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13
Chapter 2
Ways the Lukomir Highlanders of Bosnia
and Herzegovina Treat Diabetes
Jonathan Ferrier, Lana Šačiragić, Eric C. H. Chen, Sabina Trakić,
Ammar Saleem, Emira Alikadić, Alain Cuerrier, Michael J. Balick,
John T. Arnason and Sulejman Redžić
© Springer Science+Business Media New York 2014
A. Pieroni, C. L. Quave (eds.), Ethnobotany and Biocultural Diversities in the Balkans,
DOI 10.1007/978-1-4939-1492-0_2
J. Ferrier () · E. C. H. Chen · A. Saleem · J. T. Arnason
Department of Biology, University of Ottawa, Ottawa, ON, K1N, 6N5, Canada
e-mail: jonathanferrier@me.com
L. Šačiragić
Department of Obstetrics and Gynecology and Newborn Care, The Ottawa Hospital,
Ottawa, ON, Canada
Emcarta Inc., 32 Lewis St., Ottawa, ON, K2P 0S3, Canada
S. Trakić · S. Redžić
Department of Biology, Centre of Ecology and Natural Resources, University of Sarajevo,
Sarajevo, Bosnia and Herzegovina
E. Alikadić
Fondation GEA+, Foundation for Sustainable Development and Environment Preservation,
Sarajevo, Bosnia and Herzegovina
A. Cuerrier
Montreal Botanical Garden, Plant Biology Research Institute, University of Montreal, Quebec
Centre for Biodiversity Science, Montreal, QC, Canada
M. J. Balick
Institute of Economic Botany, The New York Botanical Garden, Bronx, NY, USA
J. Ferrier
Institute of Economic Botany, The New York Botanical Garden, Bronx, NY, USA
Emcarta Inc., 32 Lewis St., Ottawa, ON, K2P 0S3, Canada
Dedicated to the memory of Sulejman Redžić.
2.1 Introduction
Today, diabetes is a pandemic disease and a top health concern in indigenous so-
cieties. The global burden of diabetes was estimated to affect 366 million people
in the year 2011; by 2030, 552 million people are expected to have diabetes (IDF
14 J. Ferrier et al.
2013). Type 2 diabetes (T2D; formerly called noninsulin-dependent or adult-onset
diabetes) results from the body’s ineffective use of insulin and is characterized by
hyperglycemia. T2D is responsible for 90 % of diabetes cases (Alberti and Zimmet
1998) and is linked to rapid social change, genetics, dietary acculturation, excess
carbohydrate consumption, physical inactivity, and excess body weight (Young
1994; Hegele et al. 1999; Young et al. 1992, 2000; Ritenbaugh and Goodby 1989;
WHO 2013a). The World Health Organization (WHO) estimated that Bosnia and
Herzegovina (B&H) had 111,000 people with diabetes in 2000 and was expected to
increase to 180,000 by 2030 (WHO 2013b).
Long-term complications of T2D include vascular disease, heart disease, stroke,
neuropathy, retinopathy, cataracts, atherosclerosis, nephropathy, and impaired
wound healing. Impaired glucose uptake affects the cells of organs that do not
require insulin for glucose uptake (the nervous system, heart, kidneys, and small
blood vessels). As a consequence, these cells have high concentrations of intra-
cellular glucose during elevated hyperglycemic periods, resulting in impaired cell
function and cell death (Ahmed 2005). These T2D complications are mediated by
the formation of advanced glycation end products (AGEs), which are a therapeutic
target with phytotherapies.
T2D incidence is three times higher in indigenous populations. These are some
of the fastest-growing yet most vulnerable populations in the world that often lack
culturally appropriate primary health care (Alberti et al. 2004; Ahmed 2005; Helin
2006). This is true for the Lukomir Highlanders of B&H, one of the last native com-
munities in Europe, located in the Bjelašnica region of the Dinaric Alps (43.6°lat,
18.1°long, 1460 m.a.s.l.), southwest of the capital city Sarajevo. Local health au-
thorities have described diabetes and heart disease as the most prevalent diseases in
Lukomir (Ferrier et al. 2013). One reason for the prevalence of diabetes in Lukomir
is due to a transition from traditional to higher-glycemic diets. Municipal water di-
version was a driver for this transition in Lukomir. This recent postwar development
project removed the source of water from Lukomir’s cereal hydro mills, which in
turn caused the collapse of the mills. This led to a nutrition shift from a traditional
organic multigrain diet to a higher glycemic diet based on soft white wheat flour
(Ferrier et al. 2013). This development has eroded the Highlanders’ traditional life-
style and exercise, transformed habitats of medical flora, and increased the preva-
lence of T2D.
