2. ENVIRONMENTAL ChEMISTRy & TEChNOLOGy 2.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Environmental Chemistry & Technology
P96 ThE bIOLOGICAL ACTIVITy OF 16α(H)-
PhyLLOCLADANE ISOLATED FROM
SLOVAK bROwN COAL
AnTOn ZUBRIK a , AnDREA LAUKOVá b , ALEnA
GáBELOVá c , ZUZAnA VALOVIČOVá c , ľUDMILA
TURČánIOVá a and JOSEF CVAČKA d
a Institute of Geotechnics, Slovak Academy of Sciences, Watsonova
45, 043 53 Košice, Slovakia,
b Institute of Animal Physiology, Slovak Academy of Sciences,
Šoltésovej 4–6, 040 01 Košice, Slovakia,
c Cancer Research Institute, Slovak Academy of Sciences,
Vlárska 7, 833 91, Bratislava, Slovakia,
d Institute of Organic Chemistry and Biochemistry v.v.i., Academy
of Sciences of the Czech Republic,
Flemingovo nám. 2, 166 10 Prague 6, Czech Republic,
zubant@saske.sk
Introduction
The coal research brings novel opportunities for nonfuel
utilization. One of the promising ways is extraction and
isolation of organic compounds (diterpenes, humic acids)
with potential biological activity. The Slovak coals (nováky
lignite and Handlová brown coal) contain high amounts of
tetracyclic diterpene hydrocarbons 1,2 , which can be valuable
precursors for pharmaceutical products. Kauranes and phyllocladanes
are important diastereoisomeric tetracyclic diterpene
biomarkers. Tetracyclic diterpene derivates isolated
from plants show a strong biological activity and they are
active components of many plant medicaments and extracts
used in folk medicine. Kauranes exhibit an anti-microbial,
anti-HIV, anti-inflamatory, anti-fertility, antiparasitic, insect
anti-feedant, antifungal and cytotoxic activities 3 . Phyllocladanes
are very rare in the nature; nevertheless, they were
also identified and isolated from several plants. Orizaterpenol
(phylloklad-15-en-6β,14βdiol) from the rice hull of Oryza
sativa shows cytotoxicity against murine leukemia cells 4 .
The use of phyllocladane diterpenoids for plant growth promotion
and/or retardation is already patented 5 .
The biological activity of tetracyclic diterpenoids isolated
from the geological samples has not been studied yet,
therefore the main goal of our work was to isolate tetracyclic
diterpane from the coal and test its biological activity in
vitro.
Experimental
I s o l a t i o n o f 1 6 α ( H ) - P h y l l o c l a d a n e
f r o m S l o v a k B r o w n C o a l
The brown coal (50 kg) was collected in several places
of the Handlová mining area (Fig. 1.) and a representative
sample was obtained by quartation.
Before the extraction; the Slovak brown coal from Handlová
was physically treated on hydrocyclone 7 to obtain
quality carbon product with A d (ash content) = 7.70 % wt.,
W a (moisture) = 12.26 % wt., C d = 61.6 %, H d = 5.6 %,
n d = 0.7 %, S d = 1.2 %, O d = 23.2 %. Further, the coal (20 g)
s534
Fig. 1. The situation map of Handlová-Nováky coal basin with
Handlová mining area in central part of Slovakia (adapted from
ref. 6 )
was extracted with dichloromethane (250 ml) in microwave
oven (Wirlpool AVM 434, 500 W, 2.45 GHz) in distilling
flask with reversing reflux system (20 min). After the extraction,
the mixture was filtered and the solvent was evaporated.
The crude extract (367 mg) was re-extracted on SPE column
(Chromabond SiOH, Macharey – nagel GmbH + Co. KG)
with n-hexane (100 ml). The solvent was evaporated and
the purified SPE extract (112 mg) was further separated by
column chromatography on silica gel (5 g of Kieselgel 60,
granularity 0.06 – 0.2 mm, Carl Roth GmbH + Co. KG) with
4 × 20 ml of hexane to four fractions denominated as F1–F4.
All fractions were analyzed by TLC and GC/MS. 16α(H)phyllocladane
was found in the F1 fraction. TLC analysis
of F1 showed presence of a single spot with R F = 0.91, that
contained three compounds: the most abundant component
(81.6 %) was 16α(H)-phyllocladane; second one was isopimarane
(11.2 %) and finally 18-norisopimarane (3.9 %).
Unambiguous identification as 16α(H)-phyllocladane was
achieved by coinjection of the sample and standards in the
laboratory of Prof. Bernd Simoneit (Oregon State University,
USA). The final isolate (36.7 mg) named as TD1 containing
81.6 % of 16α(H)-phyllocladane was used for further biological
tests.
B a c t e r i a l S t r a i n s
All tested Gram + and Gram – bacteria (Enterococcus spp.,
β-hemolytic streptococci, Staphylococcus aureus CCM 3953,
S. aureus SA5, Listeria innocua LMG 13568 Enterobacter
cloacae 5139, 19259, 4417, Klebsiella pneumoniae 1962,
12506, 5182, 1366, Serratia marcescens 19194, Salmonella
enterica serovar Duesseldorf SA31, S. enterica ser. enteritidis
PT4, Shewanella putrefaciens, E. coli K88, E. coli 6295,
5765) were of human or veterinary origin (isolated from the
various wounds, blood, sputum and faeces). The strains were
maintained on the appropriate selective media according to
ISO.