. Ecol. 14, 109–114. Bramley, G., Utteridge, T., 2015. The Kew Tropical Plant Families Identification Handbook, 2nd ed. Royal Botanic Gardens, Kew. Chase, M.W., Hills, H.H., 1991. Silica gel: an ideal material for preservation of leaf samples for DNA studies. Taxon 40, 215–220. Chaveerach, A., Mokkamul, P., Sudmoon, R., Tanee, T., 2006. Ethnobotany of the genus Piper (Piperaceae) in Thailand. Ethnobot. Res. Appl. 4, 223–231. Chaveerach, A., Sudmoon, R., Tanee, T., Mokkamul, P., 2008. The species diversity of the genus Piper from Thailand. Acta Phytotaxonomica et Geobotanica 59, 105–163. Chew, W.L., 1972. The genus Piper (Piperaceae) in New Guinea, Solomon Islands and Australia. J. Arnold Arbor. 53 (1), 1–25. Costion, C., Ford, A., Cross, H., Crayn, D., Harrington, M., Lowe, A., 2011. Plant DNA barcodes can accurately estimate species richness in poorly known floras. PLoS One 6 (11). Dapar, M.L.D., Demayo, C.G., 2017. Folk medical uses of Lunas Lunasia amara Blanco by the Manobo people, traditional healers and residents of Agusan del Sur, Philippines.
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M.L.G. Dapar, et al. tortoise and the hare II: relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis. Am. J. Bot. 92 (1), 142–166. Sucher, N., Carles, M., 2008. Genome-based approaches to the authentication of medicinal plants. Planta Med. 74, 603–623. Suwanphakdee, C., Chantaranothai, P., 2011. A new species and three taxonomic changes in Piper (Piperaceae) from Thailand. Blumea 56, 235–239. Suwanphakdee, C., Simpson, D.A., Hodkinson, T.R., Chantaranothai, P., 2016. Taxonomic notes 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 Cocos nucifera 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.
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Molecular confirmation, constituents and cytotoxicity evaluation of two medicinal Piper species used by the Manobo tribe of Agusan del Sur, Philippines
T
Mark Lloyd G. Dapara,d,*, Cesar G. Demayoc, Ulrich Meved, Sigrid Liede-Schumannd, Grecebio Jonathan D. Alejandroa,b,d a
The Graduate School and Research Center for the Natural and Applied Sciences, University of Santo Tomas, España Boulevard, 1015 Manila, Philippines College of Science, University of Santo Tomas, España Boulevard, 1015 Manila, Philippines Department of Biological Sciences, College of Science and Mathematics, Mindanao State University - Iligan Institute of Technology, 9200 Iligan City, Philippines d Department of Plant Systematics, University of Bayreuth, Universitätsstr. 30, D-95447 Bayreuth, Germany b c
ARTICLE INFO
ABSTRACT
Keywords: BLASTn Cytotoxicity ITS Lunasia Phytochemicals Piper aduncum Piper decumanum
The Manobo tribe in the Philippines is culturally rich in ethnomedicinal practices and known to use popular plants locally named as “Lunas” (meaning “cure”). One of these is “Lunas-bagon tapol” which was previously identified as Lunasia sp. (Rutaceae) based on vegetative morphology. The other species is “Lunas-buyo” which is similar in ethnomedicinal use and morphology. This broadly-based study verifies these two “Lunas”-named specimens and evaluates the phytochemicals present and cytotoxic properties. The sequences of nuclear ITS, and plastid rbcL, and matK were investigated for species identification. Molecular confirmation using BLASTn nucleotide database query revealed that the two confused “Lunas”-named specimens were members of Piperaceae and not Rutaceae. Phylogeny of Asian Piper using ITS sequences revealed “Lunas-bagon tapol” as Piper decumanum L. and “Lunas-buyo” as Piper aduncum L. with strong support (BS = 100 %). Both Piper species similarly showed the presence of alkaloids, flavonoids, steroids, tannins and fatty acids but the absence of cyanogenic glycosides. Also, P. decumanum has moderate amount of saponins while P. aduncum contains moderate amount of anthraquinones. A cytotoxic activity test using trypan blue exclusion method against normal lymphocytes from human 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 practical approach of molecular-based taxon identification and provide the biochemical and biological basis as to the constituents present and cytotoxic properties of these medicinal Piper species for future pharmacological research, conservation priorities and ecological management.