Since Lukomir has no primary health-care facility, our objective was to identify
the plants the Lukomir Highlanders use for the treatment of diabetes and highly as-
sociated symptoms. The plants were ranked using the syndromic importance value
(SIV) function developed by Leduc et al. (2006) for a study of Cree traditional
medicines. To assist future ethnobotanical studies, we present physician-ranked
diabetes symptom weights ( w) required for completing Leduc’s SIV function. As a
pilot pharmacology and phytochemistry study, we investigated the bioactivity and
phytochemistry of one genus that was prominent in the Lukomir pharmacopoeia
and cross-culturally used by our Eeyou Istchee Cree partners.
This ethnobotanical study of plants was conducted between 2007 and 2013 with
the Lukomir community in Bosnia and Herzegovina, and J.F. and L.Š.’s organi-
zation, Cross-cultural Health Initiative (CHI). An international collaboration was
developed with the Partnerships for Tomorrow Program, Phase II (PTP) funded by
152 Ways the Lukomir Highlanders of Bosnia and Herzegovina Treat Diabetes
the Canadian International Development Agency (CIDA). Members of CHI were
hosted by the University of Sarajevo and Foundation GEA+. Research permits were
issued by the Municipality of Konjic and University of Ottawa (H05-09-07), with
prior informed consent from Lukomir’s leaders and informants.
2.2 Methods
2.2.1 Field Research
2.2.1.1 Partnership, Permits, and Prior Informed Consent
This ethnobotanical study of plants was conducted between 2007–2013 with the
Lukomir community in Bosnia and Herzegovina, and J.F. and L.Š.’s organization,
Cross-cultural Health Initiative (CHI). An international collaboration was devel-
oped with the Partnerships for Tomorrow Program, Phase II (PTP) funded by the
Canadian International Development Agency (CIDA). Members of CHI were host-
ed by the University of Sarajevo and Foundation GEA+. Research permits were
issued by the Municipality of Konjic and University of Ottawa (H05-09-07), with
prior informed consent from Lukomir’s leaders and informants.
Fig. 2.1 Lukomir and nearby villages in the Bjelašnica region of Bosnia and Herzegovina.
Taxa in Table 2.1 were collected within black dotted perimeter (~ 26 km2). White dashes indi-
cate roads leading to Lukomir. Numerals indicate meters above sea level (m.a.s.l.). Bobovica
( B = 1300 m.a.s.l.), Čuhovici ( Č = 1330 m.a.s.l.), Donji Lukomir ( DL = 1250 m.a.s.l.), Gornji
Lukomir ( GL = 1460 m.a.s.l.), Studeni Potok ( SP = 1420 m.a.s.l.)
16 J. Ferrier et al.
2.2.1.2 Study Site: Lukomir, Municipality of Konjic, Bosnia
and Herzegovina
A consensus ethnobotany was conducted in the Bjelašnica area of the Dinaric Alps,
in Lukomir, with the Lukomir Highlanders of Bosnia and Herzegovina (Fig. 2.1).
This area is classified as an alpine biogeographic region that is closely bordered
by Mediterranean and continental biogeographic regions (European Environment
Agency (EEA) 2012). Many community members are descendants of a Bogomil
lineage who first settled in Donji Lukomir (Lower Lukomir; 43.632 lat, 18.194 long,
1200 m.a.s.l.) and eventually moved to Gornji Lukomir (Upper Lukomir, com-
monly referred to as Lukomir; 43.637 lat, 18.182 long, 1460 m.a.s.l.). Lukomir’s
informants included spiritual leaders, elders, younger women, and men. Informants
described plants on field trips, garden tours, while shepherding, or in comfortable
settings of their choice. We earned our interview time with Lukomir’s healers by
volunteering our time to shepherd, harvest food, and stack hay. This allowed for
participatory observation, and gave informants more time on field collection trips
and interviews. Notes and photos were taken when participants displayed prepara-
tion methods of plant and natural product remedies.
