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Medicinal plants of Papua New Guinea's Miu speaking population and a focus on their use of plant–slaked lime mixtures
Author’s Accepted Manuscript Medicinal plants of Papua new Guinea’s Miu speaking population and a focus on their use of plant-slaked lime mixtures Thomas A.K. Prescott, Marie Briggs, Robert Kiapranis, Monique S.J. Simmonds www.elsevier.com/locate/jep
PII: DOI: Reference:
S0378-8741(15)30085-4 http://dx.doi.org/10.1016/j.jep.2015.08.019 JEP9684
To appear in: Journal of Ethnopharmacology Received date: 2 July 2015 Revised date: 17 August 2015 Accepted date: 18 August 2015 Cite this article as: Thomas A.K. Prescott, Marie Briggs, Robert Kiapranis and Monique S.J. Simmonds, Medicinal plants of Papua new Guinea’s Miu speaking population and a focus on their use of plant-slaked lime mixtures, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2015.08.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Journal of Ethnopharmacology, 30th June 2015
Medicinal plants of Papua New Guinea’s Miu speaking population and a focus on their use of plant-slaked lime mixtures
Thomas A.K. Prescotta, *, Marie Briggsa, Robert Kiapranisb, Monique S.J. Simmondsa a
Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, UK
b
Papua New Guinea Forest Research Institute, Lae, Papua New Guinea ∗
Corresponding author. Tel.: +44 208 332 5393; fax: +44 208 332 5310. E-mail address: [email protected] (T.A.K Prescott).
Keywords: Ethnobotany; Papua New Guinea; New Britain; Miu; Ficuseptine; tropical ulcer
Abstract Ethnopharmacological relevance Here we present the results of an ethnobotanical survey of the medicinal plants used by the Miu, a virtually unresearched ethnolinguistic group who live in the mountainous interior of Papua New Guinea’s West New Britain Province. We compare the findings for those previously reported for the neighbouring inland Kaulong speaking population. Three species, Trema orientalis, Spondias dulcis and Ficus botryocarpa are used in combination with locally prepared slaked lime to produce intensely coloured mixtures which are applied to dermatological infections. Their effects on dermal fibroblast viability with and without slaked lime are examined. The sap of F. botryocarpa which is used to treat tropical ulcers was examined further with assays relevant to wound healing.
Materials and methods Focus groups and semi-structured interviews were used to acquire information on the uses of plants, vouchers of which were collected and identified by comparison with authentic herbarium specimens. LC-MS and NMR were used to identify chemical components. Cell viability assays were used to examine the effects of added slaked lime on dermal fibroblasts. For the sap of F. botryocarpa, fibroblast stimulation assays and antibacterial growth inhibition with Bacillus subtilis were carried out. Results The survey identified 33 plants and one fungal species, and clear differences with the inland Kaulong group despite their close proximity. Added slaked lime does not greatly increase the cytotoxicity of plant material towards dermal fibroblasts. The sap of F. botryocarpa contains the alkaloid ficuseptine as a single major component and displays antibacterial activity. Conclusions The results demonstrate the potential for variation in medicinal plant use amongst Papua New Guinea’s numerous language groups. The addition of slaked lime to plant material does not appear to present a concern for wound healing in the amounts used. The sap of F. botryocarpa displays antibacterial activity at concentrations that would occur at the wound surface and could be used as a highly accessible alternative to conventional antiseptics for remote communities in Papua New Guinea.
1. Introduction New Guinea contains some of the last large areas of continuous tropical forest remaining in the world, possessing biodiverse forests that are relatively little studied botanically and inhabited by diverse ethnolinguistic groups speaking over 1000 different languages (Foley, 2000; Iamo, 2010). In previous work we surveyed the medicinal plants used by the inland Kaulong who live in the mountainous interior of the island of New Britain (Prescott et al., 2012), in the present study we examine the medicinal ethnobotany of the neighbouring Miu speaking population. The Miu inhabit the foot hills of the Whiteman Range in the Kandrian inland region in Papua New Guinea’s West New Britain Province (Fig. 1); 1980’s census figures suggest a population of approximately 400 people (Throop, 1980). The Whiteman Range forms part of the central mountain range that bisects the island of New Britain. There are no detailed ethnographic studies of the Miu; the anthropologist Ann Chowning who lived amongst the neighbouring Kaulong and Sengseng groups in
the 1960’s describes the Miu at the time as not yet under government control and “pacification” (Chowning, 1985). Archaeological evidence from sites 25 km to the east suggest the interior of New Britain may have been inhabited as early as 35 000 years ago, making it amongst the longest inhabited rainforests in the world (Palvides, 1994). In the southern part of their territory the Miu live in permanent villages situated in relatively flat ground amongst degraded forest. The Miu territory extends further northwards into the Whiteman Range whereupon the terrain gives way to mountainous limestone karst permeated with numerous sinkholes and covered with primary rainforest. Surface water is scarce as rivers run under the ground through limestone caves. In this more rugged terrain, the Miu live in small hamlets or temporary encampments often consisting of single family units. The Miu share certain customs with other groups of the Kandrian inland region, notably the use of blow pipes to hunt small game animals, the practice of blackening their teeth with local mineral deposits and the now outlawed custom of infant head binding which produces a distorted elongated adult head shape (Chowning, 1980; Goodale, 1985). The area receives little attention from the provincial government due to the difficulty in accessing the steep rugged terrain. Apart from a few aid posts in villages to the south, access to government healthcare is extremely limited. As with the other populations in New Britain, common dermatological infections such as the tropical ulcer and tinea imbricata are widespread. The tropical ulcer is a debilitating condition caused by a polymicrobial infection of spirochaeate and fusiform bacteria (Adriaans, 1988; Falkler et al., 1989). Initial trauma in the form of a scratch develops into a circular lesion which is extremely painful and in some cases may require surgical intervention (Adriaans and Drasar, 1987). Treating tropical ulcers causes a considerable drain on government aid posts with some spending a third of their time and half their budgets treating the condition (Morris et al., 1989). By comparison tinea imbricata is a less debilitating condition, it is characterised by concentric rings of scaly plaques on the skin surface (Satter, 2009). The underlying cause would appear to be the dermatophyte fungus Trichophyton concentricum to which there is evidence of a genetically inherited susceptibility (Ravine et al., 1980). Like the tropical ulcer, tinea imbricata consumes a considerable amount of time of government aid posts with some communities experiencing infection rates reaching 30% (Jamison, 2006). For populations such as the Miu, the inaccessibility of aid posts means that conditions such as the tropical ulcer and tinea imbricata are more likely to have reached a chronic state of infection before treatment is sought. Unlike conventional treatments prescribed in aid posts, plant based medicines are readily available in the surrounding environment. They therefore have a greater potential to treat the early stages of infection than conventional medications located many miles away in primary healthcare centres. Papua New Guinea has developed a national health plan which calls for the integration of effective plant medicines into the national health system with the aim of reducing healthcare costs and preserving traditional knowledge (Mann, 2007). Such medicines need to fall
within acceptable parameters of safety. With this in mind we decided to evaluate plant-slaked lime mixtures used by the Miu to evaluate their effects on fibroblasts viability.