1. Introduction The Philippines is a multi-cultural country with more than a hundred ethnolinguistic groups (Philippine Statistics Authority (PSA, 2016) classified according to its own unique identity, language, socio-political systems, and practices (National Commission on Indigenous Peoples (NCIP, 2010). The Manobo indigenous group among others is one of the most numerous Philippine ethnic groups occupying a wide area of distribution in Mindanao, Philippines using several medicinal plants for disease treatment and medication due to poor access of healthcare services. The Manobo tribe found in Agusan provinces is known as the Agusan Manobo inhabiting communities near Agusan River Valley and highlands 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 river people, coined from the “man” (people) and the “suba” (river). Given their location as a significant habitat for most of their indigenous (medicinal) plants, their traditional medical practice of using medicinal plants has long been practiced until these days for their primary disease treatment and healthcare. Some of their notable medicinal plants are locally known as “Lunas” in Bisaya and Manobo dialects which means “cure” with several species associated under this name. An example of which is the “Lunas-bagon tapol” 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
Corresponding author at: The Graduate School, University of Santo Tomas, España Boulevard, 1015 Manila, Philippines. E-mail address: [email protected] (M.L.G. Dapar).
https://doi.org/10.1016/j.phytol.2020.01.017 Received 23 September 2019; Received in revised form 17 December 2019; Accepted 16 January 2020 1874-3900/ © 2020 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
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uncertain, because vegetatively, Rutaceae can be confused easily with Piperaceae. Both families comprise woody or non-woody climbers, and also some trees, shrubs, and herbs, with alternate or opposite and aromatic leaves bearing essential oils (Bramley and Utteridge, 2015). Likewise, both families share similar biomedical properties as antibacterial and cytotoxic against cancer cells, as proven for Lunasia species (Prescott et al., 2007) and for Piper species (Wang et al., 2014). Both families were also reported rich in alkaloid content as potent cytotoxic 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 an effective treatment against cancer, stomach troubles, gastralgia, diarrhea, vomiting, hypertension, food poisoning and snakebites as reported by Macabeo and Aguinaldo (2008) for L. amara, and Salehi et al. (2019) for Piper species. As a result, Dapar and Demayo (2017) and Dapar et al. (2018) previously identified the “Lunas-bagon tapol” as one possible new variety of Lunasia amara in addition to the two varieties L. amara var. amara and L. amara var. babuyanica (Merr.) Hartley in the Philippines. The Agusan Manobo ethnomedicinally claimed “Lunasbagon” vine species as traditional source of anti-inflammatory, antimotility, antihistamine, antiparasitic, antibacterial, antitoxin, and antiviral medicines (Dapar and Demayo, 2017). Another medicinal plant, which is classified by the Agusan Manobo tribe to be closely related with “Lunas-bagon tapol” in terms of medicinal uses and morphology, is locally known as “Lunas-buyo”. As a consequence, the identity of these two “Lunas”-named specimens (“Lunas-bagon tapol” and “Lunas-buyo”) poses uncertainty in terms of their true familial and generic affinities. Piper is diverse in habit and inhabits diverse habitat (Sanderson and Donoghue, 1994), and has a widespread distribution occurring in American tropics with 700 spp. and in Southern Asia with 300 spp. (Jaramillo and Manos, 2001). Gardner (2006) recognized 15 climbing Philippine Piper species with only one taxon (P. myrmecophilum C.DC.) accepted as endemic while Pelser et al. (2011 onwards) reported 65 Piper species occurring in the Philippines. Piper nigrum L. (black pepper) and P. betle L. (betel leaf) are some of the economically important species 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 of Piper species, vegetative identification up to the species level is difficult. Meticulous investigation of its reproductive parts like fruits and infructescence are very important for species identification (Suwanphakdee and Chantaranothai, 2011). The unique composition of the genetic material for each species makes DNA-based identification a useful tool for accurate species identification. Therefore, correct identification of a medicinal plant should be examined using a molecular approach (Sanubol et al., 2014) for consistency of species and pharmacological investigations of natural products (Thomford et al., 2018). Although plant-based drug discovery in the ethnobotanical approach provides future drug leads,
