Natural Science, 2017, 9, 31-41
http://www.scirp.org/journal/ns
ISSN Online: 2150-4105
ISSN Print: 2150-4091
Genetic Analysis of Itchy Leaves (Laportea, sp)
in Papua for Herbal Medicinal Products as
Development of Studying Economics Value
Elda Kristiani Paisey1, Yaved Muyan1, Desi Natalia Edowai2, Muhammad Dailami3
1
Agriculture Faculty, University of Papua, Manokwari, Indonesia
Faculty of Agriculture Technology, University of Papua, Manokwari, Indonesia
3
Faculty of Mathematics and Sciences, University of Papua, Manokwari, Indonesia
2
How to cite this paper: Paisey, E.K., Muyan, Y., Edowai, D.N. and Dailami, M.
(2017) Genetic Analysis of Itchy Leaves (Laportea, sp) in Papua for Herbal Medicinal
Products as Development of Studying Economics Value. Natural Science, 9, 31-41.
https://doi.org/10.4236/ns.2017.92004
Received: October 27, 2016
Accepted: February 24, 2017
Published: February 27, 2017
Copyright © 2017 by authors and
Scientific Research Publishing Inc.
This work is licensed under the Creative
Commons Attribution International
License (CC BY 4.0).
http://creativecommons.org/licenses/by/4.0/
Open Access
Abstract
Papua is rich in biodiversity. The indigenous Papuans utilize most of endemic
plants as food, board and medicines. One of the plants used as medicine is Laportea, sp, that locally known as Daun Gatal. The plant however, has not been
developed commercially; this might due to lack of scientific providence of medicine content in this plant. The aims of this research is to (1) explore the types of Laportea growing in Papua, (2) conduct bio molecular character, which
observes phylogeny relationship or genetic morphology in order to obtain data on the number of species found in Manokwari and Nabire. Bio molecular
analysis is performed by PCR. Genetic analysis in this study was conducted
through several phases, as follow: (a) Isolation of genomic total, (b) amplification of genes that encoded Rbcl, (c) sequencing the nucleotide sequence of the
gene Rbcl of Laportea, (d) phylogeny analysis. The results showed that the
DNA quality was good enough to do the next analysis. Of the four samples,
two obtained haplo types are Haplo Type 1 (DT.NBX.H, DT.NBX.U and
DR.MKW.H) and Haplo Type 2 (DR.NBX.L). The results of the analysis of phylogeny trees showed samples at 1 in the same haplo type, clade sequences of
the genus Dendrocnide spp, Discocnide, and Laportea, while the sample at 2
in the same haplotype clade with Laportea interupta, Laportea ruderalis and
Urera sp.
Keywords
Genetics, Medicinal Herbs, Daungatal (Laportea, sp)
1. Introduction
Indonesia is a country that is extremely rich in biodiversity of medicinal plants
DOI: 10.4236/ns.2017.92004
February 27, 2017
E. K. Paisey et al.
and it is potential to be developed. There are about 30.000 species of plants of
the world’s total of 40.000 plant species and 940 types are medicinal plants. This
amount represents 90% of the medicinal plants in Asia. Many types of new drugs
20% - 22% plants were cultivated, while around 78% was obtained through direct retrieval (exploration) of forest [1].
Papua is part of east Indonesia. It obtains a huge number of biological state,
which is about 20.000 - 25.000 species of plants and 142 species of them are endemic Papua [2]. The indigenous people of Papua utilize these plants as food,
board and medicines. One of the plants used as medicine is Laportea, sp, the Papuan people called daun gatal and scientifically known as Laportea, sp.
The results showed that laportea Decumana (Roxb) Wed was positive for alkaloids, glycocides, steroids/triterpenoids and negative for saponis, flavonoids and
tannins [3]. Then formic acid was influencing the regardless pore dilation thus stimulating blood circulation to relieve aches. In addition to the tribe, some of La-
portea are also used to relieve nausea or dizziness pat on the head, and relief pain
during child birth.
This plant is utilized by nearly all the tribes in Papua. This plant grows in the
highlands and lowlands. Utilization is only through direct exploration of nature
without a process of cultivation. Based on research of population under the plant,
there are three types of laportea, but until now there has been no study of bio molecular about all three types of plants, while according to Esiett, et al. [4], the Family of Urticaceae consists of 22 species. Therefore, it is necessary to conduct a
study to analyze the molecular of Laportea in Papua. This study aims to (1) perform the ekspolrasi types of Laportea that grows in Papua, (2) do character the
biomolecular, (3) obtain teaching materials for lectures stratum-1 and (4) disseminate the results of research in the national journal accredited or international
Journal.