2. Materials and Methods 2.1 Ethnobotanical data collection Consent for this study was obtained from local participants and the government of Papua New Guinea prior to commencement. Informal semi-structured interviews and rainforest walks were conducted with key informants in the village of Miupark to establish a preliminary list of plant names with associated uses. A focus group was used to extend the list of names and collect more detailed use descriptions. This information was then verified against information collected in a separate focus group located several km north from which members of the first group were absent. All interviews were conducted in Neo-Melanesian language. As described previously, care was taken not to overly rely on data from any single informant (Etkin, 1993). 2.2 Collection and identification of plant material Voucher specimens were collected from forest surrounding the village of Miupark in the presence of key informants and preserved in 70% methanol before pressing and drying. Identification of vouchers was carried out by comparison with authentic herbarium specimens at the Royal Botanic Gardens, Kew, UK and the National Herbarium, Lae, PNG where vouchers are lodged. In addition to voucher specimens, plant material for laboratory tests was collected separately from three species. For T. orientalis and S. dulcis material from several leaves of each plant was collected and a mulch produced by hand, this material was divided equally and one half mixed with slaked lime by a key informant according to traditional methods, both portions were then then dried. F. botryocarpa sap was collected early in the morning when informants indicated the sap could easily be tapped. Peduncles were removed from the figs and the resulting white latex allowed to drip into 2 ml plastic vials. 2.3 Extraction of plant material and LC-MS of plant extracts For T. orientalis and S. dulcis, dried plant samples were ground to a powder and 100 mg of each extracted for 1 hour with 1 ml de-ionised water. The resulting extracts along with F. botryocarpa sap were then centrifuged twice in a microfuge at 13,000 rpm to remove debris before filter sterilising through a 0.2 µm filter syringe (Millipore). Liquid chromatography-mass spectrometry (LC-MS) was performed on a Thermo Scientific system consisting of an ‘Accela’ liquid chromatograph and an ‘LTQ-orbitrap XL’ mass spectrometer fitted with an electrospray source. Chromatography was achieved on a 150 mm × 3 mm × 3 ȝm Luna C18(2) column (Phenomenex) using a 400 ȝl/min
mobile phase gradient of 0:90:10 to 90:0:10 (water/acetonitrile/acetonitrile + 1% formic acid) over 60 mins. The mass spectrometer recorded high resolution (30 k) MS1 spectra in positive polarity (m/z 250–2000). 2.4 Isolation and NMR characterisation of ficuseptine A paste consisting of 2 ml F. botryocarpa sap mixed with slaked lime was extracted with 10 ml water three times and freeze dried. The resulting powder was then extracted with MeOH several times and after solvent removal yielded 5.34 mg of a grey amorphous solid. NMR spectra were obtained on a Bruker Avance 400 spectrometer equipped with a BBO probe at 30 °C, using standard Bruker microprograms. Chemical shift values were referenced to residual solvent signals (įH 7.26 and įC 77.0 ppm for CDCl3; įH 3.307 and įC 49.1 ppm for CD3OD). 2.5 Cell viability with human adult dermal fibroblasts Adult dermal human fibroblasts (Lonza) were maintained in Bulletkit medium (Lonza) at 37°C in a 5% CO2 environment according to standard methods. Cells were trypsinised and plated out in transparent 384 well microplates and left to attach. Medium was then changed for fresh medium containing serial dilutions of extracts or compounds alongside vehicles only controls. The cells were incubated for a further 48 h after which CellTiter reagent (Promega) was added 5 µl per well and the plate read at 490 nm in a Tecan Infinite M200 plate reader. For fibroblast stimulation assay, cells were plated out as above and medium subsequently changed for basal medium containing 0.5% FBS whereupon test substances were added. Cell proliferation over 48 h was measured relative to cells cultured in complete medium (non-serum starved). 2.6 Inhibition of B. subtilis B. subtilis NCTC 10400 was grown overnight in Isosensitest liquid medium (Oxoid) to produce a stationary phase culture which was then diluted with liquid medium to an OD600 of 0.02. Dilutions of test samples were made in medium and 100 µl added to wells of a 96 well plate. The same volume of diluted culture was then added to each well and the plate incubated at 37°C for 24 h before inspecting visually.
3. Results 3.1 Medicinal plants of the Miu language group The ethnobotanical survey identified 33 species of plants and one fungal species used as medicines by the Miu. These are detailed in Table 1 along with the number of informants from which the information was acquired. Data was acquired from focus groups carried out in two separate locations where members of each were absent from the other.
Species and
Family
Local name
Description of use
Anacardiaceae
Waringa
The tree trunk is cut and the resulting black
voucher number Semecarpus cf. rostrata Valeton
sap is applied to tinea imbricata skin
103
infections (2 informants focus group 1). Alternatively the black sap may be applied to teeth to produce a desired cosmetic darkening effect. Local minerals are also used to achieve this end (2 informants focus group 2).
Spondias dulcis
Anacardiaceae
Baklum
The leaves are crushed and squeezed by
Parkinson 124
hand to produce a green mulch, then slaked lime prepared from local limestone is added resulting in a colour change from green to yellow. This preparation is then added to tinea imbricata skin infections (3 informants focus group 1). Alternatively the leaves are treated as above without slaked lime and applied to skin to treat localised pain (2 informants focus group 2).
Alstonia cf.
Apocynaceae
Sipa
The sap is drunk to treat a condition similar
scholaris (L.)
in description to asthma (2 informants
R.Br. 116
focus group 1, 2 informants focus group 2).
Trema orientalis
Cannabaceae
Mopo
The leaves are crushed and squeezed by
(L.) Blume 115
hand to produce a green mulch, then slaked lime prepared from local limestone is added resulting in a colour change from green to yellow. This preparation is then added to tinea imbricata skin infections (2 informants focus group 1). Alternatively wood shavings made by scraping the sapwood with a knife blade are squeezed and the resulting fluid is drunk as a cure all for any illness (2 informants focus group 2).
Garcinia dulcis
Clusiaceae
Ekap
The sap is applied to tropical ulcers (3
(Roxb.) Kurz 134
informants focus group 1, 2 informants focus group 2).
Merremia peltata
Convolvulaceae Enovio
Sap is applied to tropical ulcers. Sap may be mixed with slaked lime to produce a
(L.) Merr.127
yellow face paint (3 informants focus group 1, 2 informants focus group 2).
Cheilocostus cf.
Costaceae
Asingi
The rhizome is scraped with a knife and the
speciosus
resulting material applied to tropical ulcers
(J.Koenig)
(3 informants focus group 1, 2 informants
C.D.Specht 128
focus group 2).
Octomeles
Datiscaceae
Aim
Wood shavings made by scraping the
sumatrana Miq.
sapwood with a knife blade are collected in a leaf, water is added to make a dark
126
coloured decoction which is drunk for a condition similar in description to asthma (3 informants focus group 1, 2 informants focus group 2).
Diospyros peekelii
Ebenaceae
Palasoup
The new leaves are chewed and the
Lauterb.
resulting mulch applied to tropical ulcers (3 informants focus group 1, 2 informants
131
Endospermum
focus group 2).
Euphorbiaceae
Urbi/Lukemom Wood shavings made by scraping the
moluccanum
sapwood with a knife blade are squeezed
(Teijsm. & Binn.)
and the resulting fluid is drunk for a
Kurz 120
condition linked to abdominal pain. The same fluid may also be applied to tropical ulcers (3 informants focus group 1). In another use the dry twigs may be burnt on a fire in such a way as to allow the smoke to go into ears to treat ear infections. Alternatively the leaves may be heated on a fire then applied to aches and pains (2 informants focus group 2).
Macaranga similis
Euphorbiaceae
Esul
Wood shavings made by scraping the
Pax. & K.Hoffm.
sapwood with a knife blade are squeezed
109
and the resulting fluid is drunk for a condition whose description is similar to dysentery (2 informants focus group 1, 2 informants focus group 2).
Gmelina
Lamiaceae
Pyvul
Wood shavings made by scraping the
moluccana
sapwood with a knife blade are squeezed
(Blume) Backer ex
and the resulting fluid is applied to large
K.Heyne 113
tropical ulcers (2 informants focus group 1, 2 informants focus group 2).
Desmodium
Leguminosae
Res
Leaves are crushed and squeezed and the
gangeticum (L.)
resulting fluid is drunk to treat diarrhoea (2
DC. 111
informants focus group 1, 2 informants focus group 2).
Gliricidia sepium
Leguminosae
Marmar
Leaves are crushed and squeezed and the
(Jacq.) Walp. 105
resulting fluid is applied to tropical ulcers (2 informants focus group 1, 2 informants focus group 2).
Pterocarpus
Leguminosae
Iku
Wood shavings made by scraping the
indicus Willd.
sapwood with a knife blade are squeezed and the resulting fluid is drunk for a
133
condition resembling dysentery. Alternatively the decoction may be rubbed onto the belly of pregnant women or applied to skin to treat back pain (1 informant focus group 1). Alternatively, wood shavings made by scraping the sapwood with a knife blade are chewed and then applied to tropical ulcers (2 informants focus group 2).
Kleinhovia hospita
Malvaceae
Engalong
Leaves are crushed and squeezed and the resulting fluid is applied to tropical ulcers
L. 104
(2 informants focus group 1). Alternatively the leaves may be prepared in the same way but the fluid drunk to treat coughs and colds (2 informants focus group 2).
Ficus cf.
Moraceae
Kek
The sap is mixed with slaked lime prepared
botryocarpa Miq.
from local limestone deposits causing the
129
sap to change from cream in appearance to bright yellow. The sap is then applied to tropical ulcers (3 informants focus group 1, 2 informants focus group 2).