Land mines were avoided by consulting with Bosnia and Herzegovina Mine Ac-
tion Center (BHMAC; http://www.bhmac.org). All plants were collected on trail
sides or in areas that were constantly traveled by sheep herds, since only parts of the
Lukomir territory were surveyed by BHMAC.
Field work followed a quantitative consensus methodology with individual sem-
istructured interviews during which L. Šačiragić, J. Ferrier, and S. Redžić collected
the following data: (1) specimen voucher number, (2) photo number, (3) common
name, (4) scientific name, (5) family, (6) GPS coordinates, (7) altitude (m.a.s.l.), (8)
habitat, (9) syntaxa, (10) flowering time and description, (11) medically active col-
lection time, (12) use, (13) use category, (14) plant part used, (15) amount used, (16)
preparation method, (17) administration method, (18) dosing regimen, (19) ethno-
graphic details, (20) informant name, and (21) date. Determinations were made us-
ing a Domac’s regional flora (1984), vouchers, and Tropicos.org (2013). Duplicate
vouchers were collected (when sustainable) and are currently held at the University
of Ottawa Herbarium (OTT) with voucher numbers reported in Table 2.1. There are
plans for a herbarium in Lukomir to assist future botanical studies. Vouchers of this
study will be placed in the Lukomir herbarium when available. Plant vouchers and
an iPad (Apple, Cupertino, USA) were used to display collections to elders who
could not venture over the mountainside, or for informant review purposes.
2.2.1.3 Elucidation of Plants for Diabetes Using Syndromic Importance
Values
The SIV function was adapted from Leduc et al. (2006), Oubré et al. (1997), and
McCune and Johns (2002, 2003). SIVs allow ranking of plant species by account-
ing for (1) the number of different symptoms for which a plant was cited, (2) the
frequency of plant citation by individual informants, and (3) the association rank
172 Ways the Lukomir Highlanders of Bosnia and Herzegovina Treat Diabetes
Scientific name †Common name Symptom V# SIV
Achillea millefolium L. –Kunica Diabetes 358 0.00725
Achillea millefolium L. –Kunica Swelling or inflammation 358 0.00725
Anthyllis vulneraria L. – Ranjenik Slow healing infections 372 0.00095
Asarum europaeum L.–Kopitnik,
kopitnjak
Slow healing infections 382 0.00089
Capsella bursa-pastoris
(L.) Medik.
–Rustemača Swelling or inflammation 398 0.00289
Cetraria islandica (L.) Ach – Islandski lišaj Heart or chest pain 403 0.00192
Cichorium intybus L. –Konjanik General weakness 411 0.00235
Cornus mas L. – Drijen Slow healing infections 384 0.00089
Crataegus monogyna Jacq. –Glog, gloginje Back or kidney pain 361 0.00553
Crataegus monogyna Jacq. –Glog, gloginje Diarrhea 361 0.00553
Elymus repens (L.) Gould – Pirika Heart or chest pain 389 0.00299
Elymus repens (L.) Gould – Pirika Increased urination 389 0.00299
Equisetum arvense L. – Preslica Back or kidney pain 367 0.00588
Equisetum arvense L. – Preslica Swelling or inflammation 367 0.00588
Gentiana lutea L. †Lincura Sore or swollen limbs 393 0.00257
Jovibarba hirta (L.) Opiz †Čuvarkuća Slow healing infections 379 0.00363
Jovibarba hirta (L.) Opiz †Čuvarkuća Swelling or inflammation 379 0.00363
Matricaria matricarioides
(Less.) Porter ex Britton
–Kamilica,
Stomaklija
Diabetes 351 0.00775
Matricaria matricarioides
(Less.) Porter ex Britton
–Kamilica,
Stomaklija
Swelling or inflammation 351 0.00775
Mentha longifolia (L.) L. – Nana Swelling or inflammation 349 0.00428
Nepeta cataria L. – Macina trava Swelling or inflammation 369 0.00321
Ononis spinosa L. –Gladišika Increased urination 377 0.00115
Phyllitis scolopendrium
(L.) Newman
–Podrebnica (♂
or ♀)
Heart or chest pain 357 0.00259
Plantago lanceolata L. –Bokvica ♀ Heart or chest pain 359 0.00304
Plantago lanceolata L. –Bokvica ♀ Slow healing infections 359 0.00304
Plantago major L. –Bokvica ♂ Heart or chest pain 360 0.00304
Plantago major L. –Bokvica ♂ Slow healing infections 360 0.00304
Polygonum bistorta L. –Srčanik Heart or chest pain 356 0.00266
Primula veris L. –Jagorčevina Diabetes 373 0.00382
Prunus spinosa L. – Trnjina Blurred vision 405 0.00428
Prunus spinosa L. – Rakija Swelling or inflammation 405 0.00428
Rubus saxatilis L. –Kupina Diabetes 407 0.00342
Salvia officinalis L. †Kadulja Heart or chest pain 348 0.00296
Sambucus wightiana Wall.
ex Wight & Arn.