Laporeta have wide deployment; this plant grows in almost all regions. But until
now, there has not been known how many species are found in Papua and which
type has the highest content. Therefore, knowing the type and content of the highest is the target of this research.
Laportea used by Papuans as pharmaceuticals but until now there has been
much research done on this plant and is not engaged in a professional manner.
Laportea Commercialized in the market at a price of 5000 rupiah for 5 - 10 pieces. Commercialized parts of the plant are the leaves of of Laportea. Laportea leaves are commercialized taken directly from nature without cultivation that need
to be developed with input cultivation of this plant fertilization can increase the
content of useful is the target of this research. Harvesting of medicinal plants directly from natural habitat has threatened the conservation of several species of
medicinal plants [5]. There are 55 species of medicinal plants are scarce in Indonesia with the status of scarcity [6].
Currently, the needs of herbal medicines is increasing, this is due to the lack of
side effects from the use of herbal remedies based on Ditjen PEN, 2014. Industrial development of medicinal plant raw material in the last 5 years has shown
32
E. K. Paisey et al.
significant growth and earnings of production during this period increased by
2.5% - 30%/year. Therefore, the lack of scientific information about this plant it is
important to conduct research on plants of Laportea assessment form that plants
in Papua.
2. Materials and Methods
2.1. Sample Collection
The leaves were collected from Manokwari (West Papua Province) and Nabire
(Papua Province). Fresh leaves were preserved and dried quickly by using silica
gel.
2.2. Genomic DNA Isolation
Total genome of the sample leaf was isolated with Genomic DNA mini kit (plant),
Geneaid, by using standard protocol of the product. Shortly, the DNA isolation
was done by taking approximately 50 - 100 mg of sample and grinding with
sterile mortar. The grinded sample was added to the lysis buffer GPX1 (400 µL).
To degrade the RNA, RNA-se was added to the solution and incubated at 65˚C
for five minutes. Before filtration, 100 µL of GP2 buffer was added then leaved
on ice for three minutes. Filtration on filter column with centrifuge at 3500 rpm
for 1 minutes. Supernatant of the filtrate was transferred to new micro centrifuge
tube 1.5 mL. Then, buffer GP3 (1.5 times volume of supernatant) was mixed with
the solution by vortex. The mixture was transferred to the GD column and centrifuge at 15,000 rpm for two minute. The GD column was washed with W1
buffer (400 µL) and wash buffer (600 µL) and each step was centrifuged at 15,000
rpm for 30 seconds. To dry the column, it was centrifuge at 15,000 rpm for three
minutes. DNA genome that attached to the column was eluted with heated elution buffer (100 µL). The column and elution buffer were incubated at room
temperature for three minutes to let the elution buffer interact with the DNA, then
centrifuged at 15,000 rpm for 30 seconds. The extract DNA was kept in freezer.
2.3. Amplification of Ribulose 1,5 Difosfat Carboxylase Large
Subunit Gene (rbcL)
Amplification of rbcL gen from gatal leaf was done with polymerase chain reaction
(PCR) by using thermal cycler machine MJ mini thermocycler, model TN 1148,
Biorad. The primer used were rbcLaF (5’-ATGTCACCACAAACAGAGACTAAAGC-3’) (Levin et al. 2003) and rbcLaR (5’-GTAAAATC AAGTCCACCRCG-3’) (Kres et al. 2009). The PCR reagents were Go-Taq-Green Master mix kit,
Promega. PCR reaction was conducted in total volume 50 µL, that consist of 18
µL ddH2O, 25 µL Go-Taq-Green master mix, 2.5 µL of primer rbcLaF, 2.5 µL of
primer rbcLaR, and 1 µL of DNA genomic extract. The temperature profile of the
PCR machine was 80˚C for 10 seconds, 94˚C for 2 minutes, then followed by 35
cycle of 3 steps which were (94˚C for 30 seconds, 50˚C for 30 seconds and 72˚C for
1 minute), the final extension at 72˚C for 5 minute, then closed with 37˚C for one
minute.