Ficus gul
Moraceae
Murkarek
Sap from fruit applied to tropical ulcers (3
K.Schum. &
informants focus group 1, 2 informants
Lauterb. 130
focus group 2).
Ficus pungens
Moraceae
Kili
Sap from new shoots is applied to eye
Reinw. ex Blume
infections/inflammation. Alternatively the
112
leaves may be heated on a fire to produce a vapour which is used to treat the same condition (2 informants focus group 1, 2 informants focus group 2).
Ficus septica
Moraceae
Maldonoo
Burm.f. 108
Leaves heated on fire then placed on forehead to treat headaches (2 informants focus group 1). Opinion from the second focus group is that this species can be used to treat many different kinds of illnesses. For example when children have burns from a fire, the leaves are chewed by an adult and then spat onto the burns. Another use is to drink the sap for a disease similar
in description to asthma. (2 informants focus group 2).
Maclura
Moraceae
Golgol
The vine is cut and the fluid that emerges is
amboinensis
drunk to treat a condition similar in
Blume 121
description to asthma (2 informants focus group 1). Alternatively the fluid is drunk for coughs and colds (2 informants focus group 2).
Prainea sp. 118
Moraceae
Mangiru
Leaves are eaten to treat headaches (2 informants focus group 1). Alternatively bark material is placed on the head to treat headaches (2 informants focus group 2).
Filoboletus cf.
Mycenaeceae
Kombo
The whole fungus is heated briefly on a fire
manipularis
and applied to tropical ulcers like a plaster
(Berk.) Singer.*
(3 informants focus group 1, 2 informants focus group 2).
Gymnacranthera
Myristicaceae
Koomoo
Wood shavings made by scraping the
paniculata var
sapwood with a knife blade are squeezed
zipelii (A.DC.)
and the resulting fluid is drunk for
Warb. 114
diarrhoea (2 informants focus group 1). Alternatively the leaves are chewed and then spat onto tropical ulcers (2 informants focus group 2).
Piper aduncum L.
Piperaceae
Pomilil
Leaves are crushed and squeezed and the
107
resulting fluid is applied to tropical ulcers (3 informants focus group 1). Alternatively
the leaves are boiled to make an infusion which is drunk for a non-descript internal illness related to perceived problems with blood and circulation (2 informants focus group 2).
Piper betel
Piperaceae
Eman
The leaves are chewed and the resulting
Blanco*
mulch applied to tropical ulcers (2 informants focus group 2).
Morinda citrifolia
Rubiaceae
Sarelipi
The fruit or roots are boiled and the
L. 125
resulting decoction drunk as a cure all (2 informants focus group 1). Alternative use description is as above only the decoction is drunk to treat a condition resembling asthma (2 informants focus group 2).
Uncaria sp. 102
Rubiaceae
Ayahong
Fresh plant material crushed and squeezed and the resulting fluid placed in ear to treat earache/ear infections (2 informants focus group 1). Alternatively, wood shavings from sapwood are applied to tropical ulcers (2 informants focus group 2).
Homalium
Salicaceae
Meles
Leaves heated on fire and then applied to
foetidum Benth.
tropical ulcers (3 informants focus group 1,
132
2 informants focus group 2).
Solanum torvum
Solanaceae
Kangong
Leaves are crushed and squeezed and the
Sw. 106
resulting green fluid is boiled before applying to tropical ulcers (3 informants focus group 1, 2 informants focus group 2).
Elatostema cf.
Urticaceae
Manil
Wood shavings made by scraping the
angulare
sapwood with a knife blade are mixed with
H.J.P.Winkl. 101
a small quantity of water and applied to tinea imbricata skin infections (3 informants focus group 1). Alternatively the leaves may be applied to tropical ulcers (2 informants focus group 2).
Pipturus argenteus
Urticaceae
Mike
Wood shavings made by scraping the
(G.Forst.) Wedd.
sapwood with a knife blade are mixed with
110
water. People described as having any persistent long lasting illness then wash with it (2 informants focus group 1). Alternatively the sap is drunk by people described as being extremely sick, to help awaken them (2 informants focus group 2).
Poikilospermum
Urticaceae
Sigu
The woody section of the vine is cut and
amboinense Zipp.
the resulting water that flows out is drunk
& Miq. 117
to treat coughs/colds (2 informants focus group 1, 2 informants focus group 2).
cf. Cayratia
Vitaceae
Mohlup
Leaves are crushed and squeezed and the
japonica (Thunb.)
resulting fluid is used to wash tropical
Gagnep. 122
ulcers (3 informants focus group 1, 2 informants focus group 2).
Table 1. Medicinal plants used by the Miu. Voucher numbers (Prescott T.A.K.) are underlined after the species name. Local plant names are given in the Miu language. Use descriptions are presented as described by informants and only assigned to a specific condition e.g. tropical ulcers where a reasonable interpretation could be made.*Indicates common species identified from photos taken in the field.
3.2 Comparison of plant uses between the Miu and inland Kaulong A previous survey of the neighbouring inland Kaulong group identified 31 species of plants used as medicines (Prescott et al., 2012) whereas the present study identified 33 species of medicinal plants and one fungal species. Of these only four species could be unequivocally identified as the same species with the same medicinal use as described for the Kaulong. A comparison of plant uses between the Miu and inland Kaulong is shown in Fig. 2a. Tropical ulcer plant medicines predominate amongst both the Miu and the Kaulong with a similar proportion used for this purpose in both groups. A comparison of mode of preparation between the two groups is shown in Fig. 2b. The use of slaked lime in combination with plant material is a feature shared by both groups.
3.3 Slaked lime does not markedly increase the cytotoxicity of plant material Slaked lime is caustic which suggests plant/slaked lime mixtures should exhibit undesirable cytotoxicity to human skin cells. To address this question, samples of each plant were prepared in the field according to traditional methods, in each case with and without the addition of locally prepared slaked lime. Aqueous extracts of this material were prepared, (for F. botryocarpa sap a filtered sap solution was used), and human adult dermal fibroblasts were grown in the presence of the extracts for 48 hours, then cell viability was measured using CellTiter reagent (Promega). The results in Fig. 3 show that with the exception of T. orientalis where there is a modest decrease in cell viability, the addition of slaked lime does not decrease cell viability relative to identical plant material prepared without slaked lime. The sap of F. botryocarpa is cytotoxic to fibroblasts with minimal cell viability detected above 3% v/v either with or without slaked lime added. Surprisingly, at sap concentrations of 1.25% v/v the slaked lime reduces the cytotoxicity of the sap.
3.4 F. botryocarpa sap contains the alkaloid ficuseptine as the single major component The LC-MS chromatogram of F. botryocarpa sap (Fig. 4) is dominated by a single major component which was isolated and its structure confirmed as the alkaloid ficuseptine through a comparison of the
chemical shift data acquired in CDCl3 (Baumgartner et al., 1990; Bracher and Daab, 2002; Snider and Neubert, 2005). Additionally, two-dimensional experiments (COSY, HSQC and HMBC) were conducted for a confirmation of the chemical shift assignments (see methods section). The structure of the cation is shown in Fig. 4. Ficuseptine has been previously reported from the related species Ficus septica Burm. f. (Baumgartner et al., 1990).