– Haptovina Heart or chest pain 376 0.00192
Sambucus nigra L. –Zova, zobovina Heart or chest pain 354 0.00266
Satureja montana L. –Vrijesak Diabetes 366 0.00408
Sedum sexangulare L. –Zednjak Slow healing infections 416 0.00086
Table 2.1 Taxa used by the Lukomir Highlanders to treat symptoms of diabetes. Dagger (†) indi-
cates endemism. Determinations followed legitimate names in Tropicos.org (2013)
18 J. Ferrier et al.
of symptoms for which a plant was cited to treat. Four physicians at The Ottawa
Hospital, Ottawa, Canada, who diagnose and treat patients with diabetes, deter-
mined the latter association rank of symptoms. Symptoms were given a weight ( w)
from 1 to 4, where 1 is a symptom highly associated with diabetes; 2, moderately
associated with diabetes; 3, weakly associated with diabetes; and 4 is not at all
associated with diabetes:
Scientific name †Common name Symptom V# SIV
Silene uniflora Roth ssp.
glareosa (Jord.) Chater &
Walters
† Puca Heart or chest pain 353 0.00741
Silene uniflora Roth ssp.
glareosa (Jord.) Chater &
Walters
† Puca Increased urination 353 0.00741
Silene uniflora Roth ssp.
glareosa (Jord.) Chater &
Walters
† Puca Slow healing infections 353 0.00741
Silene uniflora Roth ssp.
glareosa (Jord.) Chater &
Walters
† Puca Swelling or inflammation 353 0.00741
Silene uniflora Roth ssp.
prostrata (Gaudin) Chater
& Walters
† Puca Heart or chest pain 350 0.00741
Silene uniflora Roth ssp.
prostrata (Gaudin) Chater
& Walters
† Puca Increased urination 350 0.00741
Silene uniflora Roth ssp.
prostrata (Gaudin) Chater
& Walters
† Puca Slow healing infections 350 0.00741
Silene uniflora Roth ssp.
prostrata (Gaudin) Chater
& Walters
† Puca Swelling or inflammation 350 0.00741
Smyrnium perfoliatum L. –Ljaljica Increased urination 380 0.00111
Solanum tuberosum L. –Krompir Swelling or inflammation 408 0.00278
Symphytum officinale L. –Gavez Slow healing infections 383 0.00089
Teucrium montanum L. – Iva Diabetes 352 0.00487
Tilia platyphyllos Scop. –Lipa Heart or chest pain 374 0.00215
Tussilago farfara L. –Podbijel (♂ or ♀) Heart or chest pain 371 0.00215
Urtica dioica L. –Žara, kopriva Heart or chest pain 362 0.00222
Urtica dioica L. –Žara, kopriva Slow healing infections 362 0.00222
Vaccinium myrtillus L. – Borovnica Diabetes 368 0.00480
Vaccinium vitis-idaea L. – Brusnica Diabetes 385 0.00441
Vaccinium vitis-idaea L. – Brusnica Slow healing infections 385 0.00441
Verbascum thapsus L. –Divizbina,
divizma
Heart or chest pain 390 0.00200
Vitis vinifera L. – Sirce Swelling or inflammation 409 0.00278
Table 2.1 (continued)
192 Ways the Lukomir Highlanders of Bosnia and Herzegovina Treat Diabetes
SIV=
22
ws wf wf
ws
S SF F
S
++
=
∑∑ ∑
∑
.
The SIV function accounts for w, the weight of the symptom; s, the symptom treated
by the species; f, the frequency of citation for the species; S, the total number of
symptoms used for the survey (used in Leduc et al. 2006, but was not indicated in
the denominator on the bottom right); and F, the total number of interviews in the
survey.