authentication of the plant material is a great challenge and opportunity (Jachac and Saklani, 2007). Chemical composition, morphology, and molecular data of some species pairs must be studied for accurate species identification (Sanubol et al., 2014). Moreover, the use of internal transcribed spacer (ITS) of nuclear ribosomal DNA is a powerful phylogenetic marker showing high interspecific divergence and greater discriminatory power over the plastid regions (Hollingsworth, 2011; Li et 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 the nuclear ITS, plastid matK and rbcL. The evaluation of the phytochemical constituents and cytotoxic activities of the extracts from the two species were included to further evaluate their similarities and differences. The information generated in this study not only resolve true species identity of the two species but also provide the basis for their ethnomedicinal use and as a potential source of medicine. This study can also pave the way for the future conservation of these two ethnomedicinal plant species as one of the critical genetic resources of the Agusan Manobo in Mindanao, Philippines. 2. Materials and methods 2.1. Sample collection Fieldwork started with consultation and meeting of the tribal community with the council of elders to discuss research intent as purely academic. As agreed, a certification from the tribal council of elders was given following ritual observation with the community as a cultural tradition to grant permission on the conduct of the study. Free prior informed consents (FPICs) from the only two community tribal healers were acquired. When the tribal certification and FPICs were secured, the national regulators duly provided certification and permit following the by-laws on the protection of the indigenous peoples and the permission to collect wild plant samples as issued from the regional government administration. The National Commission on Indigenous Peoples of CARAGA Administrative Region (NCIP-CARAGA) issued certification (no. R13-2019-01), and the Department of Environment and Natural Resources of CARAGA Administrative Region (DENRCARAGA) granted wildlife gratuitous permit (no. R13-2019-12) for this study. All necessary permit and certifications were reviewed by the University of Santo Tomas Graduate School - Ethics Review Board (USTGS-ERC) and granted ethics approval (protocol no. GS-2019PN007) before plant collection. The collection of the plant samples was followed through guided field walks with the two tribal healers who are knowledgeable of their medicinal plants. Both tribal healers verified the collected plant materials and similarly classified both collected plant samples with the initial word “Lunas” but different second names denoting different plant species accordingly and locally named one as “Lunas-bagon tapol” and the other as “Lunas-buyo”. Samples of “Lunasbagon 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). 25
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Table 1 Gene 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, and ITS2) rbcL
P17F 26S-82R rbcL_aF rbcL_aR 3F_KimF IR_KimR
Alejandro et al. (2005) Bafeel et al. (2012)
5'-CTACCGATTGAATGGTCCGGTGAA-3ʹ 5ʹ-TCCCGGTTCGCTCGCCGTTACTA-3ʹ 5ʹ-ATGTCACCACAAACAGAGACTAAAGC-3ʹ 5ʹ-GTAAAATCAAGTCCACCRCG-3ʹ 5ʹ-CGTACAGTACTTTTGTGTTTACGAG-3ʹ 5ʹ-ACCCAGTCCATCTGGAAATCTTGGTTC-3ʹ
97 °C 90 s; 35 cycles of 97 °C 20 s, 72 °C 90 s, 72 °C 30 s; 72 °C 7 min 95 °C 1 min; 35 cycles of 95 °C 30 s, 51 °C 30 s, 68 °C 1 min; 68 °C 5 min 94 °C 5 min; 35 cycles of 94 °C 30 s, 52 °C 30 s, 72 °C 50 s; 5 min at 72 °C
matK
Costion et al. (2011)
Agusan del Sur (coded MGD-002) while “Lunas-buyo” (Fig. 1B&C) was collected in Mt. Pinagalaan, Bayugan City, Agusan del Sur (coded 18AG-002). Voucher specimens were deposited in the University of Santo Tomas Herbarium (USTH). Leaf samples were placed in ziplocked bags containing silica gel for molecular analysis (Chase and Hills, 1991). Mature stems were collected from each “Lunas”-named specimens for phytochemical and cytotoxicity analyses since this is the plant part used for cure by the tribal community. Collected stems were washed with water, air-dried, and pulverized into a fine powder using a blender. The preparation and extraction procedure followed the protocol used by Uy et al. (2019) to obtain the viscous crude extracts which were finally stored in vials for phytochemical screening and cytotoxicity testing.