3.5 F. botryocarpa sap inhibits B. subtilis but does not stimulate dermal fibroblast proliferation Previous work reported that ficuseptine inhibits the growth of B. subtilis, Escherichia. coli, Micrococcus luteus and Penicillium oxalicum, (Baumgartner et al., 1990). Although conventional minimal inhibitory concentrations were not reported these results are indicative of broad spectrum antimicrobial activity. B. subtilis is not involved in tropical ulcer pathogenesis, however other Bacillus species have been isolated from tropical ulcers (Woodruff, 1987) although these are not easily cultured in vitro. B. subtilis is a model organism and a useful substitute. F. botryocarpa sap with and without slaked lime were tested at a range of concentrations with B. subtilis NCTC 10400. The minimal inhibitory concentration for the sap alone was 1.5% v/v compared to the sap with slaked lime which was 3% v/v. This mirrors the data with human fibroblasts where the added slaked lime increased cell viability relative to the sap alone at concentrations of 1.25% v/v. Thus slaked lime slightly reduces the activity of ficuseptine or other active principles in the sap. The direct application of sap to the infected tropical ulcer wound surface means these concentrations are easily achievable in vivo. Stimulating fibroblast proliferation may help heal chronic wounds, and although the sap is inhibitory to fibroblasts there remained the possibility that it could stimulate fibroblast proliferation at sub-inhibitory concentrations by diffusing deeper into the wound tissue. Human dermal fibroblasts were treated with the F. botryocarpa sap/slaked lime mixture at sub-inhibitory concentrations for 48 hours, then cell numbers quantified using the CellTiter reagent and compared to non-serum starved cells. The sap/slaked lime mixture does not stimulate fibroblast proliferation (Fig. 4c) suggesting any therapeutic benefits are related to its antibacterial activity. 4. Discussion and conclusion Comparison with data for the inland Kaulong identified only four species used for the same purpose suggesting a high degree of variation in plant use between the two groups. This is surprising given the close proximity of the two groups and their similar language and customs. As the two groups inhabit near identical forest and terrain these differences cannot be attributed to availability of plants and are instead likely to reflect cultural differences. There was also variation in plant use within the Miu language group with one key informant describing medicinal plant use as clan specific rather than language group specific. This suggests a model of plant knowledge in which each clan or large family
group have their own preferred medicinal plants. Thus although there is very frequent intercommunity exchange between populations living in the Whiteman Range, a lack of sharing of medicinal plant knowledge between individual clans has prevented a completely homogeneous system of medicinal plant use from developing. It should be noted however, that as with most ethnobotanical studies, limits on time spent in the field prevent a complete picture of plant use and knowledge from being acquired. Papua New Guinea has developed a national health plan which calls for the integration of effective plant medicines into the national health system with the aim of reducing healthcare costs and preserving traditional knowledge (Mann, 2007). The addition of slaked lime to medicinal plants is found in both the Miu and inland Kaulong groups, however the caustic nature of slaked lime makes such plant preparations less likely to be considered as potential candidates that could gain acceptance in primary healthcare in Papua New Guinea. However, the experiments with dermal fibroblasts revealed that for three species, added slaked lime does not result in a great decrease in fibroblast cell viability when aqueous extracts of the plant/slaked lime mixtures were tested at concentrations up to 10% v/v. This suggests that the quantities of slaked lime added are insufficient to cause a reduction in viable cell numbers by themselves. In practice higher concentrations than this may be reached when such preparations are applied directly to the skin or a tropical ulcer wound surface, however these would be difficult to model in vitro. The sap of F. botryocarpa is mixed with slaked lime and the resulting brightly coloured yellow mixture applied directly to the tropical ulcer wound surface. The sap which consists mainly of the alkaloid ficuseptine is antibacterial. The abundance of sap that flows from the stem of a single fruit when picked from the tree provides a convenient way of applying an antibacterial substance to tropical ulcers. Considering the remote location that some members of the Miu speaking population live in this makes F. botryocarpa sap an attractive alternative to conventional antiseptics that are generally out of reach for them. Although F. botryocarpa sap does not stimulate fibroblast proliferation, it is possible that other plant-based tropical ulcer treatments exhibit antibacterial activity at the wound surface but also promote wound healing by modulating cytokine expression in tissue beneath the wound surface where plant metabolites would be present at sub-antibacterial/cytotoxic concentrations. Further work should be carried out to identify if plants or fungi with such a property exist.
Acknowledgements Funding for this work was provided by the Davis Expedition Fund, University of Edinburgh. We are grateful to Nigel Veitch and Tetsuo Kokubun for structure elucidation of ficuseptine and Dr Brian Spooner for fungal identification. We would also like to acknowledge the help provided by members of the Miu speaking population of New Britain.
References Adriaans, B., 1988. Tropical ulcer – a reappraisal based on recent work. Transactions of the Royal Society of Tropical Medicine and Hygiene 82, 185–189. Adriaans, B., Drasar, B.S., 1987. The isolation of fusobacteria from tropical ulcers. Epidemiology and Infection 99, 361–372. Baumgartner, B., Erdelmeier, C.A.J., Wright, A.D., Rali, T., Sticher, O., 1990. An Antimicrobial Alkaloid from Ficus septica. Phytochemistry 29, 3327–3330. Bracher, F., Daab, J., 2002. Total synthesis of the indolizidinium alkaloid ficuseptine. European Journal of Organic Chemistry, 2288–2291. Chowning, A., 1980. Culture and biology among the Sengseng of New Britain. Journal of the Polynesian Society 89, 7–31. Chowning, A., 1985. Rapid lexical change and aberrant Melanesian languages: Sengseng and its neighbours. Pacific Linguistics C–88, 169–198. Etkin, N.L., 1993. Anthropological methods in ethnopharmacology. Journal of Ethnopharmacology 38, 93–104. Falkler, W.A., Jr., Montgomery, J., Nauman, R.K., Alpers, M., 1989. Isolation of Fusobacterium nucleatum and electron microscopic observations of spirochetes from tropical skin ulcers in Papua New Guinea. American Journal of Tropical Medicine and Hygiene 40, 390–398. Foley, W., 2000. The Languages Of New Guinea. Annual Review of Anthropology 29, 357–404. Goodale, J., 1985. Pig's teeth and skull cycles: both sides of the face of humanity. American Ethnologist 12, 228–244. Iamo, W., 2010. Papua New Guinea’s Fourth National Report to the Convention on Biological Diversity. https://www.cbd.int/doc/world/pg/pg-nr-04-en.pdf Jamison, D., 2006. Disease Control Priorities in Developing Countries, 2nd edition. World Bank, Washington, D.C. Mann, N., 2007. National policy on traditional medicine, Department of Health, Waigani Drive, Port Moresby, Papua New Guinea. Morris, G.E., Hay, R.J., Srinivasa, A., Bunat, A., 1989. The diagnosis and management of tropical ulcer in East Sepik Province of Papua New Guinea. Journal of Tropical Medicine and Hygiene 92, 215–220. Palvides, C.G., C, 1994. 35,000-year-old sites in the rainforests of West New Britain, Papua New Guinea. Antiquity 68, 604–610. Prescott, T.A., Kiapranis, R., Maciver, S.K., 2012. Comparative ethnobotany and in–the–field antibacterial testing of medicinal plants used by the Bulu and inland Kaulong of Papua New Guinea. Journal of Ethnopharmacology 139, 497–503.
Ravine, D., Turner, K.J., Alpers, M.P., 1980. Genetic inheritance of susceptibility to tinea imbricata. Journal of Medical Genetics 17, 342–348. Satter, E.K., 2009. Tinea imbricata. Cutis 83, 188–191. Snider, B.B., Neubert, B.J., 2005. Syntheses of ficuseptine, juliprosine, and juliprosopine by biomimetic intramolecular Chichibabin pyridine syntheses. Organic Letters 7, 2715–2718. Throop, C.T., Throop, L., 1980. The languages and communities of the Kandrian inland region. In: Johnston, R. (ed.), Language, communication and development in New Britain. Summer Institue of Linguistics, Ukarumpa, PNG. Woodruff, A.W.,Wright, S.G., 1987. A Synopsis of Infectious and Tropical Diseases, 3rd ed. IOP Publishing Ltd, Bristol.
Figures
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Fig. 1. Location of the Miu speaking population on the island of New Britain
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Fig. 2. Comparison of medicinal use categories for the Miu with those previously reported for the inland Kaulong (Prescott et al., 2012) using the same use categories (A). Comparison of mode of preparation with the inland Kaulong for medicinal uses (B). Total number of species for Kaulong is 32 compared with 34 for the Miu population. N.B. compared with the inland Kaulong group, the Miu use descriptions are more varied with more than one use for a plant frequently given.
Fig. 3. Human adult dermal fibroblast cell viability after 48 hours treatment with aqueous plant extracts with and without slaked lime. T. orientalis, S. dulcis and F. botryocarpa aqueous extracts without slaked lime (A) and with slaked lime (B). In each graph T. orientalis is top line, S. dulcis middle line F. botryocarpa bottom line. Relative cell numbers were measured using the CellTiter reagent. Error bars show S.E.M. (N=3).
Fig. 4. Orbitrap LC-MS analysis of positive ions of F. botryocarpa sap without slaked lime (A) and with slaked lime added (B). The major component eluting at 16 mins was isolated and identified by NMR, the structure of the cation of the alkaloid ficuseptine is shown (C). F. botryocarpa sap with slaked lime does not stimulate human dermal fibroblast proliferation (D). Cells were incubated for 48 hours with the extract in minimal media. Cell numbers relative to complete medium were then determined with the CellTiter reagent. Error bars (S.E.M N=3) are smaller than data points.