33
E. K. Paisey et al.
2.4. Electrophoresis
The visualization of PCR product was done by electrophoresis gel agarose 1% with
sodium boric buffer and voltage 100 V for 30 minutes. Each well consist of 4 µL
PCR product and 1 µL of loading dye. As the marker, Generuller 1 Kb DNA ladder
(Thermoscientific) was used. The dying of DNA was conducted by immersing the
gel on ethidium bromide solution for 20 minutes and visualize with UV-transilluminator. The gel was documented by digital camera.
2.5. Sequencing
The sequencing of rbcL genes is conducted by dideoxy termination sanger. The
process was done by company service, PT. Genetika Science Indonesia, Jakarta.
The electropherogram in AB1 format sent by email.
2.6. Data Analysis
The sequence of rbcL gene was proof read with the electropherogram with MEGA
5 software [7]. Basic local search alignment tools (BLAST) from NCBI was used to
compare our data with the GenBank data (NCBI). Phylogenetic tree analysis
(neighbor joining and maximum likelihood) was made with MEGA 5 software.
The bootstrap support in each branch was calculated by bootstrap methods with
1000 replication.
3. Results and Discussion
3.1. The Spread Pattern of Laportea Spin Nabire and Manokwari
Regency
Based on the results of research conducted in the regency of Manokwari and Nabire result that daungatal or by the Latin name is Laportea sp, there is widespread in lowland up to the highland, the deployment begins at a height of 50
meters above sea level to 1500 meters above sea in the Manokwari regency. Laportea plants live in secondary forest and primary forest. In the primary forest,
these plants live under stands of large trees with wet environmental conditions but
dry land and have plenty of humus. This is consistent with the statement of the
WHO [8] that this Laportea plant habitats in the shade and grows well in wet areas
but with the dry land. In addition, this plant has been cultivated by some local
communities in Manokwari in the yard of the house, where in addition to the
needs of family medicine this plant is also traded in the market for profit. Similarly, in the Nabire regency, based on the exploration results obtained plants Laportea
were living in primary forests and secondary forests that spread from the lowlands
to the highlands of the area Wanggar-Lagari until the Topo-Dogiyai, which
ranges at an altitude of 65 masl to 1300 masl. This plant in Nabire is not cultivated by the community because this plant grows wild and naturally so that the
beneficiaries are taken directly from nature.
From the results of exploration in Manokwari and Nabire obtained some kind
of Laportea. Based on altitude, in the district of Manokwari in the lowlands, three
plant species of Laportea are found by the local name Consist thin daungatal, red
34
E. K. Paisey et al.
and the Laportea pig. While in the highlands also found that living plants of the
Laportea that leaves itchy scratchy thin and red leaves. Based on the exploration
results in Nabire, in the lowlands found three types of laportea living is based on
local names gatel leaves, leaf green and Laportea scratchy purple, while living on
the plateau that leaves itchy scratchy green and purple leaves. Leaf morphology
itchy red and green leaves and flowers are shown in Figure 1.
3.2. Molecular Genetic Analysis
3.2.1. Genomic DNA of Laportea, Isp
A total of six samples of Laportea from Nabire and Manokwari (Table 1), has isolated its genome DNA by using genomic geneaid isolation kit (plant). The success
of the isolation of genomic DNA is shown with their band on a 1% agarose gel
electrophoresis. Sightings DNA bands of the six samples are presented in Figure
2.
Based on the results of electrophoresis in Figure 2, seen all the DNA samples
had white ribbons lengthwise. The quality of the sample genome DGR.MKW.H
are very good with a very thick color, which indicates the concentration of DNA
obtained is very high. Other samples had sufficient quality to be used in the PCR
process.
3.2.2. Amplicon Gene Ribulose-1,5-Bisphosphate-Carboxylase (Rbcl)
Gen Rbcl of Laportea in the leaf samples in vitro amplification using polymerase
chain reaction (PCR). Primary used is Rbcl-Ar [9] and Rbcl-Af [10]. Electrophoresis was performed using 1% agarose gel. As the marker (marker) used DNA
marker 1 kb. Long amplicons obtained had a size of about 400 bp. With DNA
quality was very good with high levels of brightness, brighter in the DNA bands in
DNA marker which has a concentration of 70 ng/uL for most bright ribbon of
DNA markers. This means that the DNA amplicon concentration higher than 70
ng/uL. This concentration is enough to be used in the sequencing stage. The results of electrophoresis of products PCR can be seen in Figure 3.