Table 2 Nucleotide sequence database accession numbers of taxa used in the ITS phylogenetic analysis. Species OUTGROUP Peperomia elongata Kunth Sarcorhachis naranjoana (C.DC.) Trel. INGROUP Piper abbreviatum Opiz P. aduncum L. (1) P. aduncum L. (2) P. albispicum C.DC. P. arborescens Roxb. P. arboricola C.DC. P. austrocaledonicum C.DC. P. baccatum Blume P. bavinum C.DC. P. betle L. P. boehmeriaefolium Wall. P. brevicuspe (Miq.) Merr. P. caninum Blume P. celtidiforme Opiz P. cordatilimbum Quisumb. P. decumanum L. (1) P. decumanum L. (2) P. densum Bl. P. fragile Benth. P. guineense Schumach. & Thonn. P. gymnostachyum C.DC. P. hancei Maxim. P. hymenophyllum Miq. P. korthalsii Miq. P. laosanum C.DC. P. lessertianum (Miq.) C.DC. P. lolot C.DC. P. macropiper Pennant P. majusculum Blume P. medinillifolium Quisumb. P. muricatum Blume P. mutabile C.DC. P. myrmecophilum C.DC. P. nigrum L. P. pendulispicum C.DC. P. penninerve C.DC. P. pierrei C.DC. P. porphyrophyllum N.E.Br. P. quinqueangulatum Miq. P. retrofractum Vahl P. sarmentosum Roxb. P. sorsogonum C.DC. P. thomsonii (C.DC.) Hook.f. P. toppingii C.DC. P. umbellatum L. P. urdanetanum C.DC. P. wallichii (Miq.) Hand.-Mazz.
2.2. DNA extraction, amplification, and sequencing The total genomic DNA was extracted from silica gel-dried leaf tissues of “Lunas-bagon tapol” and “Lunas-buyo” samples following the protocols of DNeasy Plant Minikit (Qiagen, Germany). The ITS (nrDNA), matK, and rbcL (cpDNA) markers were used for this study. The gene regions, primer pairs, sequences, and PCR parameters, as shown in Table 1 were used for amplification reactions using Biometra Thermocycler T-Gradient. Amplicons were purified using Qia-Quick PCR Purification Kit (Qiagen, Germany) following the manufacturer’s protocol and sent to MACROGEN Inc., Seoul, South Korea, for bidirectional sequencing. 2.3. Sequence and phylogenetic analysis The newly generated sequences of each sample were assembled and edited using Codon Code Aligner v8.0.2, trimmed and checked for ambiguous nucleotides. Species identity was evaluated using BLASTn search algorithm available in the GenBank (www.ncbi.nim.nih.gov). Available sequences of all reported Philippine and other Asian Piper species using the three markers (ITS, matK and rbcL) were initially checked for inclusion in the phylogenetic analysis. Of the three markers, only ITS was subjected to phylogenetic analysis since it has sufficient 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 and Manos (2001) and Jaramillo et al. (2008) were also included in the analysis. A total of forty-seven accessions of Piper species including two recognized non-native Philippine species (Piper aduncum L. and Piper umbellatum L.) and two outgroups (Peperomia elongata Kunth and Sarcorhachis naranjoana (C.DC.) Trel.) were included in parsimony analysis of the ITS marker (Table 2). Multiple alignment of sequences used MUSCLE (Edgar, 2004) implemented in MEGA v.7.0.18 (Kumar et al., 2016) and was finally edited by visual inspection. Parsimony analysis was done using TBR search method with 100 random addition of initial trees and examined using 1000 bootstrap replicates. Consistency index (CI; Kluge and Farris, 1969) and retention index (RI; Farris, 1989) were calculated to estimate homoplasy.