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PII: DOI: Reference:
S0378-8741(15)30085-4 http://dx.doi.org/10.1016/j.jep.2015.08.019 JEP9684
To appear in: Journal of Ethnopharmacology Received date: 2 July 2015 Revised date: 17 August 2015 Accepted date: 18 August 2015 Cite this article as: Thomas A.K. Prescott, Marie Briggs, Robert Kiapranis and Monique S.J. Simmonds, Medicinal plants of Papua new Guinea’s Miu speaking population and a focus on their use of plant-slaked lime mixtures, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2015.08.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Journal of Ethnopharmacology, 30th June 2015
Medicinal plants of Papua New Guinea’s Miu speaking population and a focus on their use of plant-slaked lime mixtures
Thomas A.K. Prescotta, *, Marie Briggsa, Robert Kiapranisb, Monique S.J. Simmondsa a
Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, UK
b
Papua New Guinea Forest Research Institute, Lae, Papua New Guinea ∗
Corresponding author. Tel.: +44 208 332 5393; fax: +44 208 332 5310. E-mail address: [email protected] (T.A.K Prescott).
Keywords: Ethnobotany; Papua New Guinea; New Britain; Miu; Ficuseptine; tropical ulcer
Abstract Ethnopharmacological relevance Here we present the results of an ethnobotanical survey of the medicinal plants used by the Miu, a virtually unresearched ethnolinguistic group who live in the mountainous interior of Papua New Guinea’s West New Britain Province. We compare the findings for those previously reported for the neighbouring inland Kaulong speaking population. Three species, Trema orientalis, Spondias dulcis and Ficus botryocarpa are used in combination with locally prepared slaked lime to produce intensely coloured mixtures which are applied to dermatological infections. Their effects on dermal fibroblast viability with and without slaked lime are examined. The sap of F. botryocarpa which is used to treat tropical ulcers was examined further with assays relevant to wound healing.
Materials and methods Focus groups and semi-structured interviews were used to acquire information on the uses of plants, vouchers of which were collected and identified by comparison with authentic herbarium specimens. LC-MS and NMR were used to identify chemical components. Cell viability assays were used to examine the effects of added slaked lime on dermal fibroblasts. For the sap of F. botryocarpa, fibroblast stimulation assays and antibacterial growth inhibition with Bacillus subtilis were carried out. Results The survey identified 33 plants and one fungal species, and clear differences with the inland Kaulong group despite their close proximity. Added slaked lime does not greatly increase the cytotoxicity of plant material towards dermal fibroblasts. The sap of F. botryocarpa contains the alkaloid ficuseptine as a single major component and displays antibacterial activity. Conclusions The results demonstrate the potential for variation in medicinal plant use amongst Papua New Guinea’s numerous language groups. The addition of slaked lime to plant material does not appear to present a concern for wound healing in the amounts used. The sap of F. botryocarpa displays antibacterial activity at concentrations that would occur at the wound surface and could be used as a highly accessible alternative to conventional antiseptics for remote communities in Papua New Guinea.
1. Introduction New Guinea contains some of the last large areas of continuous tropical forest remaining in the world, possessing biodiverse forests that are relatively little studied botanically and inhabited by diverse ethnolinguistic groups speaking over 1000 different languages (Foley, 2000; Iamo, 2010). In previous work we surveyed the medicinal plants used by the inland Kaulong who live in the mountainous interior of the island of New Britain (Prescott et al., 2012), in the present study we examine the medicinal ethnobotany of the neighbouring Miu speaking population. The Miu inhabit the foot hills of the Whiteman Range in the Kandrian inland region in Papua New Guinea’s West New Britain Province (Fig. 1); 1980’s census figures suggest a population of approximately 400 people (Throop, 1980). The Whiteman Range forms part of the central mountain range that bisects the island of New Britain. There are no detailed ethnographic studies of the Miu; the anthropologist Ann Chowning who lived amongst the neighbouring Kaulong and Sengseng groups in
the 1960’s describes the Miu at the time as not yet under government control and “pacification” (Chowning, 1985). Archaeological evidence from sites 25 km to the east suggest the interior of New Britain may have been inhabited as early as 35 000 years ago, making it amongst the longest inhabited rainforests in the world (Palvides, 1994). In the southern part of their territory the Miu live in permanent villages situated in relatively flat ground amongst degraded forest. The Miu territory extends further northwards into the Whiteman Range whereupon the terrain gives way to mountainous limestone karst permeated with numerous sinkholes and covered with primary rainforest. Surface water is scarce as rivers run under the ground through limestone caves. In this more rugged terrain, the Miu live in small hamlets or temporary encampments often consisting of single family units. The Miu share certain customs with other groups of the Kandrian inland region, notably the use of blow pipes to hunt small game animals, the practice of blackening their teeth with local mineral deposits and the now outlawed custom of infant head binding which produces a distorted elongated adult head shape (Chowning, 1980; Goodale, 1985). The area receives little attention from the provincial government due to the difficulty in accessing the steep rugged terrain. Apart from a few aid posts in villages to the south, access to government healthcare is extremely limited. As with the other populations in New Britain, common dermatological infections such as the tropical ulcer and tinea imbricata are widespread. The tropical ulcer is a debilitating condition caused by a polymicrobial infection of spirochaeate and fusiform bacteria (Adriaans, 1988; Falkler et al., 1989). Initial trauma in the form of a scratch develops into a circular lesion which is extremely painful and in some cases may require surgical intervention (Adriaans and Drasar, 1987). Treating tropical ulcers causes a considerable drain on government aid posts with some spending a third of their time and half their budgets treating the condition (Morris et al., 1989). By comparison tinea imbricata is a less debilitating condition, it is characterised by concentric rings of scaly plaques on the skin surface (Satter, 2009). The underlying cause would appear to be the dermatophyte fungus Trichophyton concentricum to which there is evidence of a genetically inherited susceptibility (Ravine et al., 1980). Like the tropical ulcer, tinea imbricata consumes a considerable amount of time of government aid posts with some communities experiencing infection rates reaching 30% (Jamison, 2006). For populations such as the Miu, the inaccessibility of aid posts means that conditions such as the tropical ulcer and tinea imbricata are more likely to have reached a chronic state of infection before treatment is sought. Unlike conventional treatments prescribed in aid posts, plant based medicines are readily available in the surrounding environment. They therefore have a greater potential to treat the early stages of infection than conventional medications located many miles away in primary healthcare centres. Papua New Guinea has developed a national health plan which calls for the integration of effective plant medicines into the national health system with the aim of reducing healthcare costs and preserving traditional knowledge (Mann, 2007). Such medicines need to fall
within acceptable parameters of safety. With this in mind we decided to evaluate plant-slaked lime mixtures used by the Miu to evaluate their effects on fibroblasts viability.