2.2.1.4 Statistics and Phylogenetic Analysis
Statistical analysis was conducted using Prism 6 software. A phylogenetic tree was
constructed using TreeGraph 2.0.47-206 beta, taxon with positive SIVs, and based
on circumscription and topology presented by Angiosperm Phylogeny Group 3
(APG 3 2009) and the Linear Angiosperm Phylogeny Group 3 (LAPG 3; Haston
et al. 2009).
2.2.1.5 Cross-Cultural Consensus
In collaboration with the Eeyou Istchee Cree and their Canadian Institute of Health
Research Team for Aboriginal Antidiabetic Medicines (CIHR TAAM), we com-
pared the Lukomir ethnobotanical taxa with the Cree CIHR TAAM’s taxa (Leduc
et al. 2006) to find consensus specimens for cross-culturally relevant pharmaco-
logical and phytochemical investigations. Both communities contain alpine and
continental-type habitats and are isolated linguistically and geographically from
each other.
Table 2.2 Vaccinium spp. and outgroup A. uva-ursi leaf samples collected in Jahorina, B&H, with
IC50 expressed in µg·mL−1. All sample vouchers were accessioned at University of Ottawa, OTT
Sample IC50 Location Alt. (m) Date Voucher Extract #
V. myrtillus 4.1 Igman, B&H 984 July 1, 2005 417 BBE 134
V. myrtillus 5.4 Jahorina, B&H 1730 July 24, 2005 418 BBE 42
V. myrtillus 17.35 Jahorina, B&H 1730 July 1, 2005 419 BBE 133
V. myrtillus 27.8 Jahorina, B&H 1730 July 5, 2005 420 BBE 125
V. vitis-idaea 48.6 Jahorina, B&H 1730 June 5, 2006 421 BBE 23
A. uva-ursi 100.2 Jahorina, B&H 1735 June 15, 2006 422 ARC 1
20 J. Ferrier et al.
2.2.2 Laboratory Research
2.2.2.1 Extraction of Consensus Specimen and Outgroup Leaf Material
Vaccinium spp. and outgroup Arctostaphylos uva-ursi (L.) Spreng. leaves were col-
lected and identified by S. Trakić, S. Redzić, and J. Ferrier, and deposited at the
OTT (Table 2.2). Samples were dried at 35 °C and ground in a Thomas Wiley mill
(1 mm mesh). Plant material was extracted twice in ethanol/water (95:5 %) at room
temperature for 24 h per phase. The phases were centrifuged at 1000 ×g, filtered,
pooled, dried using a Speed Vac (Savant, Halbrook, NY), and lyophilized (Edwards
Super Modulyo Freeze Drier, Fisher Scientific, Ottawa, Canada).
2.2.2.2 Inhibition of Advanced Glycation End Products by Consensus
Genera Species
Inhibition of AGE formation was assessed as described by Farsi et al. (2008) with
modifications. Bovine serum albumin (BSA; 1 mg mL−1) was incubated with
100 mmol L–1 glucose/ 100 mmol L–1 fructose in sodium phosphate monobasic
monohydrate buffer (pH 7.4) with extract (experimental treatment), ethanol/water
(4:1) (negative control), or quercetin, an antioxidant against glycation by way of
phenolic hydroxyl groups in the flavonoid structure (24 µg mL−1 in assay; Sengupta
et al. 2006), which served as a positive control. To control for fluorescence of ex-
tracts, a treatment without BSA was included. To control for fluorescence of BSA, a
treatment with BSA and vehicle was included. Stock solutions of extracts were seri-
ally diluted and tested at five concentrations that were optimized for dissolution and
a linear concentration response (40, 20, 10, 5, and 2.5 µg mL−1 in well concentra-
tion). Three replicates were tested in sterile opaque polystyrene 96-well clear bot-
tom plates (Corning Inc., New York, NY, USA). Plates were covered, sealed with
Parafilm, and incubated for 7 days at 37 °C while shaking. Following incubation,
fluorescence was measured using a microplate reader (SpectraMax M5; Molecular
Devices, Sunnyvale, CA, USA) at excitation and emission wavelengths of 355 and
460 nm. Glucose/fructose and ethanol/water fluorescence was subtracted from all
results, and percent inhibition and IC50 values were calculated as previously de-
scribed (Farsi et al. 2008).