GenBank accession numbers AF275213 AF275210 MH493098 AF275157 EF060061 AY572317 AF275202 AY572319 MH493124 MH493135 AF275199 AF275201 AF275204 AY572321 AF275195 AF275205 AY572323 AF275203 MH493337 AY615963 MH493373 MH493383 AY572325 EF450272 AY572327 AF275208 AY572326 MH493410 AY326208 MH493418 MH493434 AY667455 EF060076 DQ868737 AY572328 AF275198 DQ868742 AF275206 AF275200 MH493522 MH493537 AF275196 MH493595 AY572320 DQ868749 AY572322 AF275174 AF275207 EF450289
2.4. Phytochemical screening and cytotoxicity test The phytochemical screening was based on the protocol described by Aguinaldo et al. (2005) with slight modification. Qualitative assessment was done on the stem ethanolic extracts to screen for phytochemicals present such as alkaloids, saponins, flavonoids, steroids, 26
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Table 3 BLASTn results of newly sequenced samples for markers ITS, matK, and rbcL. Local Name
“Lunas-bagon tapol” “Lunas-buyo”
Sample code/ Voucher
Place of Collection
MGD-002/ USTH 015544 18AG-002/ USTH 015181
Mt. Ararat, Bayugan City Agusan del Sur Mt. Pinagalaan, Bayugan City, Agusan del Sur
(nrDNA) ITS
(cpDNA) matK
(cpDNA) rbcL
BLAST ID
e-value
% identity
BLAST ID
e-value
% identity
BLAST ID
e-value
% identity
Piper decumanum Piper aduncum
0.0
99.68 %
0.0
97.79 %
99.85 %
99.69 %
0.0
98.67 %
Piper macropiper Piper aduncum
0.0
0.0
Piper decumanum Piper aduncum
0.0
95.98 %
tannins, fatty acids, anthraquinones, and cyanogenic glycosides. Positive results were qualitatively observed as indicated in each of the methods of phytochemical screening used. The findings were recorded using a 3-point scale [+ turbid, ++ moderate and +++ heavy] in scoring based on the Handbook of Philippine Medicinal Plants by De Padua et al. (2005). Cell viability testing using trypan blue exclusion assay following the protocol used by Dela Peña et al. (2019) was performed to determine the number of viable normal lymphocytes subjected to each of the plant's ethanolic extracts at a maximum concentration (1000 μg/ml). The trypan blue dye exclusion assay is a quantitative method to determine the percentage viability of viable cells and damaged nonviable cells. Viable cells have an intact cellular membrane with a clear appearance for not taking up the blue dye, whereas nonviable cells are stained blue for taking up the dye as cell membranes are damaged. The lymphocyte culture was prepared following the procedure of GE Healthcare (2007) from the Biological Research and Services Laboratory of the Natural Sciences Research Institute of the University of the Philippines in Diliman. The supplemented RPMI medium (RPMI with fetal bovine serum, PenicillinStreptomycin, Amphotericin B), Triton X-100 (0.1 %), and DMSO in PBS (2 %) were used as the negative, positive and vehicle control in triplicates, respectively. The number of live and dead lymphocytes were counted, and the cell density is computed.