2. Materials and Methods 2.1 Ethnobotanical data collection Consent for this study was obtained from local participants and the government of Papua New Guinea prior to commencement. Informal semi-structured interviews and rainforest walks were conducted with key informants in the village of Miupark to establish a preliminary list of plant names with associated uses. A focus group was used to extend the list of names and collect more detailed use descriptions. This information was then verified against information collected in a separate focus group located several km north from which members of the first group were absent. All interviews were conducted in Neo-Melanesian language. As described previously, care was taken not to overly rely on data from any single informant (Etkin, 1993). 2.2 Collection and identification of plant material Voucher specimens were collected from forest surrounding the village of Miupark in the presence of key informants and preserved in 70% methanol before pressing and drying. Identification of vouchers was carried out by comparison with authentic herbarium specimens at the Royal Botanic Gardens, Kew, UK and the National Herbarium, Lae, PNG where vouchers are lodged. In addition to voucher specimens, plant material for laboratory tests was collected separately from three species. For T. orientalis and S. dulcis material from several leaves of each plant was collected and a mulch produced by hand, this material was divided equally and one half mixed with slaked lime by a key informant according to traditional methods, both portions were then then dried. F. botryocarpa sap was collected early in the morning when informants indicated the sap could easily be tapped. Peduncles were removed from the figs and the resulting white latex allowed to drip into 2 ml plastic vials. 2.3 Extraction of plant material and LC-MS of plant extracts For T. orientalis and S. dulcis, dried plant samples were ground to a powder and 100 mg of each extracted for 1 hour with 1 ml de-ionised water. The resulting extracts along with F. botryocarpa sap were then centrifuged twice in a microfuge at 13,000 rpm to remove debris before filter sterilising through a 0.2 µm filter syringe (Millipore). Liquid chromatography-mass spectrometry (LC-MS) was performed on a Thermo Scientific system consisting of an ‘Accela’ liquid chromatograph and an ‘LTQ-orbitrap XL’ mass spectrometer fitted with an electrospray source. Chromatography was achieved on a 150 mm × 3 mm × 3 ȝm Luna C18(2) column (Phenomenex) using a 400 ȝl/min
mobile phase gradient of 0:90:10 to 90:0:10 (water/acetonitrile/acetonitrile + 1% formic acid) over 60 mins. The mass spectrometer recorded high resolution (30 k) MS1 spectra in positive polarity (m/z 250–2000). 2.4 Isolation and NMR characterisation of ficuseptine A paste consisting of 2 ml F. botryocarpa sap mixed with slaked lime was extracted with 10 ml water three times and freeze dried. The resulting powder was then extracted with MeOH several times and after solvent removal yielded 5.34 mg of a grey amorphous solid. NMR spectra were obtained on a Bruker Avance 400 spectrometer equipped with a BBO probe at 30 °C, using standard Bruker microprograms. Chemical shift values were referenced to residual solvent signals (įH 7.26 and įC 77.0 ppm for CDCl3; įH 3.307 and įC 49.1 ppm for CD3OD). 2.5 Cell viability with human adult dermal fibroblasts Adult dermal human fibroblasts (Lonza) were maintained in Bulletkit medium (Lonza) at 37°C in a 5% CO2 environment according to standard methods. Cells were trypsinised and plated out in transparent 384 well microplates and left to attach. Medium was then changed for fresh medium containing serial dilutions of extracts or compounds alongside vehicles only controls. The cells were incubated for a further 48 h after which CellTiter reagent (Promega) was added 5 µl per well and the plate read at 490 nm in a Tecan Infinite M200 plate reader. For fibroblast stimulation assay, cells were plated out as above and medium subsequently changed for basal medium containing 0.5% FBS whereupon test substances were added. Cell proliferation over 48 h was measured relative to cells cultured in complete medium (non-serum starved). 2.6 Inhibition of B. subtilis B. subtilis NCTC 10400 was grown overnight in Isosensitest liquid medium (Oxoid) to produce a stationary phase culture which was then diluted with liquid medium to an OD600 of 0.02. Dilutions of test samples were made in medium and 100 µl added to wells of a 96 well plate. The same volume of diluted culture was then added to each well and the plate incubated at 37°C for 24 h before inspecting visually.
3. Results 3.1 Medicinal plants of the Miu language group The ethnobotanical survey identified 33 species of plants and one fungal species used as medicines by the Miu. These are detailed in Table 1 along with the number of informants from which the information was acquired. Data was acquired from focus groups carried out in two separate locations where members of each were absent from the other.
Species and
Family
Local name
Description of use
Anacardiaceae
Waringa
The tree trunk is cut and the resulting black
voucher number Semecarpus cf. rostrata Valeton
sap is applied to tinea imbricata skin
103
infections (2 informants focus group 1). Alternatively the black sap may be applied to teeth to produce a desired cosmetic darkening effect. Local minerals are also used to achieve this end (2 informants focus group 2).
Spondias dulcis
Anacardiaceae
Baklum
The leaves are crushed and squeezed by
Parkinson 124
hand to produce a green mulch, then slaked lime prepared from local limestone is added resulting in a colour change from green to yellow. This preparation is then added to tinea imbricata skin infections (3 informants focus group 1). Alternatively the leaves are treated as above without slaked lime and applied to skin to treat localised pain (2 informants focus group 2).
Alstonia cf.
Apocynaceae
Sipa
The sap is drunk to treat a condition similar
scholaris (L.)
in description to asthma (2 informants
R.Br. 116
focus group 1, 2 informants focus group 2).
Trema orientalis
Cannabaceae
Mopo
The leaves are crushed and squeezed by
(L.) Blume 115
hand to produce a green mulch, then slaked lime prepared from local limestone is added resulting in a colour change from green to yellow. This preparation is then added to tinea imbricata skin infections (2 informants focus group 1). Alternatively wood shavings made by scraping the sapwood with a knife blade are squeezed and the resulting fluid is drunk as a cure all for any illness (2 informants focus group 2).
Garcinia dulcis
Clusiaceae
Ekap
The sap is applied to tropical ulcers (3
(Roxb.) Kurz 134
informants focus group 1, 2 informants focus group 2).
Merremia peltata
Convolvulaceae Enovio
Sap is applied to tropical ulcers. Sap may be mixed with slaked lime to produce a
(L.) Merr.127
yellow face paint (3 informants focus group 1, 2 informants focus group 2).
Cheilocostus cf.
Costaceae
Asingi
The rhizome is scraped with a knife and the
speciosus
resulting material applied to tropical ulcers
(J.Koenig)
(3 informants focus group 1, 2 informants
C.D.Specht 128
focus group 2).
Octomeles
Datiscaceae
Aim
Wood shavings made by scraping the
sumatrana Miq.
sapwood with a knife blade are collected in a leaf, water is added to make a dark
126
coloured decoction which is drunk for a condition similar in description to asthma (3 informants focus group 1, 2 informants focus group 2).
Diospyros peekelii
Ebenaceae
Palasoup
The new leaves are chewed and the
Lauterb.
resulting mulch applied to tropical ulcers (3 informants focus group 1, 2 informants
131
Endospermum
focus group 2).
Euphorbiaceae
Urbi/Lukemom Wood shavings made by scraping the
moluccanum
sapwood with a knife blade are squeezed
(Teijsm. & Binn.)
and the resulting fluid is drunk for a
Kurz 120
condition linked to abdominal pain. The same fluid may also be applied to tropical ulcers (3 informants focus group 1). In another use the dry twigs may be burnt on a fire in such a way as to allow the smoke to go into ears to treat ear infections. Alternatively the leaves may be heated on a fire then applied to aches and pains (2 informants focus group 2).
Macaranga similis
Euphorbiaceae
Esul
Wood shavings made by scraping the
Pax. & K.Hoffm.
sapwood with a knife blade are squeezed
109
and the resulting fluid is drunk for a condition whose description is similar to dysentery (2 informants focus group 1, 2 informants focus group 2).
Gmelina
Lamiaceae
Pyvul
Wood shavings made by scraping the
moluccana
sapwood with a knife blade are squeezed
(Blume) Backer ex
and the resulting fluid is applied to large
K.Heyne 113
tropical ulcers (2 informants focus group 1, 2 informants focus group 2).
Desmodium
Leguminosae
Res
Leaves are crushed and squeezed and the
gangeticum (L.)
resulting fluid is drunk to treat diarrhoea (2
DC. 111
informants focus group 1, 2 informants focus group 2).
Gliricidia sepium
Leguminosae
Marmar
Leaves are crushed and squeezed and the
(Jacq.) Walp. 105
resulting fluid is applied to tropical ulcers (2 informants focus group 1, 2 informants focus group 2).
Pterocarpus
Leguminosae
Iku
Wood shavings made by scraping the
indicus Willd.
sapwood with a knife blade are squeezed and the resulting fluid is drunk for a
133
condition resembling dysentery. Alternatively the decoction may be rubbed onto the belly of pregnant women or applied to skin to treat back pain (1 informant focus group 1). Alternatively, wood shavings made by scraping the sapwood with a knife blade are chewed and then applied to tropical ulcers (2 informants focus group 2).
Kleinhovia hospita
Malvaceae
Engalong
Leaves are crushed and squeezed and the resulting fluid is applied to tropical ulcers
L. 104
(2 informants focus group 1). Alternatively the leaves may be prepared in the same way but the fluid drunk to treat coughs and colds (2 informants focus group 2).
Ficus cf.
Moraceae
Kek
The sap is mixed with slaked lime prepared
botryocarpa Miq.
from local limestone deposits causing the
129
sap to change from cream in appearance to bright yellow. The sap is then applied to tropical ulcers (3 informants focus group 1, 2 informants focus group 2).
Ficus gul
Moraceae
Murkarek
Sap from fruit applied to tropical ulcers (3
K.Schum. &
informants focus group 1, 2 informants
Lauterb. 130
focus group 2).