2.2.2.3 HPLC–MS Analysis of Vaccinium Species
Stock solutions of extracts were prepared to 10 mg mL−1 in ethanol/water (80 %:20 %)
and filtered through a 0.2 μm PTFE nonsterile filter (Chromatographic Specialties
Inc.). High-performance liquid chromatography (HPLC) analyses were performed
using an Agilent 1100 chromatographic system (Agilent Technologies Inc.) consist-
ing of an autosampler, quarternary pump, and diode array detector (DAD). Sol-
vents were of HPLC grade (Fisher Scientific), and trifluoroacetic acid (TFA) was of
212 Ways the Lukomir Highlanders of Bosnia and Herzegovina Treat Diabetes
analytical grade (J.T. Baker). A Syngeri RP-Polar column (150 mm × 3 mm; 4 μm
particle size) was kept at 53 °C and a flow rate of 0.5 mL min−1 was maintained. The
mobile phase consisted of aqueous TFA (0.05 %; solvent A), acetonitrile with TFA
(0.05 %; solvent B), and methanol (solvent C). Initial conditions were set at 95:5:5 %
(A:B:C) and changed following a linear gradient of 5–9.2 % B and 5–17.5 % C in
25 min. The column was then washed by increasing solvent B to 100 % over 5 min
and returned to initial conditions in the next 5 min. The column was allowed to re-
equilibrate for 5 min, resulting in a total run time of 40 min. Ten microliters of each
10 mg mL−1 leaf extract were injected for each run, and the elution profiles were
monitored at 350 , 280 , and 230 nm with bandwidth kept at 4.
Phytochemical constituents were identified based on comparison to retention
times and ultraviolet (UV) spectra of pure standards (95 % purity) relative to a pro-
grammed library of known UV spectra, and further confirmed by mass spectrometry
(MS) fragmentation patterns. Standard compounds used to monitor leaf extracts
were (+)-catechin, chlorogenic acid, quercetin-3-O-glucoside, (Extra-synthèse),
rutin, quercetin-3-O-rhamnoside (Sigma) paracoumeric acid, taxifolin, quercetin-
3-O-galactoside, and myricetin (source unknown).
2.3 Results and Discussion
We were able to interview 25 informants who described plant uses on mountain
and canyon field trips by volunteering to shepherd, collect food, and hay with the
Highlanders between 2008 and 2010. Informants provided information on diabetic
symptoms and did not initially provide any diabetes use reports, but during subse-
quent conversations on casual visits to Lukomir, many healers agreed that pana-
cea remedies should be used to treat diabetes. Highlanders described eight panacea
treatments, which were subsequently treated as a diabetes SIV use report.
All eight species used as “diabetes” treatments were cited as čaj (infusions).
In one household’s case, we noticed that some of these taxa were present in their
čaj collection, but were not cited as medicine: Teucrium montanum L., Salvia of-
ficinalis L., and one specimen never mentioned, Phyllitis scolopendrium (L.) New-
man, ( podrebnica1). When we noticed this čaj collection, we asked why they were
not mentioned. The response: “We drink čaj for our health.” These plants were for
their “medicine pot,” that were prepared with a prayer after meal consumption. This
household also has one family member with T2D managed by prescription medi-
cines. Perhaps these plants are best referred to as Pieroni and Quave (2006) describe
1 Redžić (2008) translated Podrebnica, “under the ribs,” and explained that the name refers to the
fern’s sori aligned in a chevron-like pattern on the ventral side of the frond, which resembles a rib
cage. This pattern spurred the doctrine of signatures for usage of P. scolopendrium as a treatment
for ailments under the ribs.
22 J. Ferrier et al.
them: “medicinal foods or food medicines”—prepared to obtain “medical action,”
consumed in a “food context,” and in this case, not always cited as medicine.
The Lukomir Highlanders used 41 species for the treatment of diabetes symp-
toms (see Table 2.1). Antidiabetic reports (in parentheses) were of plants situated
in five general habitats: grassland (21), village and shepherd trails (12), mountain
slopes (9), riparian zones (6), rocklands (6), deciduous forest (4), and cultivated (2).
Antidiabetic preparation methods were infusion (46), poultice (7), food (3), bever-
age (2), ear drops (2), juice (1), foot bath (1), eye wash (1), and tincture (1). The
frequency of taxa to treat diabetes and associated symptoms were heart and chest
pain (15), swelling or inflammation (13), slow healing infections (12), diabetes/
panacea (8), increased urination (5), back or kidney pain (2), diarrhea (1), blurred
vision (1), general weakness (1), and sore or swollen limbs (1).