Electrophoresis results showed that there were four samples were successfully
Figure 1. Morphological of Plant Laportea sp living in primary forests Okabai, Topo district of Nabire Regency.
35
E. K. Paisey et al.
Table 1. List of samples used in molecular genetic analysis.
ID Sampel
Jenis
Lokasi
DGT.NBX.H
Daun gatal hijau
Dataran Tinggi Nabire
DGT.NBX.U
Daun gatal ungu
Dataran Tinggi Nabire
DGR.NBX.L
Daun gatal lateng
Dataran Tinggi Nabire
DGR.MKW.B
Daun gatal babi
Dataran Rendah Manokwari
DGR.MKW.H
Daun gatal hijau
Dataran Rendah Manokwari
Figure 2. Ribbon Genome DNA from leaves itchy origin Nabire and Manokwari.
amplified by using primer Rbcl-Ar and Rbcl-Af, are sample DT.NBX.H, DT.
NBX.U, DR.NBX.L and DR.MKW.H, meanwhile, samples DR.MKW.B not succeed in amplification (results not shown). The length amplicon obtained about 800
base pairs. It is seen by aligning the position of the sample DNA with DNA markers of known length.
3.3. Phylogenetic Analysis
Sequencing of the four samples of Laportea, is derived nucleotide sequences that
encode genes Rbcl. The sequence is checked and adjusted (proof reading) with
elektroferogram obtained, to ensure the correct sequence. The process of proof
reading is based on the forward and reverse sequences of each sample. The quality
of the sequences obtained was excellent, as indicated by the high peaks and clean
from electroferogram.
The results alignment and the primary cutting edge, obtained along the 552
nucleotide base pairs. Of the four samples of Laportea, 17 points polymorphism
obtained. The point of polymorphism is a nucleotide sequence that differs from
one sequence to another. Based on the polymorphism, four samples can be classified into two haplotypes, a DNA sequence is said to be different haplotype when
36
E. K. Paisey et al.
Figure 3. Results of electrophoresis of PCR products from leaf itch gene Rbcl origin Nabire and Manokwari.
there are one or more nucleotides different when compared with other sequences. The first consists of a sample haplotype DT_NBX_H, DT_NBX_U and
DR_MKW_H, while the second is a sample DR_NBX_L haplotype. There were
no nucleotide variations between samples of genes Rbcl green Laporeorigin of
Nabire with leaves of Manokwari, like wise for Laporta leaf green and purple.
Nucleotide variation is found (in the genes Rbcl) between the leaves with Lapor-
tea lateng others.
Nucleotide variation is found (in the genes Rbcl) between the itch lateng leaves and the others of itch leaf. The variations are the result of a point mutation
in the form of transition (Ts) and Transvertions (Tv) (Table 2). Transition mutations are DNA mutations that occur due to changes in nucleotides of purine
bases (adenine (A), guanine (G)) be a purine or pyrimidine bases (Cytosin (C),
Thymine (T)) be a pyrimidine. While transversion mutation occurs due to a mutation of purine bases (adenine (A), guanine (G)) into pyrimidine (Cytosin (C),
Thymine (T)) or reverse. Details of the mutated nucleotide position transitions
and tranversi presented in Table 2.
Analysis of the phylo genetic tree created using MEGA software 5 [7]. Based on
morphological analysis, obtained the basic information that Laportea samples in
this study belong to the genus of Laportea. Therefore, as a reference in the manufacture of the phylogenetic tree, use some sequences of genes Rbcl from NCBI.
According to Wu et al. [11], uriticaceae family members can be grouped into three
major clades. One clade of Laportea genus in which there is a third clade, in which
there are genus Nanocnide,Uritica, Girardinia, Dendrocnide, Discocnide, Poiki-
lospermum, Urera, Touchardia, Obetia and Laportea. Therefore, the phylo genetic
tree made using gene Rbcl of genus-the genus are available at NCBI. A total of 420
base pairs of nucleotides that encode genes Rbcl of 89 sequences (4 samples of leaves itchy, one sequence out group, and 83 sequences from the family Uriticaceae
selected from NCBI). The phylo genetic tree was made by the method of maxi37
E. K. Paisey et al.
Table 2. Polymorphisms point of the fourth samples of Daun gatal.