which conforms with the only two reported non-native Philippine Piper species (Pelser et al., 2011 onwards). The two molecularly confirmed Piper species were concurrent with their morphology as compared to the diagnostic characters concisely described by Gardner (2003, 2006, 2013) and from comprehensive revision of Chew (1972). The identified P. decumanum is a dioecious, completely glabrous climber; it possesses an ovate, often rugose lamina with acute to bluntly acuminate apex, asymmetrically cordate to auriculate base, and often shorter than the auricular base, sheathing at the lower half petioles. On the other hand, the identified P. aduncum, coinciding with its diagnostic characters, constitutes monoecious shrubs with ovate, broad lamina with long acuminate apex, lightly asymmetrical cordate base, and slightly longer than the sinus of the lamina base but shorter than the peduncular stalk petioles; and often bisexual, usually curved, as long as leaves, with flowers borne in dense spirals inflorescences. 3.3. Phytochemical constituents and cytotoxic activities The results of the phytochemical screening of the ethanolic extracts of the two “Lunas”-named specimens revealed the presence of variable amounts of constituents like alkaloids, flavonoids, steroids, tannins, and fatty acids but none of cyanogenic glycosides (Table 5). Piper decumanum has moderate to heavy amounts of these phytochemicals when compared to P. aduncum which showed turbid to moderate quantities. Also, Piper decumanum lacks anthraquinones while saponins are missing in P. aduncum. Evaluation of cytotoxic effect of the stem ethanolic extracts of the two “Lunas”-named specimens showed low (91–100 % viable cells; Dela Peña et al., 2019) for P. decumanum and mild toxicity (81–90 % viable cells; Dela Peña et al., 2019) for P. aduncum to normal lymphocytes from human blood (Table 6). This cytotoxicity result may be attributed to the absence of cyanogenic glycosides in the extracts from two plants (Dela Peña et al., 2019; Uy et al., 2019).
3. Results 3.1. Nucleotide analysis The BLASTn results of successfully sequenced ITS, matK, and rbcL of the two “Lunas”-named specimens identified these as members of the genus Piper (Piperaceae) with e-values = 0 and %identity of ≥95.98 % (Table 3). Multiple sequence alignment of Piper species using ITS sequences contained 781 positions, of which 266 were parsimony informative (Table 4). In the ITS consensus phylogenetic tree (Fig. 2), sample MGD-002 (“Lunas-bagon tapol”) grouped with Piper decumanum L. while sample 18AG-002 (“Lunas-buyo”) grouped with Piper aduncum L. with strong support (BS = 100 %). The CI and RI are 0.53 and 0.73, respectively (Table 4).
4. Discussion The use of molecular sequences to verify species identity of medicinal plants with species problem due to similar local names and confusing identity was found to be very useful. The two “Lunas”-named specimens were molecularly confirmed as the indigenous Piper decumanum for “Lunas-bagon tapol”, and the introduced P. aduncum for
3.2. Distribution and morphological comparisons The two identified Piper species were compared with the distribution and morphology as indicated in the Co's Digital Flora of the Philippines (Pelser et al., 2011 onwards) and with the respective type specimens housed in B and P. Piper aduncum is a widely naturalized and invasive non-native species in the Philippines, contrary to P. decumanum which is a native species of Mt. Hilong-hilong (previously known as Mt. Urdaneta), a medium elevated forest in Agusan del Norte, Philippines (Pelser and Barcelona, 2017). The Royal Society Expedition of Chew (1972) to the Solomon Islands in 1965 recorded the distribution of P. decumanum in Philippine Islands, Celebes, Moluccas, and New Guinea while P. aduncum is distributed in Mexico, Central America, northern South America, West Indies, and naturalized in many places of Malesian floristic regions including the Philippines. The two neotropics Piper (P. aduncum and P. umbellatum) grouped together (BS = 95 %)
Table 4 Characteristics of molecular dataset used in this study.
27
Parameters
ITS
Number of species Number of individuals Alignment length (bp) Variable sites Parsimony-informative characters Consistency index (CI) Retention index (RI)
47 51 781 412 266 0.53 0.73
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Fig. 2. Strict consensus tree derived from 34 equally parsimonious trees based on the phylogenetic analysis of ITS sequence data. Length = 938 steps; consistency index = 0.53; retention index = 0.73. Branch lengths are drawn to scale. Percentage of 1000 bootstrap replicates is given when higher than 50 %. Highlighted clades in 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 figure legend, the reader is referred to the web version of this article.)