The Lukomir Highlanders mentioned five endemic species (†, Fig. 2.2). Endemic
taxa account for 20 % of use reports: Jovibarba hirta (L.) Opiz (Crassulaceae),
Silene uniflora ssp. glareosa (Jord.) Chater & Walters, Silene uniflora Roth ssp.
prostrata (Gaudin) Chater & Walters (Caryophyllaceae), Salvia officinalis L. (La-
miaceae), and Gentiana lutea L. (Gentianaceae) were also listed as endangered (see
Fig. 2.2).
2.3.1 Ways the Lukomir Highlanders Treat Diabetes
and Associated Symptoms
In order to rank taxa from interviews with the SIV function, the weight ( w) of
diabetes and 15 associated symptoms were given association to diabetes values by
four physicians: increased thirst (1), slow healing infections (1), increased urination
(1.25), foot numbness or foot sores (1.25), blurred vision (1.75), diarrhea (2.25),
heart or chest pain (2.25), abscesses or boils (2.25), frequent headaches (2.75),
general weakness (2.75); increased appetite (2.75) and sore or swollen limbs (3);
arthritis or rheumatism (3.25) and back or kidney pain (3.25); and swelling and/or
inflammation (3.25) and diabetes (4).
SIVs of species were presented in Table 2.1. The top five SIV factors (in paren-
theses) belonged to Matricaria matricarioides (Less.) Porter ex Britton (0.0078),
Silene uniflora ssp. glareosa (0.0074), Silene uniflora ssp. prostrata (0.0074),
Achillea millefolium L. (0.0073), and Equisetum arvense L. (0.0059). Each spe-
cies’ SIV was multiplied by 100,000 and presented as wedges to infer phylogenetic
importance at various taxonomic levels (see Fig. 2.2). The top five families with
the highest average SIVs were Caryophyllaceae (0.0074), Equisetaceae (0.0059),
Asteraceae (0.0049), Ericaceae (0.0046), and Rosaceae (0.0044). Considering the
factors and framework of the SIV function, families and genera in Fig. 2.2 with large
SIV wedges have potential in delivering new medicines for diabetes. Furthermore,
studies have indicated that targeting closely related taxa is a sufficient strategy for
bioactivity screening (Cox and Balick 1994; Rønsted et al. 2008; Saslis-Lagoudakis
et al. 2011). However, taxa that have strong phylogenetic signal (see Fig. 2.2) plus
cross-cultural consensus have been demonstrated to contain more bioactive plants
than random samples (Saslis-Lagoudakis et al. 2012).
232 Ways the Lukomir Highlanders of Bosnia and Herzegovina Treat Diabetes
2.3.2 An Intriguing Cross-Cultural Consensus Among
the Lukomir Highlanders and the Cree of Eeyou Istchee
Two consensus families emerged when Leduc’s study (2006) was compared with
Table 2.1: Ericaceae and Rosaceae. Vaccinium of the Ericaceae was the only genus
with cross-cultural consensus. There was one consensus species, V. vitis-idaea L.,
which was used by three Cree healers and two Lukomir healers for slow-healing
infections and urinary infections. Vaccinium myrtillus L. was used by four High-
lander healers as a panacea. V. angustifolium Ait. was used by three Cree healers for
Fig. 2.2 Clustering of taxa used to treat diabetes and associated symptoms of diabetes. Average
SIVs for each family are presented and daggers (†) indicate endemic taxa. Double daggers (‡)
represent orders not delimited by the APG 3 (APG 3 2009). Topology is based on LAPG 3 (Haston
et al. 2009). Branch lengths are not to scale
24 J. Ferrier et al.
frequent headaches. SIV factors from Lukomir ranked V. myrtillus as 8/41 plants
and V. vitis-idaea as 9/41 plants (see Table 2.1). SIV factors from the Cree survey
ranked V. angustifolium as 11/15, and V. vitis-idaea as 13/15. SIVs indicate that
Vaccinium spp. are more important for the Highlanders than the Cree for treating
diabetes.