Nucleotida position
ID Sample
Nama of Sample.
75
114
150
263
327
366
393
396
400
DT_NBX_H
D. Gatal Hijau
C
A
T
T
T
C
A
C
T
DT_NBX_U
D. Gatal Ungu
C
A
T
T
T
C
A
C
T
DR_NBX_L
D. Gatal Lateng
T
C
A
A
G
G
C
T
G
DR_MKW_H
D. Gatal Hijau
C
A
T
T
T
C
A
C
T
Ts
Tv
Tv
Tv
Tv
Tv
TV
Ts
Tv
Mutation
Nucleotida position
ID Sample
Name of Sample
406
407
408
417
435
471
522
537
DT_NBX_H
D. Gatal Hijau
A
C
T
C
T
C
G
A
DT_NBX_U
D. Gatal Ungu
A
C
T
C
T
C
G
A
DR_NBX_L
D. Gatal Lateng
G
T
C
T
C
T
A
T
DR_MKW_H
D. Gatal Hijau
A
C
T
C
T
C
G
A
Ts
Ts
Ts
Ts
Ts
Ts
Ts
Tv
Mutation
mum likelihood parameter K2P + G (Kimura 2 parameter and Gamma distribution). The meter is chosen from the test results the appropriate evolutionary
model by using model test [10] in the MEGA 5. Bayesian Inference model with the
smallest value is selected as the best model [12]. Bootstrapt 1000 replication method used to test the phylo genetic trees were created. Making the results presented
in the phylo genetic tree in Figure 4, value of each branch boot strapt written on
each base branching. This value indicates the support or the confidence level of
each branch formed 1000 repetitions building the tree.
Phylogenetic tree in Figure 4 indicates that three samples of leaves itchy with
ID DT.NBX.H, DT.NBX.U and DR.MKW.H are in the same clade. This indicates
that based on data from three samples Rbcl gene is one species to the same. Although the morphology of bone samples DT.NBX.U has leaves and the underside
of leaves are purple, but the nucleotide sequence of its gene Rbcl exactly, in addition, in the clade seen the clade of the genus group of Dendrocnidaespp, Lapor-
teasp and Descocnidemexicana. Value the support of the establishment of
branches between the sample and sequences spp Dendrocnidae very small at only
31%. With the presence of several species are joined into one large clade and
formed politomi, namely branching comb, accompanied by a low boot strapt indicates that the kinship of the three groups did not seen any real difference. This means that, Rbcl gene which is a DNA barcoding for plants could not
distinguish between the third genus.
Samples DR.NBX.L, in phylogenetic, are in a clade with Laportea interupta, L.
ruderalis and Urera sp. with bootstrapt 66%. The third ramification of this species
38
E. K. Paisey et al.
Figure 4. Phylogenetic tree.
39
E. K. Paisey et al.
also are politomi, so the kinship of all three cannot be distinguished by using gene
Rbcl. Based on morphological analysis, it appears that this sample is a sample of
the species Laportea interupta. With the results of phylogenetic tree analysis shows
that this sample are in a clade with L. interupta, so it can be sure that the species is
L. intruptadarisampel, although in the clade there are other species. Therefore, it is
necessary to do further research that examines the kinship and systematics of the
group leaves from Papua itch by using some other loci, both chloroplast and nuclear DNA locus.
4. Conclusions
1) The nucleotide sequence of the gene Rbcl Laportea origin Nabire and Manokwari successfully determined the sequence with a total length reaching 552
base pairs.
2) Of the four samples, two obtained haplotype are Haplo Type 1 (DT.NBX.H,
DT.NBX.U, DR.MKW.H) and Haplo Type 2 (DR.NBX.L).
3) The results of the phylo genetic tree analysis show samples at 1 in the same
haplo type and clade sequences of the genus Dendrocnidespp, Discocnide, and
Laportea, while the sample at 2 in the same haplotype clade with Laportea interupta, Laportea ruderalisand Urera sp.
4) Gen Rbcl of the results of this study cannot explain the systematic relationships of these samples to the extent of the species. Multigene analyses or multilocus necessary are able to explain the systematics of the group leaves itchy.
Acknowledgements
Thanks to the Project of Competitive Research Grant (Hibah Bersaing) Fiscal Year
2016 for providing and supporting the research financials. Gratitude is also extended to all who are involved in this work.
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