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Table 5 Results 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 Saponins Flavonoids Steroids Tannins Fatty acids Anthraquinones Cyanogenic Glycosides
Mayer’s Test Froth Test Bate-Smith and Metcalf Test Keller-Kiliani Test Gelatin Test Fatty acids test Borntrager’s Test Guignard Test
Whitish or cream colored precipitate Honeycomb froth Shades of red Reddish brown ring obtained at the interface Precipitate Transparency on filter paper Pink violet or red color in the ammoniacal layer Shades of red
++ ++ +++ +++ +++ +++ – –
+ – ++ ++ ++ ++ ++ –
(+) indicates present: +turbid, ++moderate, +++heavy; (-) indicates absent.
“Lunas-buyo” based on the nuclear ITS sequences. The result of molecular confirmation of these two Piper species reinforced traditional knowledge of Agusan Manobo on their ethnoclassification as different species which are only named similarly on its first word due to its homologous ethnomedicinal uses. This molecular verification is supported by the morphological diagnostic characteristics of Piper species in the Philippines by Chew (1972) and Gardner (2003, 2006, 2013). Relatively, P. decumanum is still unresolved species with a low confidence level (The Plant List, 2013) but integrative molecular approach coupled with assessment of available data from morphology and ethnoclassification could provide a more accurate species identification. Vernacular names are essential for preserving traditional knowledge and recording ethnotaxa (Ghorbani et al., 2017) but reliance on them often result to erroneous identification resulting to research invalidation (Bennett and Balick, 2014; Rivera et al., 2014). This molecular confirmation study could serve as useful future reference for proper identification of unresolved species and even unidentifiable collected plant materials. Correct identification of plant species can be verified accurately by means of integrative molecular approach for all pharmacological studies and natural product researches. Vegetative identification of P. decumanum could be difficult because this species is most of the time sterile according to the Manobo residents and upon collection. Most dioecious Piper species like P. decumanum have resemblances among and within species (Chaveerach et al., 2008). In fact, Sanubol et al. (2014) recommended the use of chemical composition and molecular data aside from morphology in the identification of Piper species. Even in P. aduncum when collected with inflorescences, morphology alone is often insufficient for identification because Piper species have small flowers with uniform vegetative and floral morphology (Suwanphakdee et al., 2016). Within and among Piper species, controversies exist regarding their true identity based solely on morphology; thus, correct identification should be corroborated by molecular data (Sanubol et al., 2014) which is demonstrated by this present work again. Integration of molecular data with the morphology of plants is also needed to test traditional limits of identification. Different DNA-based molecular markers are applied in various fields, and their application is tremendously increasing for species characterization of medicinal plants (Shaw et al., 2005; Sucher and Carles, 2008). The BLASTn method 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 relatedness and significance of matches in the genus Piper. The result of this molecular confirmation further supported the use of ITS sequences for phylogenetic analysis of Piper species in Asia, the South Pacific and the Neotropics by Jaramillo and Manos (2001) and Jaramillo et al. (2008) as well as the phylogeny of Malesian-Pacific Piper species by Asmarayani (2018). Moreover, the presence of constituents such as alkaloids, flavonoids, steroids, tannins, fatty acids and anthraquinones are medically important bioactive compounds which scientifically validated the biochemical and biological basis of the ethnomedicinal use of Piper species among the indigenous group of Agusan Manobo against cancer, stomach troubles, gastralgia, diarrhea, vomiting, liver and urological problems, fever, hypertension, food poisoning, skin diseases, snakebites and for wound healing. The comprehensive review of Salehi et al. (2019) on the phytochemistry, biological activities and applications of Piper species presented several phytochemicals and essential oils present that are strongly antioxidant and potentially antibacterial or antifungal agents against human pathogens. Furthermore, Piper species were observed to have a significant amount of Piplartine, an amide alkaloid which is a potent 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 in the extracts further differentiated the two plant species. Differences in the level of toxicity were also observed where “Lunas-bagon tapol” has low toxicity (91–100 %) when compared to mild toxicity (81–90 %) of “Lunas-buyo”. Although trypan blue exclusion assay has its limitations since trypan blue emits fluorescence when complexed with proteins which could be hard to distinguish unstained living cells from fluorescent dead cells, combining this cell viability analysis with flow cytometry technique could be an alternative tool for a quick and reliable result (Avelar-Freitas et al., 2014). However, this cytotoxicity result is similar to studies done on nine Piper species with betel-like scents crude extracts showing significant genotoxicity in HeLa cells but generally non-toxic in leukocytes (Sanubol et al., 2017). This result may also validate the effective and safe ethnomedicinal use of medicinal Piper species used by the Agusan Manobo lending credence on their folk medicine.