2.3.3 Inhibition of AGEs
Both V. myrtillus and V. vitis-idaea showed linear concentration-dependent inhi-
bition of AGEs with activity higher or comparable to the positive control quer-
cetin (see Table 2.2). V. myrtillus ( n = 3 at 50 µg mL−1 initial concentration and
n = 1 at 40 µg mL−1 concentration) had an average IC50 of 12.4 µg mL−1, and V.
vitis-idaea ( n = 1) had an IC50 of 48.6 µg mL−1. The data for the most active species,
V. myrtillus, are shown (Fig. 2.3). Vaccinium spp. herein had approximately 61 %
greater activity than outgroup A. uva-ursi (IC50 = 100.2 µg mL−1). These results were
comparable to IC50’s from wild samples of V. angustifolium Aiton (McIntyre et al.
2009) ranging from 4.8 to 10.6 µg mL−1 with a seasonal mean of 6.3 µg mL−1.
Vaccinium poasanum Donn. Sm. was the most active taxon (IC50 = 4.2 µg mL−1) in
an environmental and date-controlled study consisting of numerous tropical speci-
mens (Ferrier et al. 2012). Considering this comparison, and knowing that AGEs
contribute to the development of retinopathy, cataracts, atherosclerosis, neuropathy,
nephropathy, diabetic embryopathy, and impaired wound healing, the Highlanders’
access to and use of V. myrtillus (Borovnica) is appropriate as a panacea remedy
and may be an effective means of preventing AGE-related insults, which should be
studied clinically.
Fig. 2.3 Inhibition of advanced glycation end products by V. myrtillus leaf extract. Half maximal
inhibitory concentration (IC50) equals 17.35 µg·mL−1
252 Ways the Lukomir Highlanders of Bosnia and Herzegovina Treat Diabetes
2.3.4 HPLC–MS: V. myrtillus and V. vitis-idaea Markers
HPLC–MS analysis of V. myrtillus and V. vitis-idaea was compared with pure
standards of (+)-catechin, chlorogenic acid, paracoumaric acid, taxifolin, quer-
cetin-3-O-galactoside, quercetin-3-O-glucoside, rutin, quercetin-3-O-rhamno-
side, and myricetin. Rutin was absent in both species (Fig. 2.4). V. myrtillus
and V. vitis-idaea were separated based on the presence of (+)-catechin in V.
myrtillus and quercetin 3-O-glucoside in V. vitis-idaea. All other metabolites
identified were common to both species. Many of these compounds are good
antioxidants and/or reported in other studies as active in antiglycation agents
(McIntyre et al. 2009).
2.4 Conclusion
This study has identified species of interest for studies of complementary T2D
medicines, while our analysis of Vaccinium spp. from Lukomir demonstrates the
high activity of at least this genus and identifies its major constituents. There are
both medicinal and many more food species utilized by the Lukomir Highlanders
that remain to be studied. For example, phenolics and saponins likely play an im-
portant role in radical scavenging and cholesterol-binding activity in the Lukomir
diet as they do in pastoral Maasai communities (Lindhorst 1998; Chapman et al.
1997; Johns et al. 1999). Eventually, clinical work is required to validate safe and
effective use. Reinforcement or revival of traditional medicines and dietary plants
Fig. 2.4 HPLC chromatogram with DAD at 280 nm of V. myrtillus leaf sample. Numbers indicate
presence of chlorogenic acid ( 2), para coumeric acid ( 3), taxifolin ( 4), quercetin-3-O-galactoside
( 5), quercetin-3-O-glucoside ( 6), quercetin-3-O-rhamnoside ( 8), and myricetin ( 9). Rutin and
(+)-catechin were absent
26 J. Ferrier et al.
with antidiabetic activity is especially important in these remote communities
where modern health care is limited but traditional complementary medicines are
abundant.
Acknowledgments We would like to thank the Lukomir Community for their hospitality and for
sharing their knowledge and physicians for their symptom assessments: Rosie Dell, Fadi Hama-
dani, Lawrie Hamilton, and Megan Wilson. Adnan Šačiragić, Ejla Salihamidžić, Elvira Jahić,
Harun Alikadić, the Kunjundzić family, Hana Zurić, Fondacija GEA+, and Michel Rapinski, for
field, language, logistical support, and manuscript review. Indspire, the Canadian International
Development Agency (CIDA), and the Alexander Graham Bell PGS award from the Natural
Sciences and Engineering Research Council of Canada (NSERC) supported Jonathan Ferrier’s
research.
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