Ficus pungens
Moraceae
Kili
Sap from new shoots is applied to eye
Reinw. ex Blume
infections/inflammation. Alternatively the
112
leaves may be heated on a fire to produce a vapour which is used to treat the same condition (2 informants focus group 1, 2 informants focus group 2).
Ficus septica
Moraceae
Maldonoo
Burm.f. 108
Leaves heated on fire then placed on forehead to treat headaches (2 informants focus group 1). Opinion from the second focus group is that this species can be used to treat many different kinds of illnesses. For example when children have burns from a fire, the leaves are chewed by an adult and then spat onto the burns. Another use is to drink the sap for a disease similar
in description to asthma. (2 informants focus group 2).
Maclura
Moraceae
Golgol
The vine is cut and the fluid that emerges is
amboinensis
drunk to treat a condition similar in
Blume 121
description to asthma (2 informants focus group 1). Alternatively the fluid is drunk for coughs and colds (2 informants focus group 2).
Prainea sp. 118
Moraceae
Mangiru
Leaves are eaten to treat headaches (2 informants focus group 1). Alternatively bark material is placed on the head to treat headaches (2 informants focus group 2).
Filoboletus cf.
Mycenaeceae
Kombo
The whole fungus is heated briefly on a fire
manipularis
and applied to tropical ulcers like a plaster
(Berk.) Singer.*
(3 informants focus group 1, 2 informants focus group 2).
Gymnacranthera
Myristicaceae
Koomoo
Wood shavings made by scraping the
paniculata var
sapwood with a knife blade are squeezed
zipelii (A.DC.)
and the resulting fluid is drunk for
Warb. 114
diarrhoea (2 informants focus group 1). Alternatively the leaves are chewed and then spat onto tropical ulcers (2 informants focus group 2).
Piper aduncum L.
Piperaceae
Pomilil
Leaves are crushed and squeezed and the
107
resulting fluid is applied to tropical ulcers (3 informants focus group 1). Alternatively
the leaves are boiled to make an infusion which is drunk for a non-descript internal illness related to perceived problems with blood and circulation (2 informants focus group 2).
Piper betel
Piperaceae
Eman
The leaves are chewed and the resulting
Blanco*
mulch applied to tropical ulcers (2 informants focus group 2).
Morinda citrifolia
Rubiaceae
Sarelipi
The fruit or roots are boiled and the
L. 125
resulting decoction drunk as a cure all (2 informants focus group 1). Alternative use description is as above only the decoction is drunk to treat a condition resembling asthma (2 informants focus group 2).
Uncaria sp. 102
Rubiaceae
Ayahong
Fresh plant material crushed and squeezed and the resulting fluid placed in ear to treat earache/ear infections (2 informants focus group 1). Alternatively, wood shavings from sapwood are applied to tropical ulcers (2 informants focus group 2).
Homalium
Salicaceae
Meles
Leaves heated on fire and then applied to
foetidum Benth.
tropical ulcers (3 informants focus group 1,
132
2 informants focus group 2).
Solanum torvum
Solanaceae
Kangong
Leaves are crushed and squeezed and the
Sw. 106
resulting green fluid is boiled before applying to tropical ulcers (3 informants focus group 1, 2 informants focus group 2).
Elatostema cf.
Urticaceae
Manil
Wood shavings made by scraping the
angulare
sapwood with a knife blade are mixed with
H.J.P.Winkl. 101
a small quantity of water and applied to tinea imbricata skin infections (3 informants focus group 1). Alternatively the leaves may be applied to tropical ulcers (2 informants focus group 2).
Pipturus argenteus
Urticaceae
Mike
Wood shavings made by scraping the
(G.Forst.) Wedd.
sapwood with a knife blade are mixed with
110
water. People described as having any persistent long lasting illness then wash with it (2 informants focus group 1). Alternatively the sap is drunk by people described as being extremely sick, to help awaken them (2 informants focus group 2).
Poikilospermum
Urticaceae
Sigu
The woody section of the vine is cut and
amboinense Zipp.
the resulting water that flows out is drunk
& Miq. 117
to treat coughs/colds (2 informants focus group 1, 2 informants focus group 2).
cf. Cayratia
Vitaceae
Mohlup
Leaves are crushed and squeezed and the
japonica (Thunb.)
resulting fluid is used to wash tropical
Gagnep. 122
ulcers (3 informants focus group 1, 2 informants focus group 2).
Table 1. Medicinal plants used by the Miu. Voucher numbers (Prescott T.A.K.) are underlined after the species name. Local plant names are given in the Miu language. Use descriptions are presented as described by informants and only assigned to a specific condition e.g. tropical ulcers where a reasonable interpretation could be made.*Indicates common species identified from photos taken in the field.
3.2 Comparison of plant uses between the Miu and inland Kaulong A previous survey of the neighbouring inland Kaulong group identified 31 species of plants used as medicines (Prescott et al., 2012) whereas the present study identified 33 species of medicinal plants and one fungal species. Of these only four species could be unequivocally identified as the same species with the same medicinal use as described for the Kaulong. A comparison of plant uses between the Miu and inland Kaulong is shown in Fig. 2a. Tropical ulcer plant medicines predominate amongst both the Miu and the Kaulong with a similar proportion used for this purpose in both groups. A comparison of mode of preparation between the two groups is shown in Fig. 2b. The use of slaked lime in combination with plant material is a feature shared by both groups.
3.3 Slaked lime does not markedly increase the cytotoxicity of plant material Slaked lime is caustic which suggests plant/slaked lime mixtures should exhibit undesirable cytotoxicity to human skin cells. To address this question, samples of each plant were prepared in the field according to traditional methods, in each case with and without the addition of locally prepared slaked lime. Aqueous extracts of this material were prepared, (for F. botryocarpa sap a filtered sap solution was used), and human adult dermal fibroblasts were grown in the presence of the extracts for 48 hours, then cell viability was measured using CellTiter reagent (Promega). The results in Fig. 3 show that with the exception of T. orientalis where there is a modest decrease in cell viability, the addition of slaked lime does not decrease cell viability relative to identical plant material prepared without slaked lime. The sap of F. botryocarpa is cytotoxic to fibroblasts with minimal cell viability detected above 3% v/v either with or without slaked lime added. Surprisingly, at sap concentrations of 1.25% v/v the slaked lime reduces the cytotoxicity of the sap.
3.4 F. botryocarpa sap contains the alkaloid ficuseptine as the single major component The LC-MS chromatogram of F. botryocarpa sap (Fig. 4) is dominated by a single major component which was isolated and its structure confirmed as the alkaloid ficuseptine through a comparison of the
chemical shift data acquired in CDCl3 (Baumgartner et al., 1990; Bracher and Daab, 2002; Snider and Neubert, 2005). Additionally, two-dimensional experiments (COSY, HSQC and HMBC) were conducted for a confirmation of the chemical shift assignments (see methods section). The structure of the cation is shown in Fig. 4. Ficuseptine has been previously reported from the related species Ficus septica Burm. f. (Baumgartner et al., 1990).