Table 6 Cytotoxic activity of P. decumanum and P. aduncum stem ethanolic extract to normal human lymphocytes. Treatment a
Supplemented RPMI Triton X-100b (0.1%) DMSOc (2%) P. decumanum (1000 μg/ml) P. aduncum (1000 μg/ml) a b c
Average No. of Live Cells
Average No. of Dead Cells
Average No. of Total Cells
Average Percent of Live Cells
61.56 0.00 49.67 60.33 57.67
4.00 56.56 6.67 5.33 7.67
65.56 56.56 56.33 65.67 65.33
93.9 0.00 88.2 91.9 88.3
Negative control. Positive control. Vehicle control. 29
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Molecular sequences inferred from the nuclear ITS with comparative morphology confirmed the identity of “Lunas-bagon tapol” as Piper decumanum while “Lunas-buyo” as Piper aduncum. Results also showed that the two confused “Lunas”-named specimens do not only differ on species identity but also in the number and amount of phytochemical constituents present and cytotoxic properties. It can be concluded that species identities can be confirmed using molecular data when morphological data is limited and inconclusive while constituent and cytotoxic evaluation can be useful in determining variations between plant species used in ethnomedicine. These scientific results also validated the tribal claims of Agusan Manobo on the ethnomedicinal uses and application of their medicinal Piper species to further conserve, protect, and investigate these important genetic resources in line with the 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 the Department of Science and Technology - Accelerated Science and Technology Human Resource Development Program - National Science Consortium (DOST-ASTHRDP-NSC) and Alexander von Humboldt Foundation as a Junior Researcher. The last author thanks the Department of Health - Philippine Institute of Traditional and Alternative Health Care (DOH-PITAHC) for the funding, and Alexander von Humboldt Foundation for a renewed research stay at the University of Bayreuth (Germany) in 2019. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online 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 of Mussaenda inferred from ITS and trnT-F data and its implication for generic limits in Mussaendeae (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., MartinsFilho, O.A., Rocha-Vieira, E., Brito-Melo, G.E., 2014. Trypan blue exclusion assay by flow 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 plants using 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 botanical 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 arboreum seeds in Brazil. J. Trop. Ecol. 14, 109–114. Bramley, G., Utteridge, T., 2015. The Kew Tropical Plant Families Identification Handbook, 2nd ed. Royal Botanic Gardens, Kew. Chase, M.W., Hills, H.H., 1991. Silica gel: an ideal material for preservation of leaf samples for DNA studies. Taxon 40, 215–220. Chaveerach, A., Mokkamul, P., Sudmoon, R., Tanee, T., 2006. Ethnobotany of the genus Piper (Piperaceae) in Thailand. Ethnobot. Res. Appl. 4, 223–231. Chaveerach, A., Sudmoon, R., Tanee, T., Mokkamul, P., 2008. The species diversity of the genus Piper from Thailand. Acta Phytotaxonomica et Geobotanica 59, 105–163. Chew, W.L., 1972. The genus Piper (Piperaceae) in New Guinea, Solomon Islands and Australia. J. Arnold Arbor. 53 (1), 1–25. Costion, C., Ford, A., Cross, H., Crayn, D., Harrington, M., Lowe, A., 2011. Plant DNA barcodes can accurately estimate species richness in poorly known floras. PLoS One 6 (11). Dapar, M.L.D., Demayo, C.G., 2017. Folk medical uses of Lunas Lunasia amara Blanco by the Manobo people, traditional healers and residents of Agusan del Sur, Philippines.
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