3.5 F. botryocarpa sap inhibits B. subtilis but does not stimulate dermal fibroblast proliferation Previous work reported that ficuseptine inhibits the growth of B. subtilis, Escherichia. coli, Micrococcus luteus and Penicillium oxalicum, (Baumgartner et al., 1990). Although conventional minimal inhibitory concentrations were not reported these results are indicative of broad spectrum antimicrobial activity. B. subtilis is not involved in tropical ulcer pathogenesis, however other Bacillus species have been isolated from tropical ulcers (Woodruff, 1987) although these are not easily cultured in vitro. B. subtilis is a model organism and a useful substitute. F. botryocarpa sap with and without slaked lime were tested at a range of concentrations with B. subtilis NCTC 10400. The minimal inhibitory concentration for the sap alone was 1.5% v/v compared to the sap with slaked lime which was 3% v/v. This mirrors the data with human fibroblasts where the added slaked lime increased cell viability relative to the sap alone at concentrations of 1.25% v/v. Thus slaked lime slightly reduces the activity of ficuseptine or other active principles in the sap. The direct application of sap to the infected tropical ulcer wound surface means these concentrations are easily achievable in vivo. Stimulating fibroblast proliferation may help heal chronic wounds, and although the sap is inhibitory to fibroblasts there remained the possibility that it could stimulate fibroblast proliferation at sub-inhibitory concentrations by diffusing deeper into the wound tissue. Human dermal fibroblasts were treated with the F. botryocarpa sap/slaked lime mixture at sub-inhibitory concentrations for 48 hours, then cell numbers quantified using the CellTiter reagent and compared to non-serum starved cells. The sap/slaked lime mixture does not stimulate fibroblast proliferation (Fig. 4c) suggesting any therapeutic benefits are related to its antibacterial activity. 4. Discussion and conclusion Comparison with data for the inland Kaulong identified only four species used for the same purpose suggesting a high degree of variation in plant use between the two groups. This is surprising given the close proximity of the two groups and their similar language and customs. As the two groups inhabit near identical forest and terrain these differences cannot be attributed to availability of plants and are instead likely to reflect cultural differences. There was also variation in plant use within the Miu language group with one key informant describing medicinal plant use as clan specific rather than language group specific. This suggests a model of plant knowledge in which each clan or large family
group have their own preferred medicinal plants. Thus although there is very frequent intercommunity exchange between populations living in the Whiteman Range, a lack of sharing of medicinal plant knowledge between individual clans has prevented a completely homogeneous system of medicinal plant use from developing. It should be noted however, that as with most ethnobotanical studies, limits on time spent in the field prevent a complete picture of plant use and knowledge from being acquired. Papua New Guinea has developed a national health plan which calls for the integration of effective plant medicines into the national health system with the aim of reducing healthcare costs and preserving traditional knowledge (Mann, 2007). The addition of slaked lime to medicinal plants is found in both the Miu and inland Kaulong groups, however the caustic nature of slaked lime makes such plant preparations less likely to be considered as potential candidates that could gain acceptance in primary healthcare in Papua New Guinea. However, the experiments with dermal fibroblasts revealed that for three species, added slaked lime does not result in a great decrease in fibroblast cell viability when aqueous extracts of the plant/slaked lime mixtures were tested at concentrations up to 10% v/v. This suggests that the quantities of slaked lime added are insufficient to cause a reduction in viable cell numbers by themselves. In practice higher concentrations than this may be reached when such preparations are applied directly to the skin or a tropical ulcer wound surface, however these would be difficult to model in vitro. The sap of F. botryocarpa is mixed with slaked lime and the resulting brightly coloured yellow mixture applied directly to the tropical ulcer wound surface. The sap which consists mainly of the alkaloid ficuseptine is antibacterial. The abundance of sap that flows from the stem of a single fruit when picked from the tree provides a convenient way of applying an antibacterial substance to tropical ulcers. Considering the remote location that some members of the Miu speaking population live in this makes F. botryocarpa sap an attractive alternative to conventional antiseptics that are generally out of reach for them. Although F. botryocarpa sap does not stimulate fibroblast proliferation, it is possible that other plant-based tropical ulcer treatments exhibit antibacterial activity at the wound surface but also promote wound healing by modulating cytokine expression in tissue beneath the wound surface where plant metabolites would be present at sub-antibacterial/cytotoxic concentrations. Further work should be carried out to identify if plants or fungi with such a property exist.
Acknowledgements Funding for this work was provided by the Davis Expedition Fund, University of Edinburgh. We are grateful to Nigel Veitch and Tetsuo Kokubun for structure elucidation of ficuseptine and Dr Brian Spooner for fungal identification. We would also like to acknowledge the help provided by members of the Miu speaking population of New Britain.
References Adriaans, B., 1988. Tropical ulcer – a reappraisal based on recent work. Transactions of the Royal Society of Tropical Medicine and Hygiene 82, 185–189. Adriaans, B., Drasar, B.S., 1987. The isolation of fusobacteria from tropical ulcers. Epidemiology and Infection 99, 361–372. Baumgartner, B., Erdelmeier, C.A.J., Wright, A.D., Rali, T., Sticher, O., 1990. An Antimicrobial Alkaloid from Ficus septica. Phytochemistry 29, 3327–3330. Bracher, F., Daab, J., 2002. Total synthesis of the indolizidinium alkaloid ficuseptine. European Journal of Organic Chemistry, 2288–2291. Chowning, A., 1980. Culture and biology among the Sengseng of New Britain. Journal of the Polynesian Society 89, 7–31. Chowning, A., 1985. Rapid lexical change and aberrant Melanesian languages: Sengseng and its neighbours. Pacific Linguistics C–88, 169–198. Etkin, N.L., 1993. Anthropological methods in ethnopharmacology. Journal of Ethnopharmacology 38, 93–104. Falkler, W.A., Jr., Montgomery, J., Nauman, R.K., Alpers, M., 1989. Isolation of Fusobacterium nucleatum and electron microscopic observations of spirochetes from tropical skin ulcers in Papua New Guinea. American Journal of Tropical Medicine and Hygiene 40, 390–398. Foley, W., 2000. The Languages Of New Guinea. Annual Review of Anthropology 29, 357–404. Goodale, J., 1985. Pig's teeth and skull cycles: both sides of the face of humanity. American Ethnologist 12, 228–244. Iamo, W., 2010. Papua New Guinea’s Fourth National Report to the Convention on Biological Diversity. https://www.cbd.int/doc/world/pg/pg-nr-04-en.pdf Jamison, D., 2006. Disease Control Priorities in Developing Countries, 2nd edition. World Bank, Washington, D.C. Mann, N., 2007. National policy on traditional medicine, Department of Health, Waigani Drive, Port Moresby, Papua New Guinea. Morris, G.E., Hay, R.J., Srinivasa, A., Bunat, A., 1989. The diagnosis and management of tropical ulcer in East Sepik Province of Papua New Guinea. Journal of Tropical Medicine and Hygiene 92, 215–220. Palvides, C.G., C, 1994. 35,000-year-old sites in the rainforests of West New Britain, Papua New Guinea. Antiquity 68, 604–610. Prescott, T.A., Kiapranis, R., Maciver, S.K., 2012. Comparative ethnobotany and in–the–field antibacterial testing of medicinal plants used by the Bulu and inland Kaulong of Papua New Guinea. Journal of Ethnopharmacology 139, 497–503.
Ravine, D., Turner, K.J., Alpers, M.P., 1980. Genetic inheritance of susceptibility to tinea imbricata. Journal of Medical Genetics 17, 342–348. Satter, E.K., 2009. Tinea imbricata. Cutis 83, 188–191. Snider, B.B., Neubert, B.J., 2005. Syntheses of ficuseptine, juliprosine, and juliprosopine by biomimetic intramolecular Chichibabin pyridine syntheses. Organic Letters 7, 2715–2718. Throop, C.T., Throop, L., 1980. The languages and communities of the Kandrian inland region. In: Johnston, R. (ed.), Language, communication and development in New Britain. Summer Institue of Linguistics, Ukarumpa, PNG. Woodruff, A.W.,Wright, S.G., 1987. A Synopsis of Infectious and Tropical Diseases, 3rd ed. IOP Publishing Ltd, Bristol.
Figures
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Fig. 1. Location of the Miu speaking population on the island of New Britain
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Fig. 2. Comparison of medicinal use categories for the Miu with those previously reported for the inland Kaulong (Prescott et al., 2012) using the same use categories (A). Comparison of mode of preparation with the inland Kaulong for medicinal uses (B). Total number of species for Kaulong is 32 compared with 34 for the Miu population. N.B. compared with the inland Kaulong group, the Miu use descriptions are more varied with more than one use for a plant frequently given.
Fig. 3. Human adult dermal fibroblast cell viability after 48 hours treatment with aqueous plant extracts with and without slaked lime. T. orientalis, S. dulcis and F. botryocarpa aqueous extracts without slaked lime (A) and with slaked lime (B). In each graph T. orientalis is top line, S. dulcis middle line F. botryocarpa bottom line. Relative cell numbers were measured using the CellTiter reagent. Error bars show S.E.M. (N=3).
Fig. 4. Orbitrap LC-MS analysis of positive ions of F. botryocarpa sap without slaked lime (A) and with slaked lime added (B). The major component eluting at 16 mins was isolated and identified by NMR, the structure of the cation of the alkaloid ficuseptine is shown (C). F. botryocarpa sap with slaked lime does not stimulate human dermal fibroblast proliferation (D). Cells were incubated for 48 hours with the extract in minimal media. Cell numbers relative to complete medium were then determined with the CellTiter reagent. Error bars (S.E.M N=3) are smaller than data points